Regenerative nanomedicine and the treatment of degenerative retinal diseases

Focus Article

Regenerative nanomedicineand the treatment of degenerativeretinal diseasesMarco A. Zarbin,1∗ Carlo Montemagno,2 James F. Leary3

and Robert Ritch4

Regenerative medicine deals with the repair or the replacement of tissues andorgans using advanced materials and methodologies. Regenerative nanomedicineuses nanoparticles containing gene transcription factors and other modulatingmolecules that allow reprogramming of cells in vivo as well as nanomaterialsto induce selective differentiation of neural progenitor cells and to createneural-mechanical interfaces. In this article, we consider some applications ofnanotechnology that may be useful for the treatment of degenerative retinaldiseases, for example, use of nanoparticles for drug and gene therapy, useof nanomaterials for neural interfaces and extracellular matrix construction forcell-based therapy and neural prosthetics, and the use of bionanotechnologyto re-engineer proteins and cell behavior for regenerative medicine. © 2011 WileyPeriodicals, Inc.

How to cite this article:WIREs Nanomed Nanobiotechnol 2012, 4:113–137. doi: 10.1002/wnan.167

INTRODUCTION

Nanotechnology involves the creation and useof materials and devices at the size scale of

intracellular structures and molecules and involvessystems and constructs on the order of <100 nm.Nanotechnology provides an important new setof tools for the diagnosis and treatment ofocular diseases, a concept that has been reviewedelsewhere.1,2

Regenerative medicine deals with the repairor the replacement of tissues and organs usingadvanced materials and methodologies. Regenera-tive nanomedicine uses nanoparticles containing genetranscription factors and other modulating molecules

∗Correspondence to: [email protected] of Ophthalmology and Visual Science, New JerseyMedical School, University of Medicine and Dentistry of NewJersey, Newark, NJ, USA2College of Engineering, University of Cincinnati, Cincinnati, OH,USA3Department of Basic Medical Sciences, Purdue University, WestLafayette, IN Purdue University, School of Veterinary Medicine,West Lafayette, IN, USA4Einhorn Clinical Research Center, New York Eye & Ear Infirmary,New York, NY, USA

that allow reprogramming of cells in vivo3 as wellas nanomaterials to induce selective differentiation ofneural progenitor cells4 and to create neural–mechan-ical interfaces.5–7 In this review, we consider someapplications of nanotechnology that may be usefulfor the treatment of degenerative retinal diseases, e.g.,use of nanoparticles for drug and gene therapy, nano-engineering of viral vectors, use of nanomaterials forneural interfaces and extracellular matrix (ECM) con-struction, and neural prosthetics.

DRUG AND GENE THERAPY

Some General Considerations RegardingNanoparticlesStrategies in the design of nanoparticles for therapeuticpurposes have been reviewed thoroughly by Petros andDeSimone.8 Nanoparticle biodistribution is affectedby particle size, shape, and surface properties. Parti-cle size, for example, influences whether the particleis internalized via phagocytosis, macropinocytosis,caveolar-mediated endocytosis, or clathrin-mediatedendocytosis, which in turn results in exposure of thenanoparticle to different intracellular environments.9

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Compacted polylysine DNA nanoparticles are takeninto cells and transported directly to the nucleus bythe cell surface receptor, nucleolin.10

Liposomes and polymer-drug conjugates are thenanoparticles used most frequently for therapy. Lipo-somes, which carry hydrophobic or hydrophilic cargo,can be coated with ligands that direct them to specificcell surface receptors (for cell targeting) as well as withpolymers that prolong their half-life in the circulatorysystem. Poly(ethylene glycol) (PEG) can be conjugatedwith different molecules to enhance solubility andstability in plasma and to reduce immunogenicity.8

Opsonization by immunoglobulin and/or complementproteins can lead to recognition of the nanoparticleas foreign and induce a hypersensitivity reaction.11,12

Coating a nanoparticle with albumin and/or PEG cancreate a hydrophilic surface that temporarily resistsprotein adsorption, thus imparting longer bioavail-ability to the particle.8,13,14

Targeting nanoparticles to specific cells can beachieved by attaching to the particle surface ligands/antibodies/peptides/aptamers for receptors/moleculesthat are abundant on the surface of the targetcell/tissue.8 However, receptor aggregation on thecell surface can induce unintended consequences,such as inducing apoptosis.15 Similarly, one canengineer the nanoparticle for a particular modeof intracellular entry depending on the choice ofnanoparticle targeting molecules, e.g., cholesterolfavors uptake via caveolin-mediated endocytosis,and transactivating transcriptional activator peptidefavors macropinocytosis.16,17 Nanoparticle surfacechemistry also can be exploited to trigger cargorelease under specific circumstances. Reductively labiledisulfide-based crosslinks between the carrier andcargo, for example, are broken when exposed toa reducing environment such as is present in thecytosol.18,19 Approaches for targeting nanoparticles toparticular subcellular organelles, e.g., mitochondria20

or the nucleus,21 have been developed.

Neurotrophic Factor TherapyNanoparticles can be used to deliver growth and neu-rotrophic factors to cells.22 Intravitreal nanoparticle-mediated basic fibroblast growth factor (bFGF)delivery, for example, provides sustained retinal rescuein Royal College of Surgeons (RCS) rats.23 RCS ratshave a mutation that prevents proper outer-segmentphagocytosis by retinal pigment epithelial (RPE) cells,which results in progressive rod and cone photorecep-tor degeneration.24 Some forms of retinitis pigmentosa(RP) in humans arise from this same mutation.25–27

Sakai et al.23 prepared bFGF nanoparticles using

acidic gelatin isolated from bovine bone collagen byan alkaline process and human recombinant bFGF.Gelatin nanoparticles were crosslinked through adehydrothermal process and ultraviolet irradiationof preprepared noncrosslinked gelatin particles. Thenanoparticle diameter, assessed using dynamic lightscattering, was ∼585 nm. The bFGF was incorporatedinto the gelatin nanoparticles by dropping 5 mg/mLbFGF solution (20 μL) onto 2 mg freeze-dried gelatinnanoparticles.

Glaucoma, the second leading cause of blind-ness worldwide, is associated with progressive reti-nal ganglion cell death and optic nerve atrophy.28

Intravitreal glial cell line-derived neurotrophic fac-tor (GDNF)-loaded biodegradable (poly)lactic-co-glycolic acid (PLGA) microspheres provide sustainedganglion cell protection in a rodent model ofglaucoma.29 Microspheres containing GDNF werefabricated using a modification of a spontaneousemulsion technique.30 Resulting GDNF microspheresexhibited average diameters of ∼8 μm. In view ofthe fact that adeno-associated virus (AAV)-mediatedGDNF secretion from glia delays retinal degen-eration in a rat model of RP,31 it seems likelythat nanoparticle-mediated GDNF delivery could beapplied to treating RP-like diseases also.

Oxidative DamageDiverse retinal diseases including age-related macu-lar degeneration (AMD), RP, diabetic retinopathy,and retinopathy of prematurity are characterized, inpart, by the presence of oxidative damage.32–37 Alter-ation in the oxidation state of cerium oxide (CeO2)nanoparticles (‘nanoceria’) creates defects in its latticestructure through loss of oxygen or its electrons. Astheir size decreases, nanoceria particles (3–5 nm diam-eter) demonstrate formation of more oxygen vacanciesin the crystal structure.38,39 Chen et al.40 posited thatengineered nanoceria particles can scavenge reactiveoxygen intermediates and demonstrated that intrav-itreal injection of nanoceria prevents light-inducedphotoreceptor damage in rodents, even if injectedafter the initiation of light damage. Vacancy engi-neered nanoceria also inhibit the development of andpromote regression of pathological retinal neovascu-larization in the Vldlr knockout mouse, which carriesa loss-of-function mutation in the very low densitylipoprotein receptor gene and whose phenotype resem-bles a clinical entity known as retinal angiomatousproliferation41,42 (Figures 1 and 2). This regressionoccurs even if nanoceria are injected intravitreallyafter the mutant retinal phenotypes are established(Figure 3). A single injection has a prolonged effect

114 © 2011 Wiley Per iodica ls, Inc. Volume 4, January/February 2012

WIREs Nanomedicine and Nanobiotechnology Regenerative nanomedicine

Saline

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FIGURE 1 | Nanoceria reduce oxidative stress in the Vldlr−/− retina. Retinal sections from saline injected WT mice (a, d, g, j); saline injectedVldlr−/− mice (b, e, h, k) and CeO2 injected (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 ROSin the normal (a), a considerable amount in the Vldlr−/− (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, e, 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, h, i) a reflection of oxidative activity due to increases in nitricoxide concentration, was highest in the Vldlr−/− retina and significantly reduced in the nanoceria injected mice. ROS-mediated damage to DNA wasindicated by the labeling of the retina with an antibody against a DNA adduct, 8-hydroxy-29-deoxyguanosine (8-OHdG; j, k, ) which showed littlelabeling 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. (Reprinted with permission from Ref 41. Copyright under Creative Commons Attributions License 2011)

(weeks) because nanoceria are a catalytic and regen-erative antioxidant. Nanoceria inhibit developmentof increased vascular endothelial growth factor levelsin this model,41 which may mean this nanoparticlewill be effective in treating macular edema in diabeticeyes and choroidal neovascularization-induced retinaledema in AMD eyes.43–45

C-60 fullerenes are cage-like structures of carbonatoms in the form of a truncated icosahedron, andthey also have antioxidant properties.46 Malonic acidC-60 derivatives (carboxyfullerenes) can eliminatesuperoxide anion and H2O2 and inhibit lipidperoxidation.8 Systemic administration of the C-3carboxyfullerene isomer delayed motor deteriorationand death in a mouse model of familial amyotrophiclateral sclerosis34,47 and therefore might be usefulin the treatment of retinal diseases associated withoxidative damage.

Iron is an essential element for enzymes involvedin the phototransduction cascade, in outer segment

disc membrane synthesis, and in the conversion ofall-trans-retinyl ester to 11-cis-retinol in the RPE.48–50

Free Fe2+ catalyzes the conversion of hydrogen per-oxide to hydroxyl radical, which is a highly reactiveoxygen species that causes oxidative damage (e.g.,lipid peroxidation, DNA strand breaks).51 Iron accu-mulation in the RPE and Bruch’s membrane is greaterin AMD eyes than in controls, including cases withearly AMD and late stages of the disease (i.e., geo-graphic atrophy, choroidal new vessels).52 Some ofthis iron is chelatable.52 Increased intracellular ironcauses oxidative photoreceptor damage.53 Althoughit is not proven that iron overload is a cause ofAMD,54–57 iron chelation might have a therapeuticeffect. Polymeric nanoparticles can be used to chelatemetals. Liu et al.58 showed that a chelator–nanoparti-cle system complexed with iron, when incubated withhuman plasma, preferentially adsorbs apolipoproteinE and apolipoprotein A-I, which should facilitatetransport into and out of the brain via mechanisms



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FIGURE 2 | Nanoceria inhibit the development of pathologic intra- and subretinal vascular lesions in the Vldlr−/− retina. Photomicrographs ofwhole mount retinas (a–c) and eyecups [retinal pigment epithelium (RPE), choroid, and sclera] (d–f) from P28 animals are shown. All retinal bloodvessels were labeled green by the vascular filling assay. Wild type (WT) retinas (a) showed the normal web-like retinal vasculature, whereas thosefrom the Vldlr−/− mice (b) showed numerous intraretinal vascular lesions or ‘blebs’ (IRN blebs). See white arrows for examples. A single injection ofnanoceria at P7 inhibited (c) the appearance of these lesions. Eyecups from WT mice (d) showed no subretinal neovascular (SRN) ‘tufts’ but thosefrom Vldlr−/− mice (e) had many bright SRN tufts. A single injection of nanoceria on P7 inhibited the appearance of these SRN tufts (f). (Reprintedwith permission from Ref 41. Copyright under Creative Commons Attributions License 2011)

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FIGURE 3 | Retinal vascular lesions in the Vldlr−/− retinas require continual production of excess reactive oxygen species (ROS). Vldlr−/− micewere injected at P28 with saline or nanoceria and killed 1 week later on P35. Analysis of VEGF levels by Western blots (a) showed a fourfoldreduction (b) within 1 week of nanoceria injection. The numbers of IRN blebs (c) and SRN tufts (d) were also dramatically reduced. *P = 0.05;**P = 0.01. (Reprinted with permission from Ref 41. Copyright under Creative Commons Attributions License 2011)

used for transporting low-density lipoprotein. Thisapproach might have utility for treating AMD eyes.

Immune Suppressive TherapyCell-based therapy might be sight-restoring forpatients with degenerative retinal diseases, such as RPand AMD. Although photoreceptors are not expectedto be immunogenic, glial cells (that might be trans-ferred as part of the transplant procedure) and RPEcells are probably immunogenic.59 Thus, in somecases, cell-based therapy for retinal disease will prob-ably require immune suppressive therapy (local or

systemic) after the transplant operation.59–61 Nan-otechnology may be useful in this regard. Nanopar-ticles, for example, are helpful in managing cornealallograft rejection in preclinical models. Yuan et al.62

manufactured 300 nm diameter rapamycin-loadedchitosan/poly(lactic acid) (PLA) nanoparticles andfound that these nanoparticles extended median allo-graft survival by 17% in rabbits compared withaqueous rapamycin eye drops. Although topical chi-tosan particles were well tolerated in this study,intraocular chitosan nanoparticles may not be welltolerated.63



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FIGURE 4 | Histopathologic features and scores of rats with experimental autoimmune uveoretinitis (EAU). Representative photographs taken7 days after treatment with saline (a), 100 μg of nonstealth nanosteroids [poly(lactic acid) (PLA)] (b), and 100 μg of stealth nanosteroid [poly(lacticacid)–poly(ethylene glycol) (PLA–PEG)] (c) are shown. Note the disruption of the inner and outer segments in all areas for saline-treated rats (a) andthe preservation of structural integrity with stealth nanosteroids (c). White arrows: retinal folds and small granuloma formation; black arrows:inflammatory cellular infiltrates in the vitreous (original magnification, ×100). The severity of EAU in rats treated with saline, nonstealth nanosteroids,or stealth nanosteroids was graded 7 days after treatment. The scores for rats treated with stealth nanosteroids (1.5 ± 0.5) were significantly lowerthan for rats treated with saline (5.5 ± 0.8, P < 0.01) and nonstealth nanosteroids (3.0 ± 0.9, P < 0.05) (d). Data are shown as the mean ± SD(n = 6 in each group). (Reprinted with permission from Ref 69. Copyright 2011 Association for Research in Vision and Ophthalmology (Arvo))

Evidence that nanoparticles can be used tomodulate the inflammatory response in the retinaand choroid comes from studies of experimentalautoimmune uveoretinitis (EAU). EAU is a T-cell-mediated autoimmune disease that targets the retinaand related tissues and serves as a model for humanautoimmune ocular diseases.64 Nanosuspensions ofrelatively insoluble glucocorticoids (developed usinga high pressure homogenization method) enhancethe rate and extent of drug absorption, the inten-sity of drug action, and the duration of drug action,compared to conventional solutions and microcrys-talline suspensions.65 PLA nanoparticles are clearedrapidly from the systemic circulation in rats withEAU.66 As noted above, PEG can be used to mod-ify the surface of the nanoparticles, which reducesopsonization and interactions with the mononu-clear phagocyte system.67 Sakai et al. prepared poly-meric nanoparticles with encapsulated betametha-sone phosphate using an oil-in-water solvent diffu-sion method to manufacture nanosteroid particles(∼120 nm diameter) composed of PLA homopoly-mer and a block copolymer of PEG.68,69 In vivoimaging of inflamed eyes of rats with EAU demon-strated greater nanoparticle accumulation and higher

betamethasone concentration in eyes of rats treatedwith PLA–PEG nanoparticles versus PLA nanopar-ticles. EAU rats treated with PLA–PEG nanosteroidsalso had lower clinical scores for ocular inflammation.These findings were confirmed with histopathology(Figures 4 and 5). The relatively stronger therapeuticeffect of PLA–PEG nanosteroids versus PLA nanos-teroids may result from prolonged blood circulationand sustained release in situ as well as due to targetingto inflamed eyes (the latter effect resulting from thesmall diameter of the nanoparticles).69 Zhang et al.70

showed that EAU responds very well to intravitrealliposomal Tacrolimus (mean diameter = 200 nm) withno side effects on retinal function or systemic cellularimmunity.

Gene Therapy: Nonviral VectorsViral vectors deliver genes efficiently but are associ-ated with risks such as immunogenicity and insertionalmutagenesis. Nonviral vectors (e.g., polymers, lipids)and other methods (e.g., electroporation, nucleo-fection) have high gene-carrying capacity, low riskof immunogenicity, relatively low cost, and greaterease of production.71 Nanoparticles can deliver genesefficiently to stem cells72 and have been explored as a



Saline Non-stealth nanosteroid Stealth nanosteroid

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FIGURE 5 | Immunohistochemistry of the retina of rats with EAU (n = 4, each group). Representative results are shown 7 days after treatmentwith saline (a), nonstealth nanosteroids [poly(lactic acid) (PLA)] (b), and stealth nanosteroids [poly(lactic acid)–poly(ethylene glycol) (PLA–PEG)] (c).Marked expression of interleukin (IL)-6 (IL-6) (green) and IL-17 (red) was observed in ocular infiltrative cells in rats treated with saline or nonstealthnanosteroids. Rats treated with stealth nanosteroids showed marked reduction of ocular infiltrative cells. Bar, 50 μm. (Reprinted with permissionfrom Ref 69. Copyright 2011 Association for Research in Vision and Ophthalmology (Arvo))

means for gene delivery in the diagnosis and treatmentof ocular disease.73–76

Electrostatic interaction of cationic polymerswith negatively charged DNA/RNA molecules resultsin condensation of the material into particles (rang-ing in size from 8 to 500 nm in diameter), pro-tection of the genes from enzymes, and mediationof cellular entry.77,78 Complexes of cationic poly-mers and plasmid DNA, termed polyplexes, canhave transfection efficiency comparable to adenovi-ral vectors.79 Polyplexes are nanometer size, havelarge vector capacity, are stable in nuclease-rich envi-ronments, and have relatively high transfectivity forboth dividing and nondividing cells.76,79 For example,nanoparticles compacted with a lysine 30-mer linkedto 10 kDa PEG-containing cytomegalovirus cysticfibrosis transmembrane conductance regulator (CMV-CFTR) cDNA were used successfully in a phase I/IIclinical trial for treatment of cystic fibrosis.80 How-ever, some particles have low-transfection efficiency,and the duration of gene expression can be short. Thetoxicity of polyplexes and nanoparticles is a reflectionof their chemistry.77

Compacted DNA nanoparticles can be targetedto different tissues in the eye by varying the injectionsite (e.g., intravitreal injection can target the cornea,trabecular meshwork, lens, and inner retina; subreti-nal injection can target the outer retina and RPE).76

Nanoparticle size and charge influence migrationthrough the vitreous cavity.81 Additional specificityin the locus of gene expression can be achieved bychoosing promoters that are cell specific. For example,the rhodopsin promoter drives expression in rod pho-toreceptors, and the human red opsin promoter drivesexpression in cone photoreceptors.82–84 Interphotore-ceptor retinoid-binding protein drives expression bothin rods and cones.85 The vitelliform macular dys-trophy promoter drives expression in RPE cells.86

Farjo et al.76 showed that after subretinal injectionof compacted lysine 30-mer DNA nanoparticles, gene

expression is observed throughout the retina and notjust at the site of the injection.

Cai et al.82,87 used a specific formulation ofDNA nanoparticles consisting of single moleculesof DNA compacted with 10 kDa PEG-substitutedlysine 30-mer peptides containing the wild-type reti-nal degeneration slow (Rds) gene, peripherin/rds,to induce cone photoreceptor rescue in an ani-mal model (rds+/−) of RP. These particles did notinduce any detectable immune response, cytotoxicity,or disruption of retinal function after injection intothe subretinal space. These compacted plasmid DNAnanoparticles are small (8–20 nm), have rod orellipsoid shape (depending on the counterion used),and have a large carrying capacity (at least up to20 kb).76,87 PLGA nanoparticles can deliver genes toRPE cells in vitro and in vivo with reasonable efficiencyand safety.88 PLGA DNA nanoparticles can be associ-ated with long-term gene expression, perhaps becauseof sustained cytosolic plasmid release.89 PLGA DNAnanoparticles tend to be larger than polylysine DNAnanoparticles,90,91 which, as noted above, may affectcellular uptake mechanism and delivery to the nucleus.PLGA DNA nanoparticles might be used to delivertherapeutic genes for conditions associated withRPE gene mutations, e.g., Best Disease92 and aform of Leber congenital amaurosis.93–95 Althoughthese results are promising, some concerns involvingnanoparticle use remain. For example, although theimmune response to polylysine-based nanoparticlesseems to be less than that for capsid proteins, the effi-ciency of gene transfer is not as high because most aredegraded in the endosomal complexes.96 As a result,one may have to use large numbers of nanoparticlesthat generate an immune response nonetheless. Also,the immune response to both nanoparticles and virusesvaries from one species to another, and the apparentlow immunogenicity observed in murine models of RPmay not be observed in human patients.96



Gene Therapy: Viral VectorsThe main barriers to successful gene therapyare (1) vector uptake, transport, and uncoating;(2) vector genome persistence; (3) sustained transcrip-tional expression; (4) the host-immune response; and(5) insertional mutagenesis and cancer.96–98 In thecase of virus-based gene therapy, immune responses,including innate, humoral, and cell-mediated, canbe directed against the vector and/or the transgeneproduct.96,99,100 As pointed out by Kay,96 primaryhumoral responses directed against the vector canlimit its capacity to deliver genes to the targetcells as well as the ability to readminister the virusto the patient (e.g., when treating the fellow eyewith a second surgical procedure). For example, animmediate innate immune response and a secondaryantigen-dependent response to intravenous admin-istration of recombinant adenoviral vectors causeddeath in a patient with ornithine transcarbamy-lase deficiency.101,102 A humoral response againstthe transgene product may neutralize the thera-peutic protein.96 A cell-mediated immune responseagainst the vector or transgene product can elimi-nate the transduced cells.96 For example, two patientswith hemophilia B developed vector dose-dependenttransaminitis that limited hepatocyte-derived factor IXexpression to <2 months because of CD8+ memory Tcells that recognized adeno-associated virus serotype2 (AAV2) capsids and eliminated AAV2-transducedhepatocytes.103,104 The innate immune response cancause local and/or systemic toxicity and amplify a sec-ondary antigen-dependent immune response.96 Thelikelihood of an immune response is influenced bythe dose of viral particles,105 which in turn is influ-enced by the efficiency of vector uptake and geneexpression, as well as by the specificity of target-ing. If dendritic cells or antigen-presenting cells takeup the vector, for example, an immune response ismore likely.

Nanoengineering of the viral capsid and trans-gene may provide effective solutions to some of theseproblems. Recombinant adeno-associated viruses(rAAVs) have been used extensively to treat pre-clinical models of human ocular disease and alsohave been used to treat humans with Leber congen-ital amaurosis.93,94,106 Modifications of the virus toimprove clinical effectiveness provide a good exampleof some of the nanoengineering strategies that havebeen employed in this area. AAVs are small (4.7 kbcarrying capacity), nonpathogenic, single-strandedDNA parvoviruses that can transduce dividing andnondividing cells.107 The genes encoding replica-tion and capsid proteins from the wild-type AAV

genome are replaced by a promoter-therapeutic trans-gene cassette flanked by the normal AAV invertedterminal repeats needed for packaging and replica-tion in rAAVs. The capsid is critical for extracellularevents related to the recognition of specific receptors,which influences cell tropism, as well as intracellularprocesses involving AAV trafficking and uncoating,which influences transduction kinetics and efficiencyof transgene expression.108,109 A significant propor-tion of the population has been exposed to variousAAV serotypes and harbors neutralizing antibodiesthat can block gene delivery.100,110,111 As noted above,administration of low doses of viral vector mightmitigate the severity of this problem. Two nano-engineering techniques have been applied to improvevector cellular tropism, transduction efficiency, andimmunogenicity: directed evolution and site-directedmutagenesis. These are discussed below. Other nano-engineering devices (i.e., DNA transposons,112 bac-teriophage recombinases113) may provide clinicallyuseful means to achieve stable, safe DNA integrationin the host genome and sustained transgene expressionin the future.

Directed EvolutionDirected evolution of AAV capsids has generatedvectors that are highly resistant to neutralizingantibodies114,115 (Figure 6). Maheshri et al.115 usederror-prone polymerase chain reaction and a stag-gered extension process116 to generate an AAV2library (>106 independent clones) with randomlydistributed capsid mutations and then used high-throughput approaches (i.e., exposure of mutantsto heparin affinity chromatography (wild-type AAV2binds to heparan sulfate) or repeated amplificationof AAV2 mutants that retain infectivity in the pres-ence of serum containing neutralizing antibodies) toidentify mutant AAV2 capsids with altered receptor-binding properties and the capacity to bind withvery low affinity to neutralizing antibodies. Thisapproach can be quite powerful. One mutagenesisand three selection steps generated mutant capsids,for example, with a threefold improved neutralizingantibody titer (vs wild-type capsid) and a ∼7.5%infectivity at serum levels that completely neutral-ized wild-type infectivity.115 Directed evolution hasbeen used to generate AAV variants that transduceMuller cells after intravitreal injection,117,118 whichmay provide a means to deliver growth factors tophotoreceptors and RPE cells. These growth factorsretard the progression of retinal degeneration in pre-clinical models of RP119,120 and possibly in humanpatients also.121



(a) Wild-type AAVs (c) Generation of capsid library

(d) Infection of cells and selection for:

(e) Production of vector and testing

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AAV-a AAV-b2AAV-b1

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• Sequence predominant capsid genes• Clone capsid genes into helper plasmids • Use reporter or therapeutic genome encapsidated by the selected capsid• Test selected vectors (in vivo) and in (vitro)Reassemble randomly

Shuffled 'wild-type AAV' library

FIGURE 6 | Adeno-associated virus capsid shuffling and directed evolution. Although the capsid sequences can be easily modified, it is difficult tomake predictions about how specific modifications in the amino acid sequence will affect the transduction parameters of the viral vector. (a) Variouscapsid DNA sequences are derived from adeno-associated viruses (AAVs) with different transduction properties (hexagons of different colors). (b) Thecapsid DNA sequences are randomly digested and then PCR ligated back into a ‘wild-type’ AAV plasmid (capS, shuffled cap gene). The AAV capsidlibrary can contain between 106 and 107 unique sequences. (c) The recombinant AAV wild-type viruses are expanded (with the addition of areplication helper virus, not shown) without any selection in cells. (d) The AAV viral library is expanded under selective pressure, allowing viruses thatsurvive the selection to be further propagated. With stronger selective pressure, the diversity of the capsid library is reduced, and select clones areenriched. (e) Selected capsid sequences that survive the selection are then cloned into a vector production system and used to pseudotype standardAAV vector genomes (containing a reporter or therapeutic expression cassette) and tested for transduction properties in cells, animals, or humans.(Reprinted with permission from Ref 96. Copyright 2011 Nature Publishing Group)

Site-directed MutagenesisEpidermal growth factor receptor protein tyrosinekinase (EGFR-PTK) signaling impairs AAV2 vec-tor transduction by impairing nuclear transportof the vectors.122 EGFR-PTK can phosphorylate

AAV2 capsids at tyrosine residues.122,123 Tyrosine-phosphorylated AAV2 vectors enter cells efficientlybut do not transduce well, in part because theAAV capsids are ubiquitinated and then degradedby the proteasome.122,124 Zhong et al.125 showed that



site-directed mutagenesis126 of the surface-exposedtyrosine residues increases vector transduction effi-ciency 30-fold in vivo at 1 log lower vector dosecompared to wild-type AAV2. The transduction effi-ciency is increased because of impaired capsid ubiq-uitination and improved intracellular trafficking tothe nucleus. Thus, the T cell response to AAV2 cap-sids seems to be manageable by using surface-exposedtyrosine mutant vectors. Another rate limiting stepin transduction efficiency is the conversion of single-stranded viral genome to double-stranded AAV DNA.This limitation has been overcome by deleting theterminal resolution site from one rAAV inverted ter-minal repeat, which prevents replication initiation atthe mutated end, to generate self-complimentary AAV(scAAV) vectors.127,128 (AAV has a tendency to pack-age DNA dimers when the replicating genome is halfthe length of the wild type.)

Ocular ApplicationsRecombinant AAV vectors have been used extensivelyto treat eye diseases in preclinical models, includ-ing preclinical models of human retinal disease,129,130

because of their relatively low immunogenicity, abilityto target many nondividing cells, and capacity for sus-tained efficient therapeutic gene expression after a sin-gle treatment.108 Site-directed mutagenesis technologyis being applied to the treatment of degenerative reti-nal disease in preclinical models. Vectors containingpoint mutations in surface-exposed capsid tyrosineresidues in AAV serotypes 2, 8, and 9 display strongand widespread transgene expression in retinal cellsafter intravitreal or subretinal delivery.131 Petrs-Silvaet al.131 showed that tyrosine-to-phenylalanine capsidscAAV2 mutants showed much greater transductionefficiency (10- to 20-fold higher transgene expression)of the entire retina (including photoreceptors) afterintravitreal injection compared with scAAV with wild-type capsids (Figure 7). Mutants of scAAV2, scAAV8,

and scAAV9 also enhanced transduction of retinalganglion cells compared to wild type AAV2 (e.g.,106-fold reduction in the number of virus particlesneeded for ganglion cell transfection with mutantscAAV2 compared to wild-type AAV2). (Previously,only AAV2 could transduce retinal ganglion cells.)Intravitreal delivery may offer an important clinicaladvantage over subretinal delivery. Subretinal virusdelivery, which has been used in clinical studies,93–95

requires pars plana vitrectomy surgery in the operat-ing room and has a higher likelihood of complications(e.g., retinal tear) than intravitreal delivery, which canbe done in an office setting under topical anesthesia.On the other hand, the subretinal space is a rela-tively immune privileged site,132 which may reducethe likelihood of an immune response after repeatvirus treatment. In fact, Li et al.133 have shown thata humoral immune response against AAV2 capsidproteins occurs after intravitreal but not after sub-retinal vector delivery. Subretinal injection of one ofthe mutant scAAVs also transduced Muller cells. Inthese experiments, the capsid tyrosine mutations didnot change the cellular tropism of the vectors rela-tive to the wild-type counterparts; they just enhancedtheir transduction potential. These studies demon-strate two strategies for reducing the immune responseto viral vectors via site directed mutagenesis: increas-ing transduction efficiency (which permits lower dosesof vector) and creation of multiple effective serotypes,which can be used sequentially for subsequent therapy.

CELL-BASED THERAPY

Degenerative Retinal DiseaseDegenerative retinal diseases such as AMD andRP cause blindness primarily through photoreceptordeath. Reactive changes in the synaptic circuitry ofsecond- and third-order neurons also occur,134 butthese changes probably will not decisively limit visual

(a) (b) (c)

FIGURE 7 | Fluorescence microscopic evaluation of enhanced green fluorescent protein (EGFP) expression in transverse sections of retinal tissue 2weeks after intravitreal injection. Immunostaining for EGFP in sections of the retina after delivery of (a) wild-type self-complementaryadeno-associated virus 2 (WT scAAV2), (b) serotype 2 tyrosine-mutant Y444F, and (c) serotype 2 tyrosine-mutant Y730F. Note intense EGFP stainingthroughout all retinal layers with Y444F mutant and predominant EGFP staining in the ganglion cell layer (GCL) with WT-2 and Y730F. Calibration bar100 μm. gcl, ganglion cell layer; ipl, inner plexiform layer; inl, inner nuclear layer; onl, outer nuclear; os, outer segment. (Reprinted with permissionfrom Ref 131. Copyright 2009 Nature Publishing Group)



recovery, particularly, if photoreceptor replacementcan be achieved before atrophy is extensive. Cell-basedtherapy may be sight-preserving and/or sight-restoringfor these patients.135–143 Fetal retina sheet transplants,for example, have been effective in preclinicalmodels.144 Also, retinal progenitor cells and evenadult photoreceptors can integrate into host retina andimprove some aspects of visual function.145,146 Thereis evidence, however, that cell isolation and bolusinjection is associated with significant cell death.146,147

Control of the ECM, which can alter cell behav-ior, may be crucial for successful retinal regeneration.Stem cells, for example, are prevented from exitingthe mitotic cycle by environments called niches,148

which comprise cellular and noncellular elements (i.e.,ECM components), and RPE survival on submacu-lar Bruch’s membrane from AMD eyes is improvedsubstantially when a provisional, ‘healthy’ ECM ispresent.149 Nanoengineered polymer scaffolds mayenable implanted cells to overcome biological obsta-cles to transplant differentiation, survival, and inte-gration with the host retina.

Engineering Scaffolds to Support CellTransplantsMicroscale topographical cues can influence retinal150

and neural151 progenitor cell attachment and dif-ferentiation independent of biochemical cues. Phys-ical features of the ECM that influence cellbehavior and phenotype include the size, lateralspacing, surface chemistry, and geometry of ECMligands.152–154 Biocompatible/biodegradable scaffoldswith the proper nanoscale features might improvetransplant efficacy by preventing anoikis (apoptosisdue to absence of cell adhesion to the ECM substrate),promoting maintenance of a differentiated phenotype,providing proper three-dimensional organization ofthe cell-ECM assembly, and promoting a supportivehost response to the transplant.155 Nanofiber scaf-folds have a high surface area:volume ratio and canpresent a high density of epitopes to cells, thus promot-ing neural progenitor cell differentiation.4 Nanofiberscaffolds might be used to create niches for stem cellself-renewal or as substrates supporting delivery ofsheets of cells.77 In addition to serving as a cell deliv-ery platform, these scaffolds can serve as a temporaryECM that maintains cell survival and differentiationwhile the transplanted cells elaborate their own ECMand degrade the scaffold. Ellis-Behnke et al.,156 forexample, reported that a designed self-assemblingpeptide nanofiber scaffold promoted axonal regen-eration through the severed optic tract of hamsters.The regenerated axons reconnected to target tissuesand promoted visual recovery.

Scaffolds for Cell Transplantation to theSubretinal SpaceHynes and Lavik have reviewed the materials, fabri-cation methods, and results of scaffold-assisted RPEand retinal cell transplantation in detail.157 Scaffoldscan maintain proper three-dimensional organizationof tissue (structural support), aid in cell delivery, influ-ence cell behavior (e.g., differentiation), and deliverdrugs or trophic molecules.157 Scaffolds can com-prise naturally occurring materials (e.g., Descemet’smembrane, lens capsule, Bruch’s membrane, amni-otic membrane), but, as Hynes and Lavik157 pointout, variation in material quality, availability, andinfectious disease concerns probably supersede theirattractive features, including biocompatibility andease of handling. Naturally occurring polymers, suchas collagen and fibrin, have the positive featuresof naturally occurring membranes and have beenused as cell scaffolds, but the problems of prod-uct consistency, allergic response, and infection riskremain. Use of synthetic polymers enables one to reg-ulate the biological properties (e.g., biodegradability,biocompatibility), mechanical properties (e.g., thick-ness, deformability), three-dimensional structure (e.g.,porosity), and distribution of bioactive molecules (e.g.,laminin, GDNF) precisely. However, synthetic scaf-folds may have undesirable features. For example,although poly(l-lactic acid)/poly(lactic-co-glycolideacid) (PLLA/PLGA) scaffolds improve cell deliv-ery 10-fold, they can have significant complicationsincluding inflammation and fibrosis.147,158 Spin-castpoly(methyl methacrylate) (PMMA) scaffolds, whilethin (6 μm) and capable of promoting retinal progen-itor cell differentiation,159 are not biodegradable. Inaddition, PMMA scaffolds require surface modifica-tion with either laminin or a combination of lamininand poly-l-lysine for retinal progenitor cells to attach.By contrast, poly(ε-caprolactone) (PCL) is biodegrad-able, biocompatible, can be spin-cast to a thin film(5 μm) with controlled microtopography (that favorscell adherence), and promotes differentiation of retinalprogenitor cells.150,160

Most synthetic scaffolds for cell transplan-tation have been manufactured using techniquesadapted from microchip fabrication methods, e.g.,photolithography and soft lithography.157 Microfab-rication permits construction of scaffolds with precisearchitecture159,161,162 [e.g., pore size (to improve cellretention), groove width (to improve cell morphol-ogy), and distribution of bioactive molecules150,159,160

(to improve cell attachment and/or differentiation)](Figures 8 and 9). Tao et al.159 compared adhesionof retinal progenitor cells to polymer, as well asmigration and differentiation in the host retina for



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FIGURE 8 | Microfabrication of poly(ε-caprolactone) (PCL) thin filmwith photolithography and soft lithography. (a) Schematic of PCL thinfilm scaffold fabrication. SU-8 photoresist is spin-cast onto a siliconwafer and exposed to ultraviolet light through a negative mask.Unexposed areas are not crosslinked and developed away, andpolydimethylsiloxane (PDMS) is cured on the wafer. After peeling thePDMS mold from the wafer, PCL is spin-cast on the mold and peeledfrom the surface. (b) A scanning electron micrograph of a PCL thin filmwith 25-μm diameter wells. (c) Profile of PCL thin film. (Reprinted withpermission from Ref 150. Copyright 2010 Springer)

PMMA scaffolds (6 μm thickness) with either smoothor porous (11 μm diameter) surface topography. Reti-nal progenitor cells were cultured under identicalconditions on smooth or porous laminin-coated poly-mer scaffolds and transplanted into the subretinalspace of C57BL/6 mice. Transplantation with non-porous scaffolds showed limited retinal progenitorcell retention, and porous scaffolds demonstratedenhanced retinal progenitor cell adherence duringtransplantation and allowed for greater process out-growth and cell migration into the host retinal layers.A related strategy involves implantation of compos-ite grafts. Redenti et al.160 studied the behavior ofmouse retinal progenitor cells cultured on laminin-coated nanowire PCL scaffolds, including the survival,differentiation, and migration of these cells into theretina of C57bl/6 and rhodospsin−/− mouse retinalexplants and transplant recipients. Retinal progenitorcells were cultured on smooth PCL and both short(2.5 μm) and long (27 μm) nanowire PCL scaffolds.Scaffolds with adherent mouse retinal progenitor cellswere then either cocultured with, or transplanted to,wild-type and rhodopsin−/− mouse retina. Robust

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FIGURE 9 | Effect of structured surface on retinal progenitor cellbehavior. (a) Attachment of retinal progenitor cells (RPCs) to substratesurfaces after 2 days growth. Substrate microtopography of 25 μm wellPCL leads to significantly more RPC attachment compared tounstructured PCL and tissue culture polystyrene surfaces (TCPS).Fluorescence images of DAPI-stained RPC nuclei attached to (b) TCPS,(c) unstructured PCL, and (d) 25-μm well PCL. *P < 0.05,Student–Newman, Keuls test. Error bars indicate SD over threeindependent experiments. (Reprinted with permission from Ref 150.Copyright 2010 Springer)

retinal progenitor cell proliferation on each type ofPCL scaffold was observed, and retinal progenitorcells cultured on nanowire scaffolds increased expres-sion of mature bipolar and photoreceptor markers.PCL-anchored retinal progenitor cells migrated intothe retina of both wild-type and rhodopsin knock-out mice. Using microfabrication processes, Sodhaet al.161 also have manufactured a novel biodegrade-able thin-film cell encapsulation scaffold in PCL as apossible cell transplantation vehicle. Individual thin-film 2–2.5-D PCL layers (<10 μm) were structuredwith varying micro and nanogeometries (protrusions,cavities, pores, particles) utilizing a modified spin-assisted solvent casting and melt templating technique.Thin-film layers were aligned and thermally bondedto form the three-dimensional cell encapsulation scaf-fold (<30 μm). These three-dimensional scaffolds pro-moted retinal progenitor cell retention and providedappropriate permeability.

NEURAL PROSTHETICS

Induced PhotosensitivityThis concept has been explored in detail elsewhere.2

The use of molecules as machines will revolutionizeneural prosthetics. For example, although rewiring



of inner retinal circuits and inner retinal neuronaldegeneration occur in association with photoreceptordegeneration in RP,134,163 it is possible to createvisually useful percepts by stimulating retinal ganglioncells electrically.164–167 Use of light-sensitive ionchannels, rather than electrodes, to stimulate retinalganglion cells provides an alternative approach toretinal cell stimulation.168–171 Induced light sensitivityhas the potential for noninvasive neuronal stimulationwith high spatial resolution.

Channelopsin-2 is a light-gated ion channel thatis sensitive to blue light and is derived from greenalgae. When its attached chromophore, all-trans reti-naldehyde, undergoes reversible photoisomerization,channelopsin-2 undergoes a conformational changethat alters its permeability to mono- and divalentcations.172 The complex of channelopsin-2 and all-trans retinal is termed channelrhodopsin-2 (ChR2).Using an AAV delivery system (AAV serotype-2) inrd1 mice, which have a null mutation in a cyclicGMP phosphodiesterase (PDE6b), and in RCS rats,which have a mutation in the tyrosine kinase, Mertk,ChR2 expression can be achieved in inner retinal neu-rons (primarily ON and OFF retinal ganglion cells).(Each of these mutations causes some forms of RP inhumans.) ChR2 converts these neurons into cells thatrespond to light with membrane depolarization.173–177

In addition, ChR2 can restore the ability of the ani-mals to encode light signals in the retina and transmitthem to the visual cortex.

Zhang et al.178 showed that expression ofhalorhodopsin (HaloR), a yellow light-activated chlo-ride ion pump from halobacteria, in inner retinalneurons converts them into OFF cells. In these experi-ments, HaloR was ∼20-fold less sensitive to light thanChR2. HaloR and ChR2 coexpressing cells can pro-duce ON, OFF, and ON–OFF responses, dependingon the illumination wavelength.178 Results in thesepreclinical models indicate that kinetics of ChR2- andHaloR-mediated light responses are compatible withtemporal processing requirements of visual infor-mation in the retina. A current limitation of thisapproach is that ChR2 and HaloR both exhibit lowlight sensitivity, with threshold activation light inten-sities ∼5–6 log units higher than those of cones.173,178

In addition, the light intensity operating range ofmicrobial rhodopsins is 2–3 log units, compared tonormal retinal dynamic range of 10 log units. Doroud-chi et al.179 have achieved stable and specific ChR2in ON bipolar cells using a recombinant AAV vec-tor packaged in a tyrosine-mutated capsid. Lightlevels that elicited visually guided behaviors werewithin the physiological range of cone photorecep-tors. There was no evidence of induced inflammation

or toxicity. Targeting ChR2 to rod bipolar cells mightpermit increased light sensitivity as well as higher spa-tial resolution because of signal convergence frombipolar cells onto retinal ganglion cells, but thisapproach may be compromised by the alterationsin synaptic circuitry that accompany photoreceptordegeneration.134,163,180–182

Greenberg et al.183 adapted a fundamentallydifferent approach to insertion of optical neuromod-ulators that retains crucial information processing(edge detection) while being independent of the stateof inner retinal circuit remodeling during degener-ation. They reconstructed an excitatory center andantagonistic surround by targeting humanized ChR2to the somata and enhanced HaloR to the dendritesof retinal ganglion cells (Figures 10 and 11). Fusion ofthe humanized ChR2 to ankyrinG polypeptide local-ized this opsin to the soma and proximal dendrites.(Ankryins couple sodium channels to the spectrin-actin network.) Fusion of enhanced HaloR to PSD-95protein targeted this opsin to the dendritic regions inganglion cells. This approach endowed ganglion cellswith differential spatial and spectral photosensitivity.Both ON and OFF-center ganglion cells could be cre-ated depending on which opsin is fused to ankyrinGand which to PDS-95. Because this approach gen-erated nonphysiological center-surround dimensions,Greenberg et al.183 preprocessed the visual imagewith Gaussian blurring, such that when convolvedwith the dimensions of the soma and dendrites, theGaussians approximated the relative dimensions ofthe ganglion cells’ center and surround receptivefields. In other words, extraction of edge informa-tion could be obtained artificially. These data andthe above considerations indicate that ChR2/HaloR-based retinal ganglion cell prosthetics will requireimage preprocessing to perform light amplification,dynamic range compression, and local gain controloperations.183

In typical RP, the rod photoreceptors degen-erate first, and cone degeneration follows.163 Thecone cell bodies remain for a time after their outersegments are lost. Busskamp et al.184 showed thatenhanced HaloR expression in light-insensitive cones(via AAV transfection) can restore light sensitiv-ity in mouse models of RP (i.e., the rd1 mouse,which models fast forms of retinal degeneration, andCnga3−/−; Rho−/−double-knockout, which models aslow form of retinal degeneration). These resensi-tized cones activate all retinal cone pathways, drivedirectional selectivity, activate cortical circuits (in rd1mice), and mediate visually guided behaviors (to agreater degree in rd1 mice than in Cnga3−/−; Rho−/−mice). Targeted expression of enhanced HaloR in



FIGURE 10 | Humanized ChR2 (hChR2) and enhancedHaloR (eNpHR) construct schematics and differentialtransgene expression in ganglion cell soma and dendritesof whole-mount rabbit retina. The calcium/calmodulin-dependent protein kinase II (CaMKIIa) promoter andwoodchuck hepatitis virus posttranscriptional regulatoryelement (WPRE) to drive high transgene expression levelsin ganglion cells were used in all constructs. (a) Schematicof untargeted hChR2-mCherry fusion. (b) UntargetedeNpHR-eGFP fusion. (c) Postsynaptic density 95 (PSD-95)targeting motif fused with hChR2-mCherry for dendriticlocalization. (d) AnkyrinG motif fused with eNpHR-eGFPfor somatic localization. (e) AnkyrinG motif fused withhChR2-mCherry. (f) PSD-95 fused with eNpHR-eGFP.(g) Confocal image of rabbit ganglion cell expressingankyrinG-hChR2-mCherry localized to the soma andproximal dendrites (red). (h) Same cell as (g) showingPSD95-eNpHR-eGFP localized primarily to the dendrites(green). (i) Merge of (g) and (h). Scale bar represents100 μm. (j) PSD95-hChR2-mCherry localized to thedendrites. (k) AnkyrinG-eNpHR-eGFP localized to the somaand proximal dendrites. (l) Merge of (j) and (k). Scale barrepresents 100 μm. (m) Untargeted hChR2-mCherry islocalized throughout the plasma membrane.(n) Untargeted eNpHR-eGFP is localized throughout theplasma membrane. (o) Merge of (m) and (n). Scale barrepresents 100 μm. (Reprinted with permission from Ref183. Copyright 2011 Elsevier)

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photoreceptors was achieved using human rhodopsin,human red opsin, and mouse cone arrestin promoters.Despite the synaptic reorganization of the inner retinathat accompanies RP progression, when stimulatedby light, HaloR-transfected photoreceptors seemed toconvey information through bipolar cells to retinalganglion cells, including both ON and OFF pathways.These effects were obtained even at time when only∼25% of cone cell bodies remained.

A further refinement of this approach to sightrestoration is illustrated by the synthesis of light-sensitive ion channels, achieved by coupling thesechannels with molecules (e.g., azobenzene) whosephotoisomerization results, ultimately, in reversibleactivation of the ion channel.168,170,171,185 In the caseof azobenzene, one end of the molecule is covalentlytethered to the ion channel (in a way that does notinterfere with the protein’s function), and to the other

end is attached an ‘active moiety’, e.g., an agonist,antagonist, or pore-blocking agent. Light absorptionby azobenzene creates a conformational change in themolecule that alters the relationship of its active moi-ety to the ion channel. The thermally relaxed transisomer is more extended (∼0.7 nm longer) than thehigher energy cis isomer. The active moiety can inter-act with the ion channel in only one of the isomericstates, which leads to a change in ion movement acrossthe cell membrane. Variants of the maleimide–azoben-zene–quaternary ammonium molecule have beendeveloped (e.g., acrylamide azobenzene quaternaryammonium), that permit affinity labeling of endoge-nous potassium channels without the need for receptormutagenesis or genetic manipulation of the target cells(e.g., ganglion cells).186 A genetically and chemicallyengineered light-gated mammalian ion channel, thelight-activated glutamate receptor (LiGluR), has been



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FIGURE 11 | Correlation of ankyrinG-hChR2 and PSD95-eNpHRlocalization and function using immunostaining and electrophysiology.(a) Endogenous ankyrinG-Cy5 (magenta) in flat-mount rabbit retinashown in the initial axon segment (arrowhead, inset) of ganglion cells.(b) Merge of ankyrinG-Cy5 and transfected ankyrinG-hChR2-mCherry(red). AnkyrinGhChR2 is localized to the soma and proximal dendrites.Colocalization of endogenous ankyrinG (arrowhead) and mCherry is notapparent. (c) Cotransfection of untargeted enhanced green fluorescenceprotein (eGFP) (green) shows the complete cellular morphology(including axon, arrows). Scale bar represents 50 μm. (d) EndogenousPSD95-Cy5 (magenta) is present in ganglion cell somata and dendriticterminals (arrowheads, inset). (e) Merge of PSD95-Cy5 and transfectedPSD95-eNpHR-eGFP (green). eNpHR-eGFP is observed to colocalizewith endogenous PSD95 in dendrites. (f) Cotransfection of untargetedmCherry (red) shows complete dendritic morphology of cell. Scale barrepresents 50 μm. (g) Illumination of ankyrinG-hChR2-mCherry (yellow)in ganglion cell soma with 50-μm blue spot (10 mW/mm2) elicits robustspiking. Untargeted eGFP (green) was cotransfected to show completemorphology. Extracellular spike recordings from whole-mount rabbitretina in the presence of l-AP4 (20 μM), CPP (10 μM), and CNQX(10 μM) cocktail designed to block all photoreceptor-driven synaptictransmission to ganglion cells. (h) Blue annulus (300 μm OD, 50 μm ID)covering only the cell dendrites and partial axon fails to elicit spiking.(i) A blue rectangular stimulus (200 × 900 μm) covering the entire axonalso fails to elicit spiking. (j) Illumination of soma in ganglion cellexpressing PSD95-eNpHR-eGFP (yellow) with 50-μm yellow spot (10mW/mm2) fails to silence spontaneous spiking. Untargeted mCherry(red) was cotransfected to show complete morphology. (k) Yellowannulus (300 μm OD, 50 μm ID) covering only the cell dendrites andpartial axon effectively silences spikes. (l) Yellow rectangular stimulus(100 × 500 μm) covering the entire axon fails to silence spiking.(Reprinted with permission from Ref 183. Copyright 2011 Elsevier)

expressed selectively in retinal ganglion cells of therd1 mouse.187 In these mice, the LiGluR restoreslight sensitivity to the retinal ganglion cells, reinstateslight responsiveness to the primary visual cortex,and restores both the pupillary reflex and a nat-ural light-avoidance behavior. One might considerthe use of ChR2 and HaloR as using molecules toreengineer cells and their behavior. Here, one is using

bionanotechnology to reengineer proteins first and toreengineer cell behavior second.

Bionic RetinaSubretinal implants that provide precisely patternedelectrical stimuli aim to replace lost photoreceptorsin patients suffering from retinal degenerative dis-ease. These devices convert a real-time video image of



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FIGURE 12 | Subretinal bionic retina. Some prostheses are placed on the retina (in direct contact with retinal ganglion cells); others are placed inthe suprachoroidal space (in contact with the choroidal vasculature), and this implant is inserted into the subretinal space as illustrated here. (a) Themicrophotodiode array (MPDA) is a light sensitive 3.0 × 3.1 mm CMOS-chip with 1500 pixel-generating elements on a 20-μm thick polyimide foilcarrying an additional test field with 16 electrodes for direct electrical stimulation (DS test field). (b) The foil exits approximately 25 mm away fromthe tip at the equator of the eyeball and is attached to the sclera by means of a small fixation pad looping through the orbit to a subcutaneoussilicone cable that connects through a plug behind the ear to a power control unit. (c) Magnification of the DS electrode array showing the 16quadruple electrodes and their dimensions. (d) Pattern stimulation via DS array (e.g., ‘U’). (e, f) switching from a triangle to a square by shiftingstimulation of a single electrode. (g) Magnification of 4 of the 1500 elements (‘pixels’), showing the rectangular photodiodes above each squaredelectrode and its contact hole that connects it to the amplifier circuit (overlaid sketch). (Reprinted with permission from Ref 191. Copyright 2011 TheRoyal Society of London)

the world into electrical signals that are transmittedto the retina through a microelectrode array(Figures 12 and 13). Many advances have occurredin the development of electrical–neural interfaces thathave permitted restoration of partial vision in other-wise blind (no light perception, bare light perception)patients.188–193 Important design issues for electrodesthat have a solid interface with cells (vs microfluidicelectrodes194) include providing electrode arrays thatare large enough to stimulate many cells while simul-taneously: (1) being thin/permeable enough to permitnutrient flow to the retina, (2) maintaining proxim-ity of the stimulating electrodes to the target tissue,(3) using currents that do not damage the target tissue,and (4) using materials that do not elicit an inflamma-tory (e.g., foreign body) response. Kotov et al.7 havereviewed the applications of nanomaterials for neuralinterfacing in detail. Nanowires, single and mul-tiwalled carbon nanotubes, nanoparticles, polymercoatings, and silicon lithographic elements might

comprise effective interfaces with neural tissue. Modu-lation of the inflammatory response to the implant canbe achieved by matching the mechanical properties ofthe target tissue and the electrodes and by reducing thesize of the neural electrodes (see Kotov et al.7 for refer-ences). Although semiconductor nanoparticles mightbe quite useful in this regard, they can induce cyto-toxicity, including changes in morphology, metabolicactivity, and oxidative damage,195–198 as well asinduction of an inflammatory response.199 Carbonnanotubes also may be neurotoxic.200 Kotov et al.7

have pointed out that nanostructures in a dispersedstate, i.e., nanocolloids, might serve as components ofneural electrodes. Kim et al.,201 for example, investi-gated the release of an antiinflammatory agent, dex-amethasone, from nanoparticles of PLGA embeddedin alginate hydrogel matrices. Dexamethasone-loadedPLGA nanoparticles were prepared using a solventevaporation technique and were characterized for size,drug loading, and in vitro release (typical particle size



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FIGURE 13 | Bionic retina location within a patient. (a) The cable from the implanted chip in the eye leads under the temporal muscle to the exitbehind the ear and connects with a wirelessly operated power control unit. (b) Position of the implant under the transparent retina.(c) Microphotodiode array (MPDA) photodiodes, amplifiers, and electrodes in relation to retinal neurons and pigment epithelium. (d) Patient withwireless control unit attached to a neckband. (e) Route of the polyimide foil (red) and cable (green) in the orbit in a three-dimensional reconstructionof CT scans. (f) Photograph of the subretinal implant’s tip at the posterior eye pole through a patient’s pupil. (Reprinted with permission from Ref191. Copyright 2011 The Royal Society of London)

ranged from 400 to 600 nm). The in vivo impedanceof chronically implanted electrodes loaded with dex-amethasone was maintained at its initial level, whilethat of the control electrode increased by threefoldby ∼2 weeks after implantation. The improved per-formance was attributed to the reduced amount ofglial inflammation in the immediate vicinity of thedexamethasone-modified neural probe. Kotov et al.7

also suggested that nanoparticle-mediated (e.g., silicananoparticles,202 liposomes203) gene delivery mightinfluence the behavior of neural and glial tissue arounda neural electrode. Nanoscale patterning of the elec-trode surface might also be exploited to coat thesurface with molecules (e.g., laminin, fibronectin, col-lagen) in a topography that favors appropriate neuralcell adhesion, orientation, and gene expression (seeKotov et al.7 for references) as well as reduced elec-trode size.204,205 Growth factors and ECM proteins

can be deposited on electrodes to improve neu-ral growth and adhesion,206,207 and layer-by-layerassembly208 has been used to deposit bioactive coat-ings on electrodes209–211 for neural stimulation. Thus,nanostructured coatings may foster improvements inneural electrodes in the areas of biocompatibility(e.g., inflammatory response, neural adhesion) andimage resolution (through manipulation of electrodearray density, charge injection capacity, and electrodeimpedance).

CONCLUSIONS

It is difficult to imagine what the full ramificationsof nanotechnology, whose origin dates to the seminalpaper of Richard Feynman,212 will be in the fieldof regenerative medicine. It seems safe to speculatethat developments in nanotechnology will result in


https://www.researchgate.net/publication/5349648_Conducting_polymers_for_neural_interfaces_Challenges_in_developing_an_effective_long-term_implant?el=1_x_8&enrichId=rgreq-ddd5bb910b6cd2131292d8b4d81e9599-XXX&enrichSource=Y292ZXJQYWdlOzUxODgwOTg0O0FTOjExMzQ5MTE1MTE2NzQ4OEAxNDA0MDY5ODA3MTI2

https://www.researchgate.net/publication/228005672_Conducting-Polymer_Nanotubes_for_Controlled_Drug_Release?el=1_x_8&enrichId=rgreq-ddd5bb910b6cd2131292d8b4d81e9599-XXX&enrichSource=Y292ZXJQYWdlOzUxODgwOTg0O0FTOjExMzQ5MTE1MTE2NzQ4OEAxNDA0MDY5ODA3MTI2


improvements in drug and gene delivery, cell-basedtherapy, and neural prosthetics. These discoveries, asthey are operationalized from preclinical models toclinical practice, are likely to have a major impact

on the development of sight-preserving and sight-restoring treatments for conditions that currently leadto irreversible blindness.

ACKNOWLEDGMENTS

Supported in part by Research to Prevent Blindness, Inc. (M.A.Z. and R.R.), the Joseph DiSepio AMD ResearchFund (M.A.Z), and the Peter J. Crowley Research Fund of the New York Eye and Ear Infirmary, New York,NY (R.R.).

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Regenerative nanomedicine and the treatment of degenerative retinal diseases