Nanomedicine in Ophthalmology

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Chapter 36 Nanomedicine in Ophthalmology

Marco A. Zarbin, James F. Leary, Carlo Montemagno, Robert Ritch, Mark S. Humayun

Nanotechnology provides an important new set of tools for the diagnosis and treatment of ocular diseases. Miniaturization of devices, chip-based technologies, and novel nanosized materials and chemical assemblies already provide novel tools that are contributing to improved healthcare in the 21st century and will impinge directly on ophthalmology.¹^–⁴ In this chapter, we review general principles of nanotechnology and nanomedicine as well as properties of nanomachines. We also consider specific and potential applications of nanotechnology to ophthalmology, including drug, peptide, and gene delivery; imaging; minimally invasive physiological monitoring; prosthetics; regenerative medicine; and surgical technology. Finally, we consider obstacles to incorporation of nanotechnology into ophthalmology. Each of these topics has been reviewed in detail previously.⁵^–⁷

General principles of nanotechnology and nanomedicine

Nanotechnology

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 <100 nm. (Recall that an average man is 1.6 meters (1.6 billion nanometers (nm) ) tall; an erythrocyte is 7 µm (7 × 10³ nm) wide; and a strand of deoxyribonucleic acid (DNA) is 2 nm wide.) Transformational opportunities in information storage, computation, and mechanical efficiency are available through nanotechnology.

Regarding information storage, Richard Feynman, who conceived of the field of nanotechnology, calculated that it was possible to write the entire 24 volumes of the Encyclopaedia Britannica on the head of a pin.⁸ If one did not simply etch the letters on to the surface of the pin but also used the interior of the material also, he calculated that one could fit all the information that humans had accumulated up to December 1959 (estimated at 10¹⁵ bits) in a cube of material 1/200 inch wide, comparable to the size of a piece of dust.⁸ Today, through nanotechnology-based precision assembly of matter, storage densities of 10¹¹ bits per cm² have been demonstrated, which closely approximates Feynman’s vision.⁹ Efficient information storage is crucial for the complexity of biological systems, as each eukaryotic cell stores an enormous amount of information. A retinal pigment epithelial (RPE) cell has a diameter of approximately 4 × 10^–4 inches (1 × 10^–3 cm), and each cell stores the blueprint to create an entire human in DNA molecules (3 billion chemical basepairs, ~25 000 genes).

Regarding computation, Feynman also noted that biological systems do not simply store information, they create measurable outputs. The human brain has the capacity to make judgments, e.g., recognize a person’s face (even if shown at different distances, under different lighting conditions, at different angles), or play chess. Feynman reasoned that if computers could have as many computational elements as our brains, they could make judgments as well.⁸ Today, sophisticated facial recognition can be accomplished with a powerful laptop computer (versus poorly in 1959, with a much larger computer), due to the development of microprocessors and sophisticated software. Defeating a grand master at chess, however, requires a supercomputer. A Cray XT5 supercomputer uses ~40 kW power/cabinet, and each cabinet measures ~81 × 23 × 57 inches³ (larger than a refrigerator) and weighs ~1530 lb (694 kg) (http://www.cray.com/downloads/CrayXT5Blade.pdf). It is remarkable that the “computer” in our cranium does not require the amount of rare elements, generate the heat, or have the energy requirements of a supercomputer. Thus, the evolution of our cognitive capacities from infancy to adulthood (derived from the interaction between a DNA template-guided, manufactured neuronal network and the external environment) is one demonstration that it is possible to develop nanoscale mechanical systems that create complex, measurable outputs.

Nanomachines are highly efficient.¹⁰ When organized in massively parallel structures, for example, nanomotors can generate large forces (e.g., muscles that move massive animals such as whales) or large electrical currents (e.g., those generated by the Hunter’s organ of electric eels). Nanomotors also can direct delicate processes such as ion transport and chromosomal migration during mitosis. Nanomachines are not only highly efficient, they typically have long operational half-lives and are mass-produced easily.

Nanomedicine

The aim of nanomedicine is the comprehensive monitoring, control, construction, repair, defense, and improvement of human biological systems at the molecular level, using engineered nanodevices and nanostructures, operating massively in parallel at the single-cell level, performing “single-cell medicine,” ultimately to achieve medical benefit.¹¹ Integration of nanoscale technologies with the practice of medicine will alter profoundly our approach to the diagnosis, treatment, and prevention of disease.¹² We will begin to diagnose and treat diseases at the single-cell level, for example, rather than just at the organ level.

General principles of nanotechnology as applied to nanomedicine include:

The functional properties of living systems arise not only from their component parts, but also from how these parts are assembled, which dictates interactions between the parts, the nature and flow of information within the system, and the outputs that the system produces.⁶ Thus, one concept from biology that may be important for development of nanomachines in medicine is that spatial control of the distribution of nanomachines directly affects the efficiency of the macromolecular assembly and nature of this assembly’s work product.²⁷ Spatial control can be achieved through the use of membranes and anchoring molecules that place enzymes and substrates in proximity (e.g., as occurs in the endoplasmic reticulum for synthesis of ECM proteins, in the mitochondrial membrane for electron transport, and on the cell surface for ECM ligand integrin-mediated changes in intracellular signaling).²⁸ This concept is exploited in the area of neural prosthetics, as described later in this chapter. Conversely, this engineering approach also permits segregation of molecules (e.g., segregation of lysosomal enzymes from the cytoplasm). Microelectromechanical systems (MEMS) or nanoelectromechanical systems (NEMS)-based techniques can be used to create engineered scaffolds (see next paragraph) that achieve this spatial control. One might also use synthesized lipid bilayer membranes, as occurs in nature.

One can produce nanomachines by assembling naturally occurring ones.^10,²⁹^–³² M/NEMS-based engineering, however, permits the construction of small devices using computer-aided design and by the repeated application of a number of procedures, including oxidation, photolithography, etching, diffusion, sputtering, chemical vapor deposition, ion implantation, and epitaxy, as illustrated by the devices described later in this chapter. Control of features down to the submicron level permits production of mechanical structures at length scales ranging from 100 nm or less to greater than 1 cm. The ability to create complex microfabricated biomaterial substrates using these techniques enables one to define surface microarchitecture, topography, and feature size. By engineering the microenvironment, one can control individual cell responses utilizing structures at the micro- and nanoscale to alter cellular attachment and motility, attenuate the foreign-body response, simulate tissue organization, and promote cell differentiation.³³^–⁴⁰

A straightforward application of nanomachines to nanomedicine is the use of error-prone polymerase chain reaction and transposons to nanoengineer novel structures (e.g., viral capsids) as vectors for gene and drug delivery (as discussed later in this chapter). A somewhat less obvious application of nanomachines exploits their capacity for energy transduction. Motor proteins, for example, transduce the following forms of energy: (1) optical and electro-osmotic; (2) optical and electronic; (3) chemical and electro-osmotic; (4) chemical and electronic; (5) mechanical and electro-osmotic; and (6) mechanical and chemical. By exploiting the transducing properties of nanomotors, one might be able to develop nanomachines that measure biological variables: oxygen tension, pH, glucose concentration, the redox state of a cell or subcellular organelle, temperature, intraocular pressure (IOP), or blood pressure. Alternatively, one can use nanomachines to induce energy sensitivity where it does not normally exist, e.g., creation of light-sensitive retinal ganglion cells (RGCs, as described later in this chapter) or to provide therapeutic molecules to cells, e.g., wild-type alleles of retinal proteins to blind mutants (described later in this chapter). Finally, one might couple the measuring and therapeutic capacity of nanomachines to create devices that measure critical biological variables (e.g., redox state) for diagnostics and provide therapy (e.g., antioxidant therapy) at the appropriate time and place and in the appropriate quantity, a concept termed theragnostics (discussed later in this chapter).

Properties of nanomachines ⁸

Physical properties

Feynman noted that, at the size scale of intracellular structures and molecules, materials acquire seemingly surprising properties that are predictable based on the principles of quantum physics.⁸ For instance, carbon becomes stronger than steel; gold melts at room temperature, and aluminum becomes highly explosive. Quantities such as weight and inertia are of relatively little importance.

Inhomogeneity of materials (e.g., metals versus plastics) might limit their utility.⁸ Magnetic properties on a very small scale are not the same as on a large scale. Lubrication is not needed if the machine is small enough because heat loss is rapid (due to the large surface area:volume ratio). Some of these properties produce unexpected results. For example, rapid heat loss might prevent gasoline from exploding, which would make a nanoscale internal combustion engine impossible.⁸

The influence of gravity on the function of true nanomachines probably is negligible because their mass is that of atoms (F_g = Gm_Am_B/r²).⁸ On the other hand, the distance between the elements is in nanometers. Because of van der Waals forces, parts of nanomachines might adhere to each other, which might not be desirable. Electrical resistance may be very large with nanocircuits, a feature that might be useful in biological systems. Another problem with nanocircuits is the inverse relationship between the size of the device and the amount of noise generated (Hooge’s rule). The development of stacked graphene sheets may provide a solution to this problem and facilitate the development of circuits much smaller than those in conventional silicone-based computer chips.⁴¹ However, when working on the scale of atoms, circuits might not be needed, and quantized energy levels might be manipulated for energy transfer according to the laws of quantum mechanics. This property is exploited in some nanoparticle-based imaging technologies.

Axial ratio is the ratio of the magnitude of the axes (e.g., length and width in the case of a two-dimensional object), the greater being divided by the lesser. Nanotubes and nanofibers have very large axial ratios. Synthetic methods exist to alter the length-to-volume ratio and thus the physical properties of the material. Conductivity, for example, is enhanced as the diameter of semiconducting nanotubes is reduced.

A key property of nanomaterials is that they are surface-rich objects in relation to volume. The following analogy illustrates this concept. A baseball filled with membrane surface-active enzymes would have only a minimal number of these extending to the surface of the ball, so the vast majority would be inactive. A pea filled with enzymes would have a greater proportion extending to the surface. A sphere 100 nm in diameter would have the majority of the enzymes it contains in an active state. This property results in a situation in which there is relatively limited unutilized surface area on to which one might attach molecules to the nanoparticle surface. The net effect is to create structures that accelerate many chemical reactions at their surfaces and act in some ways like very active enzymes.

Vacancy-engineered mixed-valence state cerium oxide (CeO₂) nanoparticles (nanoceria) illustrate the useful properties that materials can develop at the nanoscale. Alteration in the oxidation state of CeO₂ creates defects in its lattice structure via loss of oxygen or its electrons. As their size decreases, nanoceria (3–5 nm diameter) demonstrate formation of more oxygen vacancies in the crystal structure.^42,⁴³ As described later in this chapter, vacancy-engineered nanoceria may function as a highly effective treatment for ocular conditions associated with oxidative damage.

Manufacture

At each step in the process of manufacturing smaller and smaller machines, one must improve the accuracy of the equipment.⁸ Feynman speculated that if devices are built on the scale of 5–10 atoms, then it should be possible to mass-produce them such that they are perfect copies of each other.⁸ One useful outcome of a “nanomanufacturing” process is that the material costs of billions of these machines would be minimal since each is so small that minimal material is used. Nanomachines can have the capacity to repair and build themselves. Indeed, most nanoparticle structures are self-assembling under the proper thermodynamic conditions, allowing for production of a large number of virtually identical nanostructures.

Some properties of nanomachines are illustrated by the design and manufacture of an axon surgery platform. Using microtechnology, electrokinetic axon manipulation (i.e., dielectrophoresis), and cell fusion (i.e., electrofusion), Sretavan et al.²⁴ developed a paradigm of direct axon repair involving the substitution of damaged axon regions with healthy segments from donor axons. This multidisciplinary group developed a multifunctional axon surgery platform that is ~1 mm³ (Figs 36.1 and 36.2). The cutting device consists of a silicon nitride knife with an ultrasharp knife-edge mounted on to a silicon-based compliant knife suspension (Fig. 36.1). The knife edge’s radius of curvature (~20 nm) is similar to the diameter of a single microtubule. Because the knife is manufactured from a silicon nitride membrane, it is nearly transparent, which permits visualization of axons during the cutting procedure. The mechanical compliance of the suspension can be varied to deliver sufficient force for cutting different tissues (e.g., single axons) or for harvesting specific cell populations from histological tissue sections. The authors envision future improvements such as sensors as well as force-generating actuation mechanisms that automatically deliver a controlled cutting stroke, and they indicate that both piezoelectric and thermal expansion actuation mechanisms can deliver forces in the range needed for axon cutting. A femtosecond laser might also be used for axotomy.⁴⁴ The goal is to develop a microcutting device with on-board sensing and actuation that can function as a semiautonomous instrument, requiring only initiating commands from the surgeon. Important limitations to the practical application of this invention remain. The authors estimate, for example, that ~20 seconds are required to repair a single axon using dielectrophoresis and electrofusion.²⁴ Cutting and fusing multiple axons simultaneously might enable relatively rapid repair of several thousand axons.

Fig. 36.1 Microfabricated axon surgery platform, an example of a nanomachine. (A) Axon knife assembled within a compliant suspension frame. The pyramidal structure in the center is an optically transparent axon knife with a 10-µm cutting edge. f, silicon suspension flexures; k, axon knife; h, handle to micromanipulator. The footprint of the frame is 1 mm². Scale = 200 µm. Devices with substantially smaller footprints can be fabricated. (B) Detail of the silicon suspension flexure on each side of the axon knife that provides mechanical compliance during cutting. The number of switchbacks in the flexure can be modified to obtain devices with different mechanical compliances that deliver a range of cutting forces. The compliance of the flexures allows it to act as a suspension, maximizing the durability of the knife edge. (C) Oblique view of the silicon nitride knife. Knives with edges from 5 to 200 µm have been fabricated and tested. A 200-µm-long knife is shown. (D) Scanning electron micrograph showing a cross-section of the silicon nitride knife at its very edge. The radius of curvature at the edge is roughly 20 nm.

(Reproduced with permission from Sretavan DW, Chang W, Hawkes E, et al. Microscale surgery on single axons. Neurosurgery 2005;57:635–46; discussion 646, and from Zarbin MA, Montemagno C, Leary JF, et al. Nanomedicine in ophthalmology: the new frontier. Am J Ophthalmol 2010;150:144–62.)

Fig. 36.2 Diagrams showing the design, assembly, and scale of a prototype multifunctional axon surgery platform. (A) Schematic representation of the space frame. Carrier holes are used for positioning the platform. (B) Modular axon repair components such as cutting devices and electrode arrays are inserted into the space frame to preposition their functional elements for efficient axon repair. (Generic components are shown here.) The space frame also contains a force generation (actuation) mechanism to execute the up–down motion of the axon knife during cutting. (C) Oblique view of platform assembled with a microcutting device and electrode arrays. (D) View from bottom. (E) Sixteen individual microfabricated parts on display on a penny. A microgripper and a microprobe are shown on the left. (F) Scanning electron microscope image of the assembled axon surgery device prototype containing a functioning axon microcutting device and supporting pieces locked in place to form a 1 mm³ superstructure. Scale bar = 200 µm.

Applications to ophthalmology

Nanomedicine will foster revolutionary advances in the diagnosis and treatment of disease. Nanomedicine is likely to have a major impact on biopharmaceuticals (e.g., drug delivery, drug discovery),⁴⁵ implantable materials (e.g., tissue regeneration scaffolds, bioresorbable materials), implantable devices (e.g., IOP monitors,⁴⁶ glaucoma drainage valves⁴⁷), and diagnostic tools (e.g., infectious disease diagnosis, genetic testing, imaging, IOP monitoring). Nanotechnology will bring about the development of regenerative medicine (i.e., replacement and improvement of cells, tissues, and organs), ultrahigh resolution in vivo imaging, microsensors and feedback devices, and artificial vision. “Regenerative nanomedicine,” a new subfield of nanomedicine, uses nanoparticles containing gene transcription factors and other modulating molecules that allow reprogramming of cells in vivo.

Delivery of drugs, peptides, and genes

General considerations regarding nanoparticles

Nanoparticles are colloidal carrier systems that can improve the efficacy of drug delivery by overcoming diffusion barriers, permitting reduced dosing (through more efficient tissue targeting) as well as sustained delivery (Fig. 36.3). These features are attractive for drug treatment of chronic conditions such as glaucoma,⁴⁸ uveitis,⁴⁹ or retinal edema (due to venous occlusion or choroidal neovascularization (CNV)) as well as for treatment of intraocular tumors and other conditions associated with cell proliferation such as capsular fibrosis after cataract surgery, ocular neovascularization, and proliferative vitreoretinopathy. Nanoscale-engineered cell substrata (e.g., nanowires) and carbon nanotubes also can be used for gene and drug delivery.⁵⁰^–⁵²

Fig. 36.3 Schematics of different nanotechnology-based drug delivery systems. Nanoparticles are small polymeric colloidal particles with a therapeutic agent either dispersed in polymer matrix or encapsulated in polymer. Polymeric micelles are self-assembled block copolymers, which in aqueous solution arrange to form an outer hydrophilic layer and an inner hydrophobic core. The micellar core can be loaded with a water-insoluble therapeutic agent. Liposomes are lipid structures that can be made “stealth” by PEGylation and further conjugated to antibodies for targeting. Dendrimers are monodispersed symmetric macromolecules built around a small molecule with an internal cavity surrounded by a large number of reactive end groups. Quantum dots are fluorescent nanocrystals that can be conjugated to a ligand and thus can be used for imaging purposes. Ferrofluids are colloidal solutions of iron oxide magnetic nanoparticles surrounded by a polymeric layer, which can be further coated with affinity molecules such as antibodies. PEG, poly(ethylene glycol).

(Reproduced with permission from Sahoo SK, Labhasetwar V. Nanotech approaches to drug delivery and imaging. Drug Discov Today 2003;8:1112–20 with permission from Elsevier, and from Zarbin MA, Montemagno C, Leary JF, et al. Nanomedicine in ophthalmology: the new frontier. Am J Ophthalmol 2010;150:144–62.)

Strategies in the design of nanoparticles for therapeutic purposes have been reviewed thoroughly by Petros and DeSimone.⁵³ Particle size, shape, and surface properties influence nanoparticle biodistribution. Particle size, for example, affects whether the particle is internalized via phagocytosis, macropinocytosis, caveolar-mediated endocytosis, or clathrin-mediated endocytosis, which in turn results in exposure of the nanoparticle to different intracellular environments.⁵⁴ The cell surface receptor, nucleolin, transports compacted polylysine DNA nanoparticles into cells and directly to the nucleus.⁵⁵

One can target nanoparticles to specific cells by attaching to the particle surface ligands/antibodies/peptides/aptamers for receptors/molecules that are abundant on the surface of the target cell/tissue.⁵³ This approach can have complications. Receptor aggregation on the cell surface, for example, can induce unintended events, such as apoptosis.⁵⁶ One can engineer the nanoparticle for a particular mode of intracellular entry depending on the choice of nanoparticle targeting molecules, e.g., cholesterol favors uptake via caveolin-mediated endocytosis, and trans-activating transcriptional activator peptide favors macropinocytosis.^57,⁵⁸ Nanoparticle surface chemistry also can be manipulated to trigger cargo release under specific circumstances. For example, when exposed to a reducing environment such as is present in the cytosol, reductively labile disulfide-based crosslinks between the carrier and cargo are broken.^59,⁶⁰ Approaches for targeting nanoparticles to particular subcellular organelles, e.g., mitochondria⁶¹ or the nucleus,⁶² also have been developed.

Liposomes and polymer–drug conjugates are among the most frequently used nanoparticles for therapeutic purposes. Liposomes, which carry hydrophobic or hydrophilic cargo, 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. Poly(ethylene glycol) (PEG) can be conjugated with different molecules to enhance their solubility and stability in plasma and to reduce immunogenicity.⁵³ Opsonization by immunoglobulin and/or complement proteins can lead to recognition of the nanoparticle as foreign and induce a hypersensitivity reaction.^63,⁶⁴ Coating a nanoparticle with albumin and/or PEG can create a hydrophilic surface that temporarily resists protein adsorption, thus prolonging the particle’s bioavailability.^53,^65,⁶⁶ This approach allows for much longer drug circulation and concomitant lowering of therapeutic-level drug doses, which in turn can reduce many unintended side-effects.

Dendrimers are synthetic, highly branched polymers that have precisely controllable nanoscale scaffolding and nanocontainer properties, which in some senses mimic the properties of macromolecules such as DNA and ribonucleic acid (RNA).⁶⁷ The diameter of poly(amidoamine) dendrimer ranges from 1.5 to 14.5 nm.⁶⁸ As generation (G) number increases, the number of active terminal groups doubles. G3 dendrimers, for example, contain 32 terminal groups, and G4 dendrimers contain 64 terminal groups. In poly(amidoamine) dendrimers, full generations (e.g., G3) have terminal amine or hydroxyl groups while half-generation dendrimers (e.g., G3.5) have carboxylic acid terminal groups. Dendrimers have been explored as vehicles for controlled drug delivery, including cancer therapy, pilocarpine, gatifloxacin, and for vascular endothelial growth factor (VEGF) inhibition.⁶⁹^–⁷² Marano et al.,⁷⁰ for example, used a lipophilic amino acid dendrimer to deliver an anti-VEGF oligonucleotide into rats’ eyes with laser-induced CNV. The dendrimer–oligonucleotide conjugate inhibited CNV development for 4–6 months by up to 95%, whereas eyes injected with oligonucleotide alone showed no treatment benefit compared to vehicle-injected controls at these times. The dendrimer–oligonucleotide conjugate was well tolerated in vivo. Ideta et al.⁷³ used dendrimer porphyrin encapsulated by a polymeric micelle to treat laser-induced CNV in rodents and found significant enhancement of photodynamic therapy efficacy with less light energy required for CNV occlusion.

Antibiotic therapy

Typically, only a small fraction (<5%) of topically administered medications is biologically available due to limited ocular penetration and rapid clearance from the aqueous humor. Because dendrimers contain surface functional groups as well as void spaces within and between their branches, they can serve as delivery vehicles for therapeutic modalities such as carboplatin.⁷² Dendrimeric polyguanidilyated translocators (DPT) are nano-sized dendrimers that translocate molecules across biological barriers efficiently. Durairaj et al.⁶⁹ used a six-guanidine group-containing dendrimer to enhance gatifloxacin solubility (fourfold) and delivery to the anterior and posterior segment of rabbits. The DPT–gatifloxacin complexes (346 nm) enhanced tissue concentration in the conjunctiva (13-fold) and cornea (twofold). A single dose resulted in sustained aqueous humor levels (t_1/2 = 9 hours), potentially allowing decreased frequency of administration (e.g., once-daily dosing). After multiple dosing, DPT–gatifloxacin achieved therapeutic levels in the vitreous humor for 12 hours (versus no drug levels detectable at 12 hours after topical gatifloxacin alone).

Antimetabolite therapy

Shaunak et al.⁷⁴ used anionic, polyamidoamine, generation 3.5 dendrimers to make novel water-soluble conjugates of d(+)-glucosamine and d(+)-glucosamine 6-sulfate with immunomodulatory and antiangiogenic properties, respectively. Dendrimer glucosamine inhibited Toll-like receptor 4-mediated lipopolysaccharide-induced synthesis of proinflammatory chemokines (i.e., macrophage inflammatory protein (MIP)-1α, MIP-1β, interleukin (IL)-8) and proinflammatory cytokines (i.e., tumor necrosis factor-α, IL-1β, IL-6) primarily from immature human monocyte-derived dendritic cells and monocyte-derived macrophages, but allowed upregulation of the costimulatory molecules CD25, CD80, CD83, and CD86. Dendrimer glucosamine 6-sulfate blocked fibroblast growth factor-2-mediated human umbilical vein endothelial cell proliferation, but not VEGF-mediated proliferation, and neoangiogenesis in human Matrigel and placental angiogenesis assays. When dendrimer glucosamine and dendrimer glucosamine 6-sulfate were used together in a validated, clinically relevant rabbit model of scar tissue formation after glaucoma filtration surgery,^75,⁷⁶ they increased the long-term success of the surgery from 30% to 80% (P = 0.029). A clinical trial of this modality to reduce scarring after trabeculectomy, however, was not successful (P Khaw, R Ritch, personal communication).

Neurotrophic factor therapy

Nanoparticles can deliver growth and neurotrophic factors to cells.⁷⁷ Intravitreal nanoparticle-based basic fibroblast growth factor (bFGF) delivery, for example, provides sustained retinal rescue in Royal College of Surgeons (RCS) rats.⁷⁸ In RCS rats, the RPE cells have a mutation that prevents proper outer-segment phagocytosis, with secondary rod and cone photoreceptor degeneration.⁷⁹ Some patients with retinitis pigmentosa (RP) have this same mutation.⁸⁰^–⁸² Sakai et al.⁷⁸ prepared bFGF nanoparticles using gelatin isolated from bovine bone collagen and human recombinant bFGF. The nanoparticle diameter, assessed using dynamic light scattering, was ~585 nm.

Glaucoma, a leading cause of blindness worldwide, is associated with progressive RGC death and optic nerve atrophy.⁸³ Intravitreal glial cell line-derived neurotrophic factor (GDNF)-loaded biodegradable (poly)lactic-co-glycolic acid (PLGA) microspheres provide sustained RGC protection in a rodent model of glaucoma.⁸⁴ Microspheres (~8 µm diameter) containing GDNF were fabricated using a modification of a spontaneous emulsion technique.⁸⁵ Since adeno-associated virus (AAV)-mediated GDNF secretion from glia delays retinal degeneration in a rat model of RP,⁸⁶ it is possible that nanoparticle-mediated GDNF delivery can be applied to treating RP-like diseases.

Antioxidant therapy

Age-related macular degeneration (AMD), RP, diabetic retinopathy, and retinopathy of prematurity are characterized, in part, by the presence of oxidative damage.⁸⁷^–⁹² As noted above, alteration in the oxidation state of CeO₂ nanoparticles creates defects in its lattice structure via loss of oxygen or its electrons. Chen et al.⁹³ posited that engineered nanoceria can scavenge reactive oxygen intermediates because the large surface area-to-volume ratio at 5 nm diameter enables CeO₂ to regenerate its activity and thereby act catalytically. (Unlike nanoceria, most free-radical scavengers require repetitive dosing.) Chen et al.⁹³ showed that intravitreal injection of nanoceria prevents light-induced photoreceptor damage in rodents, even if injected after the initiation of light exposure. Vacancy-engineered nanoceria also inhibit the development of and 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 whose phenotype resembles a clinical entity known as retinal angiomatous proliferation (Figs 36.4 and 36.5).^94,⁹⁵ This regression occurs even if nanoceria are injected intravitreally after the mutant retinal phenotypes are established (Fig. 36.6). Because nanoceria are a catalytic and regenerative antioxidant, a single injection has a prolonged effect (measured in weeks). Nanoceria inhibition of increased VEGF levels in this model⁹⁴ may mean that CeO₂ nanoparticles will be effective in treating macular edema in diabetic eyes and CNV-induced retinal edema in AMD eyes.⁹⁶^–⁹⁸

Fig. 36.4 Nanoceria reduce oxidative stress in the Vldlr–/– retina. Retinal sections from saline-injected wild-type (WT) mice (panels A, D, G, J); saline-injected Vldlr–/– mice (panels B, E, H, K), and CeO₂-injected (panels C, F, I, L) Vldlr–/– mice are shown as imaged by confocal microscopy. The 2′,7′-dicholoro-dihydro-fluorescein-diacetate (DCF) assay (A–C) visualizes reactive oxygen species (ROS) as punctuate fluorescence and demonstrates a very low level of ROS in the normal retina (A), a considerable amount in the Vldlr–/– retina (B), and a greatly reduced amount in the retina of the Vldlr–/– mice injected with CeO₂ (C). Similar results were obtained with the other three assays. NADPH-oxidase (P47-phox; D–F), a major producer of ROS, was very high in the Vldlr–/– retina and almost reduced to control levels in the CeO₂-injected mice. Nitrotyrosine (G–I), a reflection of oxidative activity due to increases in nitric oxide concentration, was highest in the Vldlr–/– retina and significantly reduced in the nanoceria-injected mice. ROS-mediated damage to DNA was indicated by the labeling of the retina with an antibody against a DNA adduct, 8-hydroxy-29-deoxyguanosine (8-OHdG; J–L), which showed little labeling in the control, significant labeling in the saline-injected Vldlr–/– retina, and a greatly reduced amount in the nanoceria-treated retina. DAPI (blue) was used to visualize the nuclei.

(Reproduced with permission from Zhou X, Wong LL, Karakoti AS, et al. Nanoceria inhibit the development and promote the regression of pathologic retinal neovascularization in the Vldlr knockout mouse. PLoS ONE 2011;6:e16733, and from Zarbin MA, Montemagno C, Leary JF, et al. Regenerative nanomedicine and the treatment of degenerative retinal diseases. Wiley Interdiscip Rev Nanomed Nanobiotechnol 2012;4:113–37.)

Fig. 36.5 Nanoceria inhibit the development of pathologic intra- and subretinal vascular lesions in the Vldlr–/– retina. Photomicrographs of whole-mount retinas (A–C) and eyecups (retinal pigment epithelium, 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 web-like retinal vasculature whereas those from the Vldlr–/– mice (B) showed numerous intraretinal vascular lesions or “blebs.” See white arrows for examples. A single injection of nanoceria at P7 (C) inhibited the appearance of these lesions. Eyecups 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).

Fig. 36.6 Retinal vascular lesions in the Vldlr–/– retinas require continual production of excess reactive oxygen species. Vldlr–/– mice were injected at P28 with saline or nanoceria and killed 1 week later on P35. Analysis of vascular endothelial growth factor (VEGF) levels by Western blots (A) showed a fourfold reduction (B) within 1 week of nanoceria injection. The numbers of intraretinal neovascular (IRN) blebs (C) and subretinal neovascular (SRN) tufts (D) were also dramatically reduced. *P = 0.05; **P = 0.01.

C-60 fullerenes are cage-like structures (truncated icosahedron) of carbon atoms with antioxidant properties.⁹⁹ Malonic acid C-60 derivatives (carboxyfullerenes) can eliminate superoxide anion and H₂O₂, and inhibit lipid peroxidation.⁸ Systemic administration of the C-3 carboxyfullerene isomer delayed motor deterioration and death in a mouse model of familial amyotrophic lateral sclerosis.^89,¹⁰⁰ It might be useful in the treatment of retinal diseases associated with oxidative damage.

Iron is an essential element for enzymes involved in the phototransduction cascade, in outer-segment disc membrane synthesis, and in the conversion of all-trans-retinyl ester to 11-cis-retinol in the RPE.¹⁰¹^–¹⁰³ Free Fe²⁺ catalyzes the conversion of hydrogen peroxide to hydroxyl radical, which is a highly reactive species that causes oxidative damage (e.g., lipid peroxidation, DNA strand breaks).¹⁰⁴ Increased intracellular iron causes oxidative photoreceptor damage.¹⁰⁵ Polymeric nanoparticles can be used to chelate metals. Liu et al.¹⁰⁶ showed that a chelator-nanoparticle system complexed with iron, when incubated with human plasma, preferentially adsorbs apolipoprotein E and apolipoprotein A-I, which should facilitate transport into and out of the brain via mechanisms used for transporting low-density lipoprotein. Iron accumulation in the RPE and Bruch’s membrane is greater in AMD eyes than in controls, including cases with early AMD and late stages of the disease (i.e., geographic atrophy, CNV).¹⁰⁷ Some of this iron is chelatable.¹⁰⁷ Although it is not proven that iron overload is a cause of AMD,¹⁰⁸^–¹¹¹ iron chelation might have a therapeutic effect. Thus, the technology developed by Liu et al.¹⁰⁶ might have utility for treating AMD eyes.

Immune-suppressive therapy

Cell-based therapy might be sight-restoring for patients with degenerative retinal diseases such as RP and AMD. An immune response to transplanted cells may depend upon the cell types included in the cellular therapy.¹¹² Nanotechnology provides immune-suppressive therapy (local or systemic) in selected cases where its need is anticipated.¹¹²^–¹¹⁴ In preclinical models, for example, nanoparticles are helpful in managing corneal allograft rejection. Yuan et al.¹¹⁵ manufactured 300-nm-diameter rapamycin-loaded chitosan/polylactic acid nanoparticles and demonstrated that they extended median allograft survival by 17% in rabbits compared with aqueous rapamycin eye drops. Topical chitosan particles were well tolerated in this study, but intraocular chitosan nanoparticles may not be well tolerated.¹¹⁶

Studies of experimental autoimmune uveoretinitis (EAU) demonstrate that nanoparticles can be used to modulate the inflammatory response in the retina and choroid. EAU is a T-cell-mediated autoimmune disease that targets the retina and related tissues and serves as a model for human autoimmune ocular diseases.¹¹⁷ Nanosuspensions of relatively insoluble glucocorticoids (developed using a high-pressure homogenization method) enhance the rate and extent of drug absorption as well as the intensity and duration of drug action, compared with conventional solutions and microcrystalline suspensions.⁴⁹ Rats with EAU clear poly(lactic acid) (PLA) nanoparticles rapidly from the systemic circulation.¹¹⁸ As noted above, PEG can be used to modify the surface of the nanoparticles, which reduces opsonization and interactions with the mononuclear phagocyte system.¹¹⁹ Sakai et al.¹²⁰ prepared polymeric nanoparticles with encapsulated betamethasone phosphate. These nanosteroid particles (~120 nm diameter) were composed of PLA homopolymer and a block copolymer of PEG.¹²¹ In vivo imaging of inflamed eyes of rats with EAU demonstrated greater nanoparticle accumulation and higher betamethasone concentration in eyes of PLA-PEG nanoparticle-treated rats versus PLA nanoparticle-treated rats. PLA-PEG nanosteroid-treated EAU rats also had lower clinical and histopathological scores for ocular inflammation. The stronger therapeutic effect of PLA-PEG nanosteroids versus PLA nanosteroids may be due to prolonged blood circulation and sustained release in situ as well as due to targeting to inflamed eyes (the latter effect resulting from the small diameter of the nanoparticles).¹²¹ EAU also responds very well to intravitreal liposomal tacrolimus (mean diameter = 200 nm) with no side-effects on retinal function or systemic cellular immunity.¹²²

Gene therapy

Nonviral vectors

Viral vectors deliver genes efficiently but can be associated with risks such as immunogenicity and insertional mutagenesis. Nonviral vectors (e.g., polymers, lipids) and other methods (e.g., electroporation, nucleofection) have high gene-carrying capacity, low risk of immunogenicity, relatively low cost, and, possibly, greater ease of production.¹²³ Nanoparticles can deliver genes efficiently to stem cells¹²⁴ and have been explored as a means for gene delivery in the diagnosis and treatment of ocular disease.^3,¹²⁵^–¹²⁷ As viruses do, nanoparticles can use transactivating sequences that allow them to deploy the host cell machinery to manufacture therapeutic molecules in situ. Because these sequences can contain an upstream biomolecular control sensor, therapeutic molecules can be manufactured in situ under tight feedback control.^3,⁴

Electrostatic interaction of cationic polymers with negatively charged DNA/RNA molecules results in condensation of the material into particles ranging from 8 to 500 nm in diameter, protection of the genes from enzymes, and mediation of cellular entry.^26,¹²⁸ Complexes of cationic polymers and plasmid DNA, termed polyplexes, can have transfection efficiency comparable to adenoviral vectors.¹²⁹ In addition to nanometer size, polyplexes have large vector capacity, are stable in nuclease-rich environments, and can have relatively high transfectivity for both dividing and nondividing cells.^127,¹²⁹ For example, nanoparticles compacted with a lysine 30-mer linked to 10 kDa PEG-containing cytomegalovirus-cystic fibrosis transmembrane conductance regulator (CMV-CFTR) cDNA were used successfully in a phase I/II clinical trial for the treatment of cystic fibrosis.¹³⁰ Some particles, however, have low transfection efficiency, and the duration of gene expression can be short. When it occurs, toxicity is related to nanoparticle chemistry.²⁶

To some degree, compacted DNA nanoparticles can be targeted to different tissues in the eye through selection of an appropriate injection site (e.g., intravitreal injection can target the cornea, trabecular meshwork, lens, and inner retina; subretinal injection can target the outer retina and RPE).¹²⁷ Nanoparticle size and charge influence migration through the vitreous cavity.¹³¹ Farjo et al.¹²⁷ demonstrated that after subretinal injection of compacted lysine 30-mer DNA nanoparticles, gene expression is observed throughout the retina and not just at the site of the injection. By choosing cell-specific promoters, one can achieve additional specificity in the locus of gene expression. The rhodopsin promoter, for example, drives expression in rod photoreceptors, and the human red opsin promoter drives expression in cone photoreceptors.¹³²^–¹³⁴ Interphotoreceptor retinoid-binding protein drives expression in both rods and cones.¹³⁵ The vitelliform macular dystrophy promoter drives expression in RPE cells.¹³⁶

Cai et al.^132,¹³⁷ used a specific formulation of DNA nanoparticles consisting of single molecules of DNA compacted with 10 kDa 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 RP. After injection into the subretinal space, these particles did not induce a detectable immune response, cytotoxicity, or disruption of retinal function. These compacted plasmid DNA nanoparticles are small (8–20 nm), have rod or ellipsoid shape (depending on the counterion used), and have a large carrying capacity (at least up to 20 kilobases).^127,¹³⁷ PLGA nanoparticles can deliver genes to RPE cells in vitro and in vivo relatively efficiently and safely,¹³⁸ and PLGA DNA nanoparticles can be associated with long-term gene expression.¹³⁹ PLGA DNA nanoparticles tend to be larger than polylysine DNA nanoparticles,^140,¹⁴¹ which may affect cellular uptake mechanism and delivery to the nucleus. PLGA DNA nanoparticles might be used to deliver therapeutic genes for conditions associated with RPE gene mutations, e.g., Best disease¹⁴² and a form of Leber congenital amaurosis.¹⁴³^–¹⁴⁵

Albumin has a highly charged amino acid content, which facilitates its action as a carrier for charged drugs and oligonucleotides. 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.¹⁴⁶

Despite these promising results, concerns involving nanoparticle use remain. Although the immune response to polylysine-based nanoparticles seems to be less than that for capsid proteins, for example, the efficiency of gene transfer is not as high since most are degraded in the endosomal complexes.¹⁴⁷ As a result, one may generate an immune response because one must use large numbers of nanoparticles to achieve therapeutic useful transfection. Also, the apparent low immunogenicity observed in murine models of RP may not be observed in human patients because the immune response to both nanoparticles and viruses varies from one species to another.¹⁴⁷

Viral vectors

Critical issues for successful gene therapy include: (1) vector uptake, transport, and uncoating; (2) vector genome persistence; (3) sustained transcriptional expression; (4) the host immune response; and (5) insertional mutagenesis and cancer.¹⁴⁷^–¹⁴⁹ Virus-based gene therapy can induce immune responses, including innate, humoral, and cell-mediated, that are directed against the vector and/or the transgene product.^147,^150,¹⁵¹ Primary humoral responses directed against the vector can limit its capacity to deliver genes to the target cells as well as the ability to readminister the virus to the patient (e.g., when treating the fellow eye with a second surgical procedure).¹⁴⁷ An immediate innate immune response and a secondary antigen-dependent response to intravenous administration of recombinant adenoviral vectors, for example, caused death in a patient with ornithine transcarbamylase deficiency.^152,¹⁵³ A humoral response against the transgene product may neutralize the therapeutic protein.¹⁴⁷ A cell-mediated immune response against the vector or transgene product can eliminate the transduced cells.¹⁴⁷ Two patients with hemophilia B, for example, developed vector dose-dependent transaminitis that limited hepatocyte-derived factor IX expression to less than 2 months due to CD8⁺ memory T cells that recognized AAV serotype 2 (AAV2) capsids and eliminated AAV2-transduced hepatocytes.^154,¹⁵⁵ The innate immune response can cause local and/or systemic toxicity and amplify a secondary antigen-dependent immune response.¹⁴⁷ The likelihood of an immune response is influenced by the dose of viral particles,¹⁵⁶ which in turn is influenced by the efficiency of vector uptake and gene expression, as well as by the specificity of targeting. If dendritic cells or antigen-presenting cells take up the vector, for example, an immune response is more likely.

Nanoengineering of the viral capsid and transgene may provide a means to solve some of these problems. Recombinant AAVs (rAAVs) have been used successfully to treat preclinical models of human ocular disease and also have been used to treat humans with Leber congenital amaurosis.^143,^144,¹⁵⁷ Modifications of the virus to improve clinical effectiveness illustrate some of the nanoengineering strategies that have been employed in this area. AAVs are small (4.7 kilobase carrying capacity), nonpathogenic, single-stranded DNA parvoviruses that can transduce dividing and nondividing cells.¹⁵⁸ The capsid is critical for extracellular events related to the recognition of specific receptors, which influences cell tropism, as well as intracellular processes involving AAV trafficking and uncoating. In turn, the latter processes influence transduction kinetics and transgene expression efficiency.^159,¹⁶⁰ Due to previous exposure to various AAV serotypes, a significant proportion of the population harbors neutralizing antibodies that can block gene delivery.^151,^161,¹⁶² Because administration of low doses of viral vector might mitigate the severity of this problem, two nanoengineering techniques have been used to improve vector cellular tropism, transduction efficiency, and immunogenicity: directed evolution and site-directed mutagenesis. These are discussed below. Other nanoengineering devices (e.g., DNA transposons,¹⁶³ bacteriophage recombinases¹⁶⁴) may provide clinically useful means to achieve stable, safe DNA integration in the host genome and sustained transgene expression in the future.

Directed evolution of AAV capsids has generated vectors that are highly resistant to neutralizing antibodies.^165,¹⁶⁶ Maheshri et al.¹⁶⁶ used error-prone polymerase chain reaction and a staggered extension process¹⁶⁷ to generate an AAV2 library (>10⁶ independent clones) with randomly distributed capsid mutations and then used high-throughput approaches (i.e., exposure of mutants to heparin affinity chromatography (wild-type AAV2 binds to heparan sulfate) or repeated amplification of AAV2 mutants that retain infectivity in the presence of serum containing neutralizing antibodies) to identify mutant AAV2 capsids with altered receptor-binding properties and the capacity to bind with very low affinity to neutralizing antibodies. This approach can be quite powerful. One mutagenesis and three selection steps generated mutant capsids, for example, with a threefold improved neutralizing antibody titer (versus wild-type capsid) and a ~7.5% infectivity at serum levels that completely neutralized wild-type infectivity.¹⁶⁶ Directed evolution has been used to generate AAV variants that transduce Müller cells after intravitreal injection,^168,¹⁶⁹ which may provide a means to deliver growth factors to photoreceptors and RPE cells. These growth factors retard the progression of retinal degeneration in preclinical models of RP^86,^170,¹⁷¹ and possibly in human patients also.¹⁷²

Zhong et al.¹⁷³ demonstrated that site-directed mutagenesis¹⁷⁴ of surface-exposed tyrosine residues increases vector transduction efficiency 30-fold in vivo at one log lower vector dose compared to wild-type AAV2. The increased transduction efficiency is due to impaired capsid ubiquitination and improved intracellular trafficking to the nucleus. (Epidermal growth factor receptor protein tyrosine kinase (EGFR-PTK) signaling impairs AAV2 vector transduction by impairing nuclear transport of the vectors¹⁷⁵; EGFR-PTK can phosphorylate AAV2 capsids at tyrosine residues,^175,¹⁷⁶ and 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.^175,¹⁷⁷) Thus, the T-cell response to AAV2 capsids seems to be manageable by using surface-exposed tyrosine mutant vectors. Another rate-limiting step in transduction efficiency, the conversion of single-stranded viral genome to double-stranded AAV DNA, has been overcome by deleting the terminal resolution site from one rAAV inverted terminal repeat, which prevents replication initiation at the mutated end, to generate self-complementary AAV (scAAV) vectors.^178,¹⁷⁹ (AAV has a tendency to package DNA dimers when the replicating genome is half the length of the wild type.)

Ocular applications

Due to their relatively low immunogenicity, ability to target many nondividing cells, and capacity for sustained efficient therapeutic gene expression after a single treatment,¹⁵⁹ rAAV vectors have been used to treat preclinical models of human retinal disease.^180,¹⁸¹ Site-directed mutagenesis technology has been used to improve the treatment of degenerative retinal disease in these preclinical models. Vectors containing point mutations in surface-exposed capsid tyrosine residues in AAV serotypes 2, 8, and 9 display strong and widespread transgene expression in retinal cells after intravitreal or subretinal delivery.¹⁸² Petrs-Silva et al.¹⁸² demonstrated that tyrosine-to-phenylalanine capsid scAAV2 mutants showed much greater transduction efficiency (10–20-fold higher transgene expression) of the entire retina (including photoreceptors) after intravitreal injection compared to scAAV with wild-type capsids (Fig. 36.7). Mutants of scAAV2, scAAV8, and scAAV9 also enhanced transduction of RGCs compared to wild-type AAV2 (e.g., 10⁶-fold reduction in the number of virus particles needed for RGC transfection with mutant scAAV2 compared to wild-type AAV2). Intravitreal delivery may offer an important clinical advantage over subretinal delivery. Subretinal virus delivery, which has been used in clinical studies,¹⁴³^–¹⁴⁵ requires pars plana vitrectomy in the operating room and has a higher likelihood of complications (e.g., retinal tear) than intravitreal delivery, which can be done in an office under topical anesthesia. On the other hand, the subretinal space is a relatively immune-privileged site,¹⁸³ which may reduce the likelihood of an immune response after repeat virus treatment. Li et al.¹⁸⁴ demonstrated that a humoral immune response against AAV2 capsid proteins occurs after intravitreal but not after subretinal vector delivery. Subretinal injection of one of the mutant scAAVs also transduced Müller cells. These studies demonstrate two strategies for reducing the immune response to viral vectors via site-directed mutagenesis: increasing transduction efficiency, which permits lower doses of vector, and creation of multiple effective serotypes, which can be used sequentially for subsequent therapy.