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Regenerative Nanomedicine for Vision Restoration

      Abstract

      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.

      Abbreviations and Acronyms:

      AAQ (acrylamide-azobenzene-quaternary ammonium), ChR2 (channelrhodopsin-2), CNV (choroidal new vessel), ECM (extracellular matrix), GDNF (glial cell line–derived neurotrophic factor), HaloR (halorhodopsin), IOP (intraocular pressure), PCL (poly(ε-caprolactone)), PEDF (pigment epithelium–derived factor), PEG (polyethylene glycol), RGC (retinal ganglion cell), RPE (retinal pigment epithelium), shRNA (short hairpin RNA), siRNA (small interfering RNA), VEGF (vascular endothelial growth factor)
      CME Activity
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      Credit Statement: Mayo Clinic College of Medicine designates this journal-based CME activity for a maximum of 1.0 AMA PRA Category 1 Credit(s).™ Physicians should claim only the credit commensurate with the extent of their participation in the activity.
      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.
      Disclosures: As a provider accredited by ACCME, Mayo Clinic College of Medicine (Mayo School of Continuous Professional Development) must ensure balance, independence, objectivity, and scientific rigor in its educational activities. Course Director(s), Planning Committee members, faculty, and all others who are in a position to control the content of this educational activity are required to disclose all relevant financial relationships with any commercial interest related to the subject matter of the educational activity. Safeguards against commercial bias have been put in place. Faculty also will disclose any off-label and/or investigational use of pharmaceuticals or instruments discussed in their presentation. Disclosure of this information will be published in course materials so that those participants in the activity may formulate their own judgments regarding the presentation.
      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.
      Method of Participation: In order to claim credit, participants must complete the following:
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        Read the activity.
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        Complete the online CME Test and Evaluation. Participants must achieve a score of 80% on the CME Test. One retake is allowed.
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      Date of Release: 12/01/2013
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      Questions? Contact [email protected] .
      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.
      • Prow T.
      • Salazar J.H.
      • Rose W.A.
      • et al.
      Nanomedicine-nanoparticles, molecular biosensors and targeted gene/drug delivery for combined single-cell diagnostics and therapeutics.
      • Prow T.
      • Grebe R.
      • Merges C.
      • et al.
      Nanoparticle tethered antioxidant response element as a biosensor for oxygen induced toxicity in retinal endothelial cells.
      • Prow T.W.
      • Bhutto I.
      • Grebe R.
      • et al.
      Nanoparticle-delivered biosensor for reactive oxygen species in diabetes.
      We have reviewed applications of nanotechnology to vision restoration previously,
      • Zarbin M.A.
      • Montemagno C.
      • Leary J.F.
      • Ritch R.
      Nanomedicine in ophthalmology: the new frontier.
      • Zarbin M.A.
      • Montemagno C.
      • Leary J.F.
      • Ritch R.
      Nanotechnology in ophthalmology.
      • Zarbin M.A.
      • Montemagno C.
      • Leary J.F.
      • Ritch R.
      Regenerative nanomedicine and the treatment of degenerative retinal diseases.
      • Zarbin M.A.
      • Montemagno C.
      • Leary J.F.
      • Ritch R.
      Nanomedicine for the treatment of retinal and optic nerve diseases.
      • Zarbin M.
      • Montemagno C.
      • Leary J.
      • Ritch R.
      Artificial vision.
      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
      • Chen J.
      • Patil S.
      • Seal S.
      • McGinnis J.F.
      Rare earth nanoparticles prevent retinal degeneration induced by intracellular peroxides.
      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.
      • Zhou X.
      • Wong L.L.
      • Karakoti A.S.
      • Seal S.
      • McGinnis J.F.
      Nanoceria inhibit the development and promote the regression of pathologic retinal neovascularization in the vldlr knockout mouse.
      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
      • Cai X.
      • Sezate S.A.
      • Seal S.
      • McGinnis J.F.
      Sustained protection against photoreceptor degeneration in tubby mice by intravitreal injection of nanoceria.
      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.
      • Kong L.
      • Cai X.
      • Zhou X.
      • et al.
      Nanoceria extend photoreceptor cell lifespan in tubby mice by modulation of apoptosis/survival signaling 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
      • Han Z.
      • Conley S.M.
      • Makkia R.S.
      • Cooper M.J.
      • Naash M.I.
      DNA nanoparticle-mediated ABCA4 delivery rescues Stargardt dystrophy in mice.
      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,
      • Cai X.
      • Nash Z.
      • Conley S.M.
      • Fliesler S.J.
      • Cooper M.J.
      • Naash M.I.
      A partial structural and functional rescue of a retinitis pigmentosa model with compacted DNA nanoparticles.
      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
      • Koirala A.
      • Makkia R.S.
      • Cooper M.J.
      • Naash M.I.
      Nanoparticle-mediated gene transfer specific to retinal pigment epithelial cells.
      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.
      • Han Z.
      • Koirala A.
      • Makkia R.
      • Cooper M.J.
      • Naash M.I.
      Direct gene transfer with compacted DNA nanoparticles in retinal pigment epithelial cells: expression, repeat delivery and lack of toxicity.
      Infiltration of neutrophils and lymphocytes has not been detected in injected retinae, and no elevations in tumor necrosis factor messenger RNA or protein have been detected after treatment.
      • Ding X.Q.
      • Quiambao A.B.
      • Fitzgerald J.B.
      • Cooper M.J.
      • Conley S.M.
      • Naash M.I.
      Ocular delivery of compacted DNA-nanoparticles does not elicit toxicity in the mouse retina.
      Liposomes have been used to improve cellular uptake of small interfering RNA (siRNA) by integrin receptor–mediated endocytosis owing to the intrinsic difficulty that free siRNA has in entering cells.
      • Pirollo K.F.
      • Chang E.H.
      Targeted delivery of small interfering RNA: approaching effective cancer therapies.
      Chen et al
      • Chen C.W.
      • Lu D.W.
      • Yeh M.K.
      • Shiau C.Y.
      • Chiang C.H.
      Novel RGD-lipid conjugate-modified liposomes for enhancing siRNA delivery in human retinal pigment epithelial cells.
      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.
      Similarly, Suen and Chau
      • Suen W.-L.L.
      • Chau Y.
      Specific uptake of folate-decorated triamcinolone-encapsulating nanoparticles by retinal pigment epithelium cells enhances and prolongs antiangiogenic activity.
      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.
      • Suen W.-L.L.
      • Chau Y.
      Specific uptake of folate-decorated triamcinolone-encapsulating nanoparticles by retinal pigment epithelium cells enhances and prolongs antiangiogenic activity.
      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.
      • Zarbin M.A.
      • Rosenfeld P.J.
      Pathway-based therapies for age-related macular degeneration: an integrated survey of emerging treatment alternatives.
      Luo et al
      • Luo L.
      • Zhang X.
      • Hirano Y.
      • et al.
      Targeted intraceptor nanoparticle therapy reduces angiogenesis and fibrosis in primate and murine 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.
      • Singh S.R.
      • Grossniklaus H.E.
      • Kang S.J.
      • Edelhauser H.F.
      • Ambati B.K.
      • Kompella U.B.
      Intravenous transferrin, RGD peptide and dual-targeted nanoparticles enhance anti-VEGF intraceptor gene delivery to laser-induced CNV.
      The nanoparticles targeted CNVs, induced CNV regression, decreased fibrotic scarring, improved visual acuity, and documented safety in 2 murine CNV models and a primate CNV model (Figure 2).
      • Luo L.
      • Zhang X.
      • Hirano Y.
      • et al.
      Targeted intraceptor nanoparticle therapy reduces angiogenesis and fibrosis in primate and murine macular degeneration.
      This delivery system provides extended-release, nonviral gene therapy that inhibits CNV formation and subretinal scarring.
      Figure thumbnail gr1
      Figure 1A single intravenous injection of targeted, biodegradable nanoparticles delivering a recombinant Flt23k intraceptor plasmid homes to neovascular lesions in the retina and regresses choroidal new vessels in primate and murine age-related macular degeneration models. Moreover, this treatment suppressed subretinal fibrosis, which is not addressed by currently approved clinical therapies. Murine vision, as tested by OptoMotry (CerebralMechanics Inc), significantly improved, with nearly 40% restoration of visual loss induced by choroidal new vessels. These findings offer a nanoparticle-based platform for targeted, vitreous-sparing, extended-release, nonviral gene therapy. PLGA = poly(lactic-co-glyocolic acid); RGD = arginine-glycine-aspartate.
      From ACS Nano,
      • Luo L.
      • Zhang X.
      • Hirano Y.
      • et al.
      Targeted intraceptor nanoparticle therapy reduces angiogenesis and fibrosis in primate and murine macular degeneration.
      with permission.
      Figure thumbnail gr2
      Figure 2A single systemic administration of arginine-glycine-aspartate (RGD)–functionalized poly(lactic-co-glyocolic acid) nanoparticles (572 nm) loaded with antiangiogenic plasmid Flt23k and conjugated with Nile Red (RGD.Flt23k.NR.NP) regressed choroidal new vessels (CNVs) in mice more than intravitreal mouse anti–vascular endothelial growth factor (VEGF) antibody. ∗ = P<.05. Data are given as mean ± SD.
      From ACS Nano,
      • Luo L.
      • Zhang X.
      • Hirano Y.
      • et al.
      Targeted intraceptor nanoparticle therapy reduces angiogenesis and fibrosis in primate and murine macular degeneration.
      with permission.
      Cationic liposomes can be used to improve therapy. Gross et al
      • Gross N.
      • Ranjbar M.
      • Evers C.
      • et al.
      Choroidal neovascularization reduced by targeted drug delivery with cationic liposome-encapsulated paclitaxel or targeted photodynamic therapy with verteporfin encapsulated in cationic liposomes.
      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.
      • Akiyama G.
      • Sakai T.
      • Kuno N.
      • et al.
      Photoreceptor rescue of pigment epithelium-derived factor-impregnated nanoparticles in Royal College of Surgeons rats.
      These rats have a sequence variation that is found in some patients with retinitis pigmentosa,
      • Gal A.
      • Li Y.
      • Thompson D.A.
      • et al.
      Mutations in MERTK, the human orthologue of the RCS rat retinal dystrophy gene, cause retinitis pigmentosa.
      and there is some evidence that sustained neurotrophic factor therapy might benefit selected patients with retinitis pigmentosa.
      • Sieving P.A.
      • Caruso R.C.
      • Tao W.
      • et al.
      Ciliary neurotrophic factor (CNTF) for human retinal degeneration: phase I trial of CNTF delivered by encapsulated cell intraocular implants.
      Nanoparticles also provide sustained delivery of basic fibroblast growth factor and rescue the retina in Royal College of Surgeons rats.
      • Sakai T.
      • Kuno N.
      • Takamatsu F.
      • et al.
      Prolonged protective effect of basic fibroblast growth factor-impregnated nanoparticles in Royal College of Surgeons rats.
      Jiang et al
      • Jiang C.
      • Moore M.J.
      • Zhang X.
      • Klassen H.
      • Langer R.
      • Young M.
      Intravitreal injections of GDNF-loaded biodegradable microspheres are neuroprotective in a rat model of glaucoma.
      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
      • Checa-Casalengua P.
      • Jiang C.
      • Bravo-Osuna I.
      • et al.
      Retinal ganglion cells survival in a glaucoma model by GDNF/Vit E PLGA microspheres prepared according to a novel microencapsulation procedure.
      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
      • Du J.
      • Zhao W.
      • Wang Y.
      • Cai Y.
      Lentivirus vector-mediated knockdown of erythropoietin-producing hepatocellular carcinoma receptors B4 inhibits laser-induced choroidal neovascularization.
      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.
      • Jani P.D.
      • Singh N.
      • Jenkins C.
      • et al.
      Nanoparticles sustain expression of Flt intraceptors in the cornea and inhibit injury-induced corneal angiogenesis.
      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
      • Owen L.A.
      • Uehara H.
      • Cahoon J.
      • Huang W.
      • Simonis J.
      • Ambati B.K.
      Morpholino-mediated increase in soluble Flt-1 expression results in decreased ocular and tumor neovascularization.
      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
      • Lai C.M.
      • Estcourt M.J.
      • Wikstrom M.
      • et al.
      rAAV.sFlt-1 gene therapy achieves lasting reversal of retinal neovascularization in the absence of a strong immune response to the viral vector.
      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
      • Cho Y.K.
      • Uehara H.
      • Young J.R.
      • et al.
      Flt23k nanoparticles offer additive benefit in graft survival and anti-angiogenic effects when combined with triamcinolone.
      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
      • Qazi Y.
      • Stagg B.
      • Singh N.
      • et al.
      Nanoparticle-mediated delivery of shRNA: VEGF-a plasmids regresses corneal neovascularization.
      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
      • Montana C.L.
      • Kolesnikov A.V.
      • Shen S.Q.
      • Myers C.A.
      • Kefalov V.J.
      • Corbo J.C.
      Reprogramming of adult rod photoreceptors prevents retinal degeneration.
      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.
      • Winkler J.L.
      • Kedees M.H.
      • Guz Y.
      • Teitelman G.
      Inhibition of connective tissue growth factor by small interfering ribonucleic acid prevents increase in extracellular matrix molecules in a rodent model of diabetic retinopathy.
      Similarly, siRNA targeted against Smad7 decreases production of ECM in the aqueous outflow pathway and may be a potential treatment for glaucoma.
      • Su Y.
      • Yang C.Y.
      • Li Z.
      • et al.
      Smad7 siRNA inhibit expression of extracellular matrix in trabecular meshwork cells treated with TGF-β2.

      Regenerative Medicine: Optogenetics and Optic Nerve Regeneration

      Optogenetics involves the use of light-sensitive ion channels (vs electrodes) to make neurons light sensitive. This approach to visual rehabilitation has been reviewed extensively.
      • Zarbin M.A.
      • Montemagno C.
      • Leary J.F.
      • Ritch R.
      Regenerative nanomedicine and the treatment of degenerative retinal diseases.
      • Zarbin M.A.
      • Montemagno C.
      • Leary J.F.
      • Ritch R.
      Nanomedicine for the treatment of retinal and optic nerve diseases.
      • Zarbin M.
      • Montemagno C.
      • Leary J.
      • Ritch R.
      Artificial vision.
      • Busskamp V.
      • Picaud S.
      • Sahel J.A.
      • Roska B.
      Optogenetic therapy for retinitis pigmentosa.
      Stimulation of RGCs or bipolar cells provides an alternative approach to retinal cell stimulation in lieu of that provided by the retinal prosthesis.
      • Ahuja A.K.
      • Dorn J.D.
      • Caspi A.
      • et al.
      Blind subjects implanted with the Argus II retinal prosthesis are able to improve performance in a spatial-motor task.
      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
      • Zhang H.
      • Morrison M.A.
      • Dewan A.
      • et al.
      The NEI/NCBI dbGAP database: genotypes and haplotypes that may specifically predispose to risk of neovascular age-related macular degeneration.
      ; ChR2 is a cation channel, and HaloR is an anion channel. Using viral delivery systems, these molecules can be expressed in vivo in RGCs, amacrine cells, biopolar cells, and photoreceptors.
      • Tomita H.
      • Sugano E.
      • Yawo H.
      • et al.
      Restoration of visual response in aged dystrophic RCS rats using AAV-mediated channelopsin-2 gene transfer.
      • Bi A.
      • Cui J.
      • Ma Y.P.
      • et al.
      Ectopic expression of a microbial-type rhodopsin restores visual responses in mice with photoreceptor degeneration.
      • Doroudchi M.M.
      • Greenberg K.P.
      • Liu J.
      • et al.
      Virally delivered channelrhodopsin-2 safely and effectively restores visual function in multiple mouse models of blindness.
      • Lagali P.S.
      • Balya D.
      • Awatramani G.B.
      • et al.
      Light-activated channels targeted to ON bipolar cells restore visual function in retinal degeneration.
      • Zhang Y.
      • Ivanova E.
      • Bi A.
      • Pan Z.H.
      Ectopic expression of multiple microbial rhodopsins restores ON and OFF light responses in retinas with photoreceptor degeneration.
      • Busskamp V.
      • Duebel J.
      • Balya D.
      • et al.
      Genetic reactivation of cone photoreceptors restores visual responses in retinitis pigmentosa.
      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.
      • Kleinlogel S.
      • Feldbauer K.
      • Dempski R.E.
      • et al.
      Ultra light-sensitive and fast neuronal activation with the Ca2+-permeable channelrhodopsin CatCh.
      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.
      • Lin J.Y.
      Optogenetic excitation of neurons with channelrhodopsins: light instrumentation, expression systems, and channelrhodopsin variants.
      A genetically and chemically engineered light-gated mammalian ion channel, the light-activated glutamate receptor, has been expressed selectively in RGCs of the rd1 mouse.
      • Caporale N.
      • Kolstad K.D.
      • Lee T.
      • et al.
      LiGluR restores visual responses in rodent models of inherited blindness.
      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
      • Polosukhina A.
      • Litt J.
      • Tochitsky I.
      • et al.
      Photochemical restoration of visual responses in blind mice.
      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.
      Figure thumbnail gr3
      Figure 3Acrylamide-azobenzene-quaternary ammonium (AAQ) restores the pupillary light reflex in mice lacking all retinal photoreceptors. A, Pupillary light responses to 5.5 × 104 mW/m2 of white light in opn4–/– rd/rd mice before and 3 hours after intravitreal injection of AAQ (1 μL of 80 mmol/L in dimethylsulfoxide). Dark images were taken 5 seconds before light stimulus; light images represent maximal pupillary constriction during 30 seconds of light exposure. Images were taken using an infrared-sensitive camera under infrared illumination. B, Irradiance dependence of pupillary light responses to white light. Irradiance response for wild-type mice (plotted as mean ± SD, n=5) and 4 opn4–/– rd/rd mice injected with AAQ (plotted individually: closed circles, open circles, closed inverted triangles, open triangles). Data were fitted using a 3-parameter Hill equation.
      From Neuron,
      • Polosukhina A.
      • Litt J.
      • Tochitsky I.
      • et al.
      Photochemical restoration of visual responses in blind mice.
      with permission.

      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
      • Pearson R.A.
      • Barber A.C.
      • Rizzi M.
      • et al.
      Restoration of vision after transplantation of photoreceptors.
      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
      • de Lima S.
      • Koriyama Y.
      • Kurimoto T.
      • et al.
      Full-length axon regeneration in the adult mouse optic nerve and partial recovery of simple visual behaviors.
      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.
      • Dai C.
      • Khaw P.T.
      • Yin Z.Q.
      • Li D.
      • Raisman G.
      • Li Y.
      Structural basis of glaucoma: the fortified astrocytes of the optic nerve head are the target of raised intraocular pressure.
      • Dai C.
      • Khaw K.T.
      • Yin C.
      • Li D.
      • Raisman G.
      • Li Y.
      Olfactory ensheathing cells rescue optic nerve fibers in a rat glaucoma model.
      The use of stem cells to treat blinding disease has been reviewed elsewhere.
      • Zarbin M.
      The promise of stem cells for age-related macular degeneration and other retinal degenerative diseases.
      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.
      • Cai S.
      • Smith M.E.
      • Redenti S.M.
      • Wnek G.E.
      • Young M.J.
      Mouse retinal progenitor cell dynamics on electrospun poly(ε-caprolactone).
      Christiansen et al
      • Christiansen A.T.
      • Tao S.L.
      • Smith M.
      • et al.
      Subretinal implantation of electrospun, short nanowire, and smooth poly(ε-caprolactone) scaffolds to the subretinal space of porcine eyes.
      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
      • Bernards D.A.
      • Lance K.D.
      • Ciaccio N.A.
      • Desai T.A.
      Nanostructured thin film polymer devices for constant-rate protein delivery.
      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.
      • Liu Y.
      • Wang R.
      • Zarembinski T.I.
      • et al.
      The application of hyaluronic acid hydrogels to retinal progenitor cell transplantation.
      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,
      • Ma J.
      • Kabiel M.
      • Tucker B.A.
      • Ge J.
      • Young M.J.
      Combining chondroitinase ABC and growth factors promotes the integration of murine retinal progenitor cells transplanted into Rho(-/-) mice.
      matrix metalloprotease 2,
      • Yao J.
      • Tucker B.A.
      • Zhang X.
      • Checa-Casalengua P.
      • Herrero-Vanrell R.
      • Young M.J.
      Robust cell integration from co-transplantation of biodegradable MMP2-PLGA microspheres with retinal progenitor cells.
      and AG1478, an investigational epidermal growth factor receptor tyrosine kinase inhibitor.
      • Robinson R.
      • Viviano S.R.
      • Criscione J.M.
      • et al.
      Nanospheres delivering the EGFR TKI AG1478 promote optic nerve regeneration: the role of size for intraocular drug delivery.
      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
      • Zhu X.
      • Su M.
      • Tang S.
      • et al.
      Synthesis of thiolated chitosan and preparation nanoparticles with sodium alginate for ocular drug delivery.
      and may serve as an excellent means to treat retinal disease
      • Jayaraman M.S.
      • Bharali D.J.
      • Sudha T.
      • Mousa S.A.
      Nano chitosan peptide as a potential therapeutic carrier for retinal delivery to treat age-related macular degeneration.
      and glaucoma and other ocular pathologic disorders.
      • Fulgêncio Gde O.
      • Viana F.A.
      • Ribeiro R.R.
      • Yoshida M.I.
      • Faraco A.G.
      • Cunha-Júnior Ada S.
      New mucoadhesive chitosan film for ophthalmic drug delivery of timolol maleate: in vivo evaluation.
      Wang et al
      • Wang T.J.
      • Wang I.J.
      • Lu J.N.
      • Young T.H.
      Novel chitosan-polycaprolactone blends as potential scaffold and carrier for corneal endothelial transplantation.
      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.

      Diagnostics

      Some ways in which nanotechnology has improved diagnostic imaging have been reviewed previously.
      • Zarbin M.A.
      • Montemagno C.
      • Leary J.F.
      • Ritch R.
      Nanomedicine in ophthalmology: the new frontier.
      Progress in this area continues. For example, monitoring active angiogenesis in neovascular eye diseases is essential for gauging a patient's disease progression and response to treatment.
      • Lalwani G.A.
      • Rosenfeld P.J.
      • Fung A.E.
      • et al.
      A variable-dosing regimen with intravitreal ranibizumab for neovascular age-related macular degeneration: year 2 of the PrONTO Study.
      Thus far, no in vivo imaging methods are available to label active angiogenesis specifically. Hua et al
      • Hua J.
      • Gross N.
      • Schulze B.
      • et al.
      In vivo imaging of choroidal angiogenesis using fluorescence-labeled cationic liposomes.
      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.
      • Barkana Y.
      • Anis S.
      • Liebmann J.
      • Tello C.
      • Ritch R.
      Clinical utility of intraocular pressure monitoring outside of normal office hours in patients with glaucoma.
      The results of frequent IOP measurement over a 24-hour period often lead to a change in glaucoma management.
      • Graham S.L.
      • Drance S.M.
      Nocturnal hypotension: role in glaucoma progression.
      • Wax M.B.
      • Camras C.B.
      • Fiscella R.G.
      • Girkin C.
      • Singh K.
      • Weinreb R.N.
      Emerging perspectives in glaucoma: optimizing 24-hour control of intraocular pressure.
      The wireless contact lens is a microelectromechanical systems–engineered, noninvasive IOP fluctuation monitor.
      • Leonardi M.
      • Pitchon E.M.
      • Bertsch A.
      • Renaud P.
      • Mermoud A.
      Wireless contact lens sensor for intraocular pressure monitoring: assessment on enucleated pig eyes.
      Mansouri et al
      • Mansouri K.
      • Medeiros F.A.
      • Tafreshi A.
      • Weinreb R.N.
      Continuous 24-hour monitoring of intraocular pressure patterns with a contact lens sensor: safety, tolerability, and reproducibility in patients with glaucoma.
      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.
      • Mansouri K.
      • Medeiros F.A.
      • Tafreshi A.
      • Weinreb R.N.
      Continuous 24-hour monitoring of intraocular pressure patterns with a contact lens sensor: safety, tolerability, and reproducibility in patients with glaucoma.
      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.
      Figure thumbnail gr4
      Figure 4Contact lens used to measure changes in intraocular pressure (Triggerfish; Sensimed AG). A, Diagram of the contact lens sensor showing the location of the sensor-active strain gauges (which are placed circumferentially to measure changes in the corneal curvature caused by intraocular pressure variations) and the sensor-passive strain gauges for thermal compensation (which are placed radially, where no strain is measured). For wireless powering and communication, a microprocessor and an antenna have been integrated into the soft contact lens. B, The contact lens sensor resting on the cornea. C, Wireless power and data transfer are achieved using a patched periorbital antenna, from which a cable is connected to a portable recorder (black box shown in the center of the photograph). Courtesy of Sonja Simon-Zoula, PhD, Sensimed AG.
      From Curr Opin Pharmacol,
      • Zarbin M.A.
      • Montemagno C.
      • Leary J.F.
      • Ritch R.
      Nanomedicine for the treatment of retinal and optic nerve diseases.
      with permission.

      Barriers to Clinical Application

      Several obstacles to the incorporation of nanotechnology into medicine are recognized.
      • Zarbin M.A.
      • Montemagno C.
      • Leary J.F.
      • Ritch R.
      Nanomedicine in ophthalmology: the new frontier.
      The biodistribution of nanoparticles and their persistence in tissues despite immune surveillance is a concern.
      • Zhang L.W.
      • Monteiro-Riviere N.A.
      Mechanisms of quantum dot nanoparticle cellular uptake.
      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.
      • Hoet P.
      • Legiest B.
      • Geys J.
      • Nemery B.
      Do nanomedicines require novel safety assessments to ensure their safety for long-term human use?.
      However, this field is nascent. In general, nanoparticle toxicity reflects the underlying chemistry.
      • Ferreira L.
      • Karp J.M.
      • Nobre L.
      • Langer R.
      New opportunities: the use of nanotechnologies to manipulate and track stem cells.
      Carbon nanotubes have toxicity that reflects their shape, size, surface coating, and concentration.
      • Magrez A.
      • Kasas S.
      • Salicio V.
      • et al.
      Cellular toxicity of carbon-based nanomaterials.
      • Zhu L.
      • Chang D.W.
      • Dai L.
      • Hong Y.
      DNA damage induced by multiwalled carbon nanotubes in mouse embryonic stem cells.

      Conclusion

      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.

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