Nanotechnology-Based Approaches for Ophthalmology Applications: Therapeutic and Diagnostic Strategies.

ANNUAL REVIEW

Nanotechnology-Based Approaches for Ophthalmology Applications: Therapeutic and Diagnostic Strategies Mehdi Ghaffari Sharaf, MSc,*Þ Sibel Cetinel, PhD,*Þ Lisa Heckler, MD,þ Karim Damji, MD,þ Larry Unsworth, PhD, PEng,*Þ§ and Carlo Montemagno, PhD*Þ§ Purpose: The purpose of this article was to review recent advances in applications of nanotechnology in ophthalmology.

Design: Literature review. Methods: Research articles about nanotechnology-based treatments for particular eye diseases and diagnostic technologies were searched through Web of Science, and the most recent advances were reported. Results: Nanotechnology enabled to improve drug and gene delivery systems, medicine solubility and short half-life in biological systems, controlled release, targeted delivery, bioavailability, diffusion limitations, and biocompatibility so far. These promising achievements are the assurance of next-generation treatment technologies. As well as treatment, nanofabrications systems such as microelectromechanical manufacturing systems removed the limitations of nanodevice generations and led the development of diagnostic tools such as intraocular pressure monitors and biosensors. Conclusions: The pursuit of personalized medicine approaches for combating ocular diseases may be possible only through the development of nanotechnology platforms that include molecular-level engineering. Nanoparticle engineering is a common thread; herein, we attempt to show unmodified nanoparticles as well as interesting and representative biomimetic strategies can be used for specific diseases. Finally, through combining microelectromechanical and nanoelectromechanical manufacturing system strategies, interesting manufacturing and sensor development can be accomplished for early detection and, in some cases, treatment of ocular diseases. Key Words: nanotechnology, keratitis, neovascularization, glaucoma, corneal wounds (Asia-Pac J Ophthalmol 2014;3: 172Y180)

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dvances in understanding the unique chemical, mechanical, electrical, and magnetic properties of nanoscale (1 dimension of 1-100 nm) materials have directly facilitated the emergence of nanoenabled technological platforms. Fabrication of these nanomaterials has allowed a diversity of applications to open up, especially in the fields of chemistry, biology, materials science, electronics, and photonics, to name a few.1,2 As might be expected, these breakthroughs in classic sciences have started to directly affect medically oriented research activities, whereby through the application of nanomaterials, true nanotechnology can be developed for the express purpose of affecting critical outcomes in patient-centered activities, viz., personalized medicine, reduced healing times, increased therapeutic advantage, and so on. The From the *Chemical & Materials Engineering, †Ingenuity Lab, and ‡Ophthalmology and Visual Sciences, University of Alberta; and §National Institute of Nanotechnology, National Research Council, Edmonton, Alberta, Canada. Received for publication March 18, 2014; accepted May 8, 2014. The authors have no funding or conflicts of interest to declare. Mehdi Ghaffari Sharaf and Sibel Cetinel contributed equally to this article. Reprints: Larry D. Unsworth, PhD, PEng, ECERF W7-070D, University of Alberta, Edmonton, AB, Canada T6G 2V4. E-mail: [email protected]; or Carlo Montemagno, PhD, Ingenuity Lab, 1-070C, 11421 Saskatchewan Dr, Edmonton, Alberta, Canada T6G 2M9. E-mail: [email protected]. Copyright * 2014 by Asia Pacific Academy of Ophthalmology ISSN: 2162-0989 DOI: 10.1097/APO.0000000000000059

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application of nanotechnology platforms to the field of medicine is far reaching and too vast to cover in a single review article, but suffice it to say that nanotechnology has dramatically affected drug discovery, drug therapeutic efficacy, drug and gene delivery systems, biocompatible implant materials, regenerative medicine, and diagnostic devices.3Y5 Herein, a review of some major applications of nanotechnology platforms will be surveyed in the context of treating serious ophthalmologic diseases. Of course, it should be noted that there may be significant overlap in platforms applied to different diseases [ie, nanoparticle (NP)Ybased treatments are ubiquitous but are a similar technology], and case studies thought to be significant and portraying the essence of the nanotechnological platform are provided, including a discussion on sensor and diagnostic strategies. Finally, these nanotechnology-based strategies are further organized for particular anterior and posterior segment eye diseases. Numerous ocular disorders have been identified that are static or dynamic that ultimately adversely affect patient vision. Even if these disorders are not immediately sight threatening, reduction in vision acuity can have a direct effect on a patient’s quality of life, where loss of vision may directly lead to loss of employment and entire families being reduced to extreme poverty. In concert with this, classic drug therapies have restricted treatment efficiency due to the presence of numerous barriers that isolate the eye from the external environment, isolate different compartments within the eye, and isolate the eye from the host itself (Fig. 1A). At every level of the eye, significant barriers exist that directly influence drug delivery. For example, the mucoaqueous tear layer decreases drug adhesion at the tissue interface and absorbed amounts by lowering residence time (Fig. 1B).6 Specialized corneal epithelium with tight junctions and desmosomes creates a physical barrier for molecules larger than 500 daltons.7 The iris blood vessels, ciliary epithelium, and retinal vessels restrict the passage of molecules via the blood-aqueous layer and blood-retina barriers, respectively.8 It is hoped that through using nanotechnology platforms some of these clinical issues may be overcome. For example, through incorporating drugs into engineered NP delivery vehicles, some of the barriers to drug delivery might be reduced. Although it is evident that NP delivery is, perhaps, the most common application of nanotechnology in ophthalmology, it is not the only one. For example, the anatomy of the eye necessitates injections for both treatment and diagnosis. Nanotechnology enables the production of microneedles [using microelectromechanical (MEMS) and nanoelectromechanical manufacturing systems (NEMS)] for appropriate administration to these regions of the eye, as well as drainage valves for glaucoma treatment.9,10 Biosensors, diagnostic labeling strategies, and detection monitors are also some of the other areas where nanoenabled technology can play a role in maintaining eye health.

NANOTECHNOLOGY THERAPEUTIC APPROACHES FOR ANTERIOR SEGMENT DISEASES The anterior segment of the eye is classically defined as that which includes the cornea, iris, ciliary body, and lens. This

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Nanotechnology in Ophthalmology Applications

FIGURE 1. Schematic presentation of the ocular structure (A) and the mucoadhesive tear film (B).

region is directly exposed to the external environment and, despite the barrier present from the continuous tear film, has a high probability of injury from foreign objects. Moreover, despite natural barriers, microorganisms may still invade the cornea and result in keratitis or conjunctivitis. These serious conditions are only exacerbated through the improper use of contact lens materials, where nonspecific protein adsorption and subsequent microbial colonization break the natural tear film barrier and increase the risks associated with infections. Also, surgeries done for cataract, glaucoma, and cornea replacements further enhance risks associated with infection.11 That said, the following are some important disease states that may affect the eye.

Inflammation-Induced Diseases Keratitis, conjunctivitis, and uveitis12 are diseases related to inflammation in the cornea, conjunctiva, and pigmented vascular regions of the eye, respectively. Induction of inflammation within these tissues may occur through several pathways including, but not limited to, an infection due to the presence of bacteria, fungi, and viruses. The most common treatment vehicle for these types of infections is topical antimicrobial eye drops. However, because of the tear film encasing these tissues, residence time at the tissue site is significantly reduced. Relatively recent work has attempted to engineer NPs so as to interact with mucins produced by the epithelia of the eye via hydrogen bonding and electrostatic interactions. Through utilizing this nanotechnology strategy, it was found that ocular residence time increased significantly, reaching upward of ~300 minutes, hence increasing bioavailability.13 Commonly used cationic mucoadhesive polymers, chitosan and Eudragit, were recently utilized as NP reservoirs for terbinafine hydrochloride and obtained an extended drug release time of ~8 hours.14 Mucoadhesive agents were also formulated in combination with thermosetting and hydrogel-forming polymers so as to enhance the sustained drug release due to the polymer swelling, erosion, and/or drug diffusion through the gel.15 In early studies, polyethylene oxide (PEO)YEudragit microspheres (Fig. 2, * 2014 Asia Pacific Academy of Ophthalmology

nanosphere structure) loaded with ofloxacin were found to increase drug bioavailability with a relatively controlled release profile up to ~300 minutes.15 In a more recent study, poloxamer/chitosan in situYforming gels, which had shown to prolong retention time 2- to 4-fold,16 was used to treat fungal keratitis in rabbits.17 Kesavan et al18 used the cyclic oligosaccharide, hydroxypropyl-A-cyclodextrin, to increase the solubility of dexamethasone and combined their formulation with the anionic mucoadhesive Carbopol. Their hydroxypropyl-A-cyclodextrinYbased, pH-induced mucoadhesive hydrogel resulted in higher bioavailability and a sustained release profile compared with conventional dexamethasone eye drops. Besides charged polymers, polyamidoamine (PAMAM) dendrimers were considered as mucoadhesive because of their strong electrostatic interactions with mucins in the tear film.19 Furthermore, acyclovir-encapsulated thiolated PAMAM dendrimers (Fig. 2, dendrimer structure) showed promising drug loading, bioadhesion, and controlled release pattern.20 The cornea and conjunctiva contain membrane transporters responsible for nutrient transport, making transportertargeted delivery a promising strategy to enhance absorption of poorly permeating drugs. Amino acid and peptide transporters are the most common targeting regions. Acyclovir (ACV), hydrophilic antiviral nucleoside used for herpes simplex virus 1Yinduced corneal keratitis, prepared in L-Glu-ACV and L-Asp-ACV prodrug forms, acts as a substrate for corneal B0,+ transporter and leads to increased corneal permeability.21,22 Further to this, dipeptide prodrugs of ACV were encapsulated into poly(lactic-co-glycolic acid) (PLGA) NPs in order to slow down the degradation of prodrugs and therefore improve the therapeutic effect upon topical administration.23 Biotinylated lipid prodrugs of ACV were designed for targeting the sodium-dependent multivitamin transporter in the cornea.24 With an extended strategy, econazoleimpregnated PLGA was encapsulated in p-HEMA contact lenses for fungal keratitis treatment, yielding (in vitro) an extended antifungal activity against Candida albicans.25 Recently, a gatifloxacin-loaded p-(HEMA-co-MAA) hydrogel was shown to www.apjo.org


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FIGURE 2. Schematic representation of some NP structures used in nanotechnology. Linear polymers could be attached to molecules by covalent conjugation. Nanospheres could be loaded with drug and ensure sustained release. Dendrimers are branched polymers, which could encapsulate or covalently conjugate drug molecules. Nanocapsules could encapsulate relatively large amount of molecules. Micelles are self-assembled amphiphilic particles that can encapsulate both lipophilic and lipophobic molecules. Liposomes are vesicles formed by lipid bilayers.

exhibit sustained release (70% in 25 hours) and favorable healing profiles (in 48 hours) using a rat model with bacterial keratitis.26

Corneal Wounds Any insult, accidental or intentional, to the cornea may result in a disturbance of the tissue itself. However, unlike other cutaneous tissues where wound healing processes usually yield scar tissue and angiogenesis, the latent properties of the corneal tissue actively inhibit this end result, which would drastically impede vision. When reviewing the literature in this area, it is obvious that the majority of testing has been conducted on animals, but there are major differences between animal models and the human cornea that should be realized when trying to apply results of one to the other. Current clinical practice involves suturing the cornea; however, this practice increases the potential for postoperative complications such as infections, astigmatism, corneal scars (sometimes necessitating further corneal procedures), and postsurgical cataracts. Therefore, adhesives are under development to replace or supplement sutures. An ideal adhesive is considered to adhere to the moist corneal surface, rapidly seal the wound, restore intraocular pressure (IOP), maintain eye structural integrity, have a refractive index close to native cornea, be biocompatible and elastic, maintain a microbial barrier, and be bioabsorbed during tissue regeneration.27 First, polymer glues have been developed such as cyanoacrylate and fibrin.28 However, these glues were not very effective because of inflexibility and the requirement for autologous blood components, although they are used for the

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conjunctiva. Nanostructured (~10-nm diameter) dendrimers are highly branched, monodisperse polymers exhibiting a 3-dimensional shape with numerous end groups. The biggest advantage afforded by dendritic systems is the controllable crosslinked networks, coupled with the high density of functional side chains in the nanovolume and macrovolume.29 Dendrimer-based hydrogels were shown to promote rapid wound healing, without scar formation and inflammation, in comparison to sutures for the leghorn chicken model.30 Another hydrogel system composed of PEO functionalized peptide-based dendrimeric crosslinker was effective in closing a 4.1-mm corneal laceration (ex vivo), while withstanding IOP prior to healing.31 Finally, through engineering the crosslinkers, degradation time for this adhesive could be tuned between hours and months. Thus, nanoscale dendrimers may be used to tailor various wound healing properties for extensive recovery periods.

Corneal Neovascularization The formation of blood vessels within the avascular corneal tissue is a critical outcome associated with inflammation, infection, and degenerative or traumatic diseases and is referred to as corneal neovascularization.32 Available therapies are corticosteroids, nonsteroidal anti-inflammatory eye drops, photodynamic therapy, photocoagulation, and antibody (bevacizumab) against vascular endothelial growth factor A (VEGF-A). Inhibition of angiogenic factors such as VEGFs, platelet-derived growth factor, basic fibroblast growth factor, metalloproteinases, and interleukins is considered the goal of impeding this process.32Y34 * 2014 Asia Pacific Academy of Ophthalmology



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Recently, hypoxia due to extended contact lens wear was shown to up-regulate VEGF and hypoxia-inducible factor 1a expressions leading to corneal angiogenesis.35 They also indicated that hypoxia-inducible factor 1a knockdown using shRNA decreased the expression of interleukin 1b and metalloproteinases 2/9 compared with negative control in mice. This study implied gene level control of angiogenesis factors as a promising strategy. Complementary work showed that plasmid-containing shRNA against VEGF-A encapsulated in PLGA tested on BALB/c mice reduced (~80%) VEGF-A protein expression compared with naked plasmid (20%) and showed a 2-fold decrease in neovascularization area after 4 weeks.36 In another study, micelles composed of copolymer and plasmid DNA expressing soluble VEGF receptor 1 (VEGFR-1) (sFit-1) were used for gene therapy.37 The expressed sFit-1 acted as a sink of VEGF and prevented activation of angiogenesis cascade. Results of the study indicated stable micelle (Fig. 2, micelle structure) nanovector injection leading to prolonged sFit-1 expression and decreased vascularization of the cornea.

NANOTECHNOLOGY THERAPEUTIC APPROACHES FOR POSTERIOR SEGMENT DISEASES The posterior segment of the eye consists of the vitreous humor, the retina, choroid, and optic nerve. Intraocular liquid circulation, the corneal epithelium limiting molecular diffusion, and the retinal-blood barrier are the primary reasons that the mobility of drugs from the anterior to the posterior segments of the eye is retarded.7,8 In fact, no easy route for administration to the posterior regions of the eye has been developed; current efforts focus on intravitreal injection, subretinal injection, transscleral administration, subconjunctival injection, and topical instillation. However, risk of postadministrative complications may increase, causing retinal toxicity, retinal detachment, or intraocular infections (including potentially devastating endophthalmitis). New treatment strategies should focus on decreasing the frequency of injections as a means of reducing the risk of postadministrative complications; where nanotechnology could be helpful is to develop strategies for increased drug biodistribution, passing retinablood barrier, as well as controlled and targeted delivery.

Neovascularization-Related Diseases Choroidal neovascularization (CNV) is characterized by the growth of choroidal capillaries under the retinal pigment epithelium. Choroidal neovascularization could lead to hemorrhage, scar formation, and retinal detachment due to exudation. Nanotechnology-based therapies inhibiting angiogenesis are the focus of sustained drug release and targeted gene therapy efforts for this work. To obtain sustained delivery, previous work has demonstrated that dexamethasone acetate was encapsulated in PLGA NPs and tested both in vitro and in a laser-induced rat model.38 The results indicated controlled release for 40 days and dose-dependent inhibitory effect on CNV. Similarly, an antiangiogenic peptide (a serpin-derived peptide SP6001) was self-assembled into NPs with a cationic polymer and then loaded in PLGA microparticles.39 This peptide-NP system succeeded in decreasing neovascularization for 14 weeks prior to single dose administration in a mouse model. In another study, an integrinantagonist peptide (C16Y) was encapsulated in polylactic acid/ polylactic acid-polyethylene oxide (PLA/PLA-PEO) NPs for sustained drug release.40 It was shown that C16Y significantly inhibits CNV in both naked and encapsulated forms. However, in longer periods, NP-peptide form was more effective in tissue healing compared with naked peptide because of the short halflife of peptide in solution. Gene therapy utilizing the plasmid for the proteolytic plasminogen kringle 5 (K5) was loaded to PLGA:chitosan NPs and administrated via intravitreous * 2014 Asia Pacific Academy of Ophthalmology


injection, yielding the inhibition of VEGF expression and a sustained suppression of CNV.41 Further work using siRNA against VEGFR1 encapsulated in PEG-LPH NPs (PEGylated liposomeY protamineYhyaluronic acid) has shown successful inhibition of VEGF expression was inhibited using siRNA.42 Gene therapy strategies obviously hold much promise for the molecular-level treatment of disease; however, it is important to acknowledge that general acceptance of genetic delivery and therapy by the general population remains to be tested. Besides the success of gene delivery to the eye, Singh et al43 went a step forward and developed NPs for targeted delivery. Poly(lactic-co-glycolic acid) NPs, coated with transferrin and an integrin-binding RGD peptide (or combinations thereof ), was used for targeted gene delivery via endocytosis.43 Peptide-coated PLGA NPs loaded with anti-VEGF intraceptor plasmid were intravenously administered to Brown Norway rats with CNV, resulting in increased retinal delivery, anti-VEGF expression in retinal vascular endothelial cells, and significantly smaller CNV areas compared with nonfunctionalized NPs. Recently, a very similar NP system was tested in 2 murine age-related macular degeneration models and a primate CNV model in order to identify whether they could regress neovascularization, decrease fibrotic scarring, improve visual acuity, and demonstrate safety profile.44 Results indicated a safe treatment strategy with restored vision by 40% and suppressed subretinal fibrosis. Retinal neovascularization is thought to be caused by angiogenesis in the retina and is associated with age-related macular degeneration, diabetic retinopathy, and other retinal diseases. Similar to corneal and CNV, growth factor antagonists and gene therapy are the treatment options.45 Besides these strategies, NPs such as cerium oxide, gold, and silicate were shown to suppress angiogenesis by different routes.46Y48 Oxidative stress is one of the factors promoting retinal neovascularization. Cerium oxide NPs (nanoceria) are thought to mimic the activity of superoxide dismutase and clear out reactive oxygen species. Intravitreal injection of nanoceria inhibited reactive oxygen species, VEGF overproduction, and neovascular lesion formation.48 On the other hand, Au NPs were found to suppress VEGFR-2 signaling pathway by blocking ERK1/2 activation. Intravitreally injected Au NPs effectively inhibited VEGF-induced neovascularization in a mouse model.47 Similar to Au NPs, silicate NPs were also found to inhibit ERK1/2 activation and suppress in vitro VEGF-induced neovascularization. Intravitreal injection of silicate NPs was shown to reduce retinal neovascularization in mice model with oxygeninduced retinopathy.46

Retinoblastoma Retinoblastoma (RB) is a prolific retinal cancer presenting in 1 in 15,000 to 20,000 live births.49 Current treatment options include enucleation, radiation therapy, and chemotherapy with carboplatin, etoposide phosphate, or vincristine sulfate with local laser therapy. The local delivery of chemotherapeutic agents to the eye significantly reduces the systemic adverse effects, where it is hoped that nanotechnology may enable targeted chemotherapeutic50,51 and gene delivery52 systems for RB treatment. Previous efforts have shown that the administration of carboplatin-encapsulated PAMAM dendrimers reduced RB tumor growth after 1-dose injection in LHA-Tag mouse model more effectively than free drug.53 In another study, PLGA-PEG-folate micelles were utilized for folate receptorYtargeted delivery and sustained release of doxorubicin for up to 14 days.54 Even if doxorubicin uptake is inhibited because of its efflux through MDR1 pumps, PLGA-PEG-folate nanosystem was not affected because it enters the cell via folate receptors.55 Hence, the Y-79 RB cell line overexpressing folate receptors showed 4 times www.apjo.org


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TABLE 1. Summary of Articles Reviewed for Nanotechnology Diagnostic Approaches Authors Piffaretti et al

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Kang et al74 Chen et al75 Ghaed et al76 Chitnis et al77 Ha et al Xue et al78 Kim et al79 Todani et al80 Laukhin et al81 Sanchez et al82 Goto et al83 Leonardi et al84

Title

Year

Major Contribution

Rollable and implantable IOP sensor for the continuous adaptive management of glaucoma A Wireless Intraocular Pressure Sensor With Variable Inductance Using a Ferrite Material Capacitive Contact Lens Sensor for Continuous Noninvasive IOP Monitoring Circuits for a Cubic-Millimeter EnergyVAutonomous Wireless Intraocular Pressure Monitor A Minimally Invasive Implantable Wireless Pressure Sensor for Continuous IOP Monitoring

2013

Rollable and implantable piezo-resistive IOP contact lens Wireless IOP pressure sensor (with ferrite material) Wireless capacitive contact lens sensor for IOP Nanowatt power levels for IOP monitoring microsystem Implantable IOP capacitive pressure sensor, with a hypodermic needle to directly access the vitreous Flexible polymer-based capacitive pressure sensor for IOP Implantable SU-8Ybased wireless pressure sensor Portable tonometer using a micro reflected air pressure sensor for IOP

2013 2013 2013

Polymer-Based Miniature Flexible Capacitive Pressure Sensor for IOP Monitoring Inside a Mouse Eye A SU-8YBased Microfabricated Implantable Inductively Coupled Passive RF Wireless Intraocular Pressure Sensor A Noncontact IOP Measurement Device Using a Micro Reflected Air Pressure Sensor for the Prediagnosis of Glaucoma Intraocular pressure measurement by radio wave telemetry

2012

Non-invasive Intraocular Pressure Monitoring With a Contact Lens Engineered With a Nanostructured Polymeric Sensing Film Prototype of a Nanostructured Sensing Contact Lens for Noninvasive Intraocular Pressure Monitoring Direct Electrochemical Detection of DNA Methylation for Retinoblastoma and CpG Fragments Using A Nanocarbon Film Wireless contact lens sensor for intraocular pressure monitoring: assessment on enucleated pig eyes

2011

higher uptake of micelle-doxorubicin, compared with doxorubicin alone. Recently, Gary-Bobo et al56 developed a bitherapy strategy where chemotherapy was combined with phototherapy in a single NP system, which also exhibits targeted delivery. The mesoporous silica NPs encapsulating a photosensitizer (for phototherapy) and camptothecin (for chemotherapy) were functionalized with mannose or galactose for receptor targeting on Y-79 cells. Their results indicated that bitherapy has a higher therapeutic effect than phototherapy alone.

Glaucoma Glaucoma is characterized by high IOP and the death of retinal ganglion cells. Recently, it has been postulated that the activation of subclinical inflammation (chronic) may lead to exfoliation glaucoma, as well as defects in microfibrils themselves may contribute to glaucoma by altering the biomechanical properties of surrounding tissue as well as affecting signaling.57,58 Increased IOP is thought to be mainly caused by resistance to outflow through the trabecular meshwork, and elevated pressure is believed to ultimately induce neuronal cell damage and vision loss.59 Thus, the main treatment strategy is based on decreasing the IOP either through suppressing the production or increasing the outflow of aqueous humor. Timolol and brimonidine are 2 commonly used IOP-lowering agents. Nanotechnology approaches are under development to obtain therapeutics with increased bioavailability, prolonged retention time, and sustained drug delivery. One such work showed that timolol-encapsulated chitosan NPs were shown to be more effective on lowering IOP compared with drug solution, in vivo, in rabbits.60 In another study, brimonidine-loaded Eudragit polymers increased the

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2012 2012

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2011 2010 2009

WIT (Implandata GmbH) implantable sensor Piezo-resistiveYbased IOP contact lens sensor Piezo-resistiveYbased IOP contact lens sensor Sensor for direct electrochemical detection of DNA methylation for RB Issues around SENSIMED Triggerfish sensor for IOP

residence time and drug release from 6 to 72 hours.61 Similarly, subconjunctival administration of timolol-encapsulated microspheres exhibited sustained drug release for up to 3 months in an in vitro study.62 For controlled and extended drug release, contact lenses with disperse PGT (propoxylated glyceryl triacylate) NPs containing timolol were developed.63 When the lens was loaded with 5% timolol-PGT, therapeutic dose continued to be released for up to 1 month under in vitro conditions. Also, reduction in IOP was observed in preliminary animal studies in beagle dogs. A nanodevice fabricated by MEMS techniques offers an alternative for IOP medication and glaucoma drainage implants.64 This nanodrainage prototype could be implanted in the sclera and creates a bypass for aqueous humor outflow.65 Together with controlling the IOP levels, regeneration of damaged neuronal cells or even neuroprotection should be also considered as treatment strategies. Administration of microparticles encapsulated with glial cell lineYderived neurotrophic factor resulted in neuroprotective effects on retinal ganglion cells in animal glaucoma models.66,67 Similar to glial cell lineYderived neurotrophic factor, heat shock proteins could also be utilized for inducing ocular neuroprotection. Jeun et al68 developed superparamagnetic NP agents (EMZF-SPNPAs) to induce heat shock proteins by local magnetic hyperthermia. They have tested both silica-coated and uncoated EMZF-SPNPAs for their toxicity and diffusion rate to retina. In this study, 5.5-nm NPs have been injected into vitreous instead of intravenous. Both coated and uncoated particles diffused to retina and did not show any toxic effect to the cell. Finally, several articles are now investigating exfoliation glaucoma using atomic force microscopy (AFM) techniques. Antibody-modified AFM tips have recently been used to define the protein aggregation * 2014 Asia Pacific Academy of Ophthalmology



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FIGURE 3. Schematic presentation of (A) the capacitive pressure sensor. An increase in the pressure of the environment of the sensor will affect the flexible thin membrane that will result in change in distance and therefore capacitance between 2 electrodes, (B) implantable IOP sensor with hypodermic needle, (C) capacitive contact lens sensor, (D) implantable ring-shaped WIT with an ASIC chip and a circular microcoil antenna.

and identify constituent proteins, showing that lysyl oxidaseYlike 1 protein is colocalized with the fibrous protein aggregates.69 Another interesting use of AFM involves looking at stiffness of tissues for understanding biomechanical implications of exfoliation, where optic nerve heads were shown to have a decreased stiffness for cases of exfoliation that may reflect tissue weakness.70

NANOTECHNOLOGY DIAGNOSTIC APPROACHES Nanotechnology has the profound potential for the development of platform technologies that may make it possible to monitor and, thus, treat ocular diseases; even at the molecular level. Microelectromechanical manufacturing system and NEMSY based engineering for developing submicron-size mechanical tools is opening the opportunities for implantable monitoring biosensors that are proving to be extremely valuable in treating chronic diseases such as glaucoma, diabetic retinopathy, and age-related macular degeneration.71 With this increased ability to detect initial disease states, it is to be hoped that the efficacy of therapeutic intervention will dramatically increase while severely reducing patient costs.72 Monitoring IOP is considered an important diagnostic tool for the onset of glaucoma; IOP is considered a critical factor in glaucoma progression (Table 1).85 Intraocular pressure is dynamic and exhibits a diurnal variation, where continuous IOP characterization at night (ie, outside the hospital setting) may have the most benefit for the management of glaucoma.71,86 Hence, minimally invasive IOP monitoring devices are actively being sought. The SENSIMED Triggerfish system (SENDIMED AG, Switzerland) is the most studied IOP monitor, utilizing a wireless Pt-Ti strain gauge that measures the changes in the area of the * 2014 Asia Pacific Academy of Ophthalmology

cornea-scleral junction, all embedded within a silicone contact lens.3,71,84,87 That said, some have concluded that this sensor cannot be used clinically for sensing an IOP decrease induced by topical glaucoma medication.88Y90 Thus, active research is ongoing in order to develop IOP monitoring systems using different methods such as capacitive pressure sensors, micromachined pressure sensor, and piezoresistive IOP sensors.3 An ultrathin, flexible, polymer-based capacitive MEMS pressure sensor (500 500 100 Km3) for IOP monitoring has been developed (Fig. 3A) and shown to have advantageous properties such as high sensitivity to pressure changes, low temperature sensitivity, and low consumed power, making it more suitable for implantation.91 Noninvasive sensor using a wireless capacitive contact lens (Fig. 3C) for continuously monitoring IOP was shown to have the capability to track pressure variation profiles with minimal lag. A minimally invasive implantable IOP sensor for continuous wireless monitoring has also been developed, using a hypodermic needle (30-gauge) to access the vitreous, a micromachined capacitive pressure sensor, and a polyimide coil, to form a parallel LC circuit whose resonant frequency is a function of IOP (in real time) (Fig. 3B).75,77 Radiowave telemetry is also used in the wireless IOP transducer (WIT) (Implandata GmbH, Hannover, Germany) that is an implantable microstructured device consisting of 2 parts, an ASIC chip and a microcoil antenna, with a pressure, temperature, an identification encoder, an analog-to-digital converter, and telemetry into a single implantable microchip (Fig. 3D). This telemetric IOP allows patients without a healthy cornea or who have received an implanted artificial cornea (keratoprosthesis) to be monitored.80 Rollable and implantable IOP measurement devices based on a piezo-resistive pressure sensor have been used to develop an www.apjo.org


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IOP contact lens sensor.73 This sensor uses a flexible highly piezo-resistive bilayer film based on a polycarbonate [poly(bisphenol-A-carbonate)] matrix containing a conducting top layer of nanocrystals of the organic molecular metal A-(ET)2I3. Changes in cornea curvature are measured and provide IOP determination.81,82 Microelectromechanical manufacturing system technology has also facilitated the development of a portable tonometer using a microreflected air pressure sensor, where a wireless IOP sensor is reported that uses variable inductance according to the external pressure.74,79 Moreover, microfabricated sensors containing a planar spiral gold coil inductor, 2-parallelgold plate (metal-insulator-metal) capacitor, and a pressuresensitive diaphragm were developed and showed high pressure sensitivities.78 Nanowatt power levels for IOP monitoring microsystem were also recently developed, which provided accuracy in measured IOP of 0.5 mm Hg.76 Other important sensors are being developed that allow for direct, molecular-level identification for therapeutic purposes. An excellent example of this has been worked on for directly detecting RB. Retinoblastoma has been linked to the inactivation of the RB gene (Rb), which is a tumor suppressor gene.49 The promoter region of the Rb gene is methylated in a significant number of primary RB tumors.83,92 A direct electrochemical detection of DNA methylation for RB has been devised using a 40-nm-thick nanocarbon film electrode that has a nanocrystalline sp2 and sp3 hybrid structure, providing a wide potential window, low background current, little surface fouling, and high electrode activity compared with boron-doped diamond and glassy carbon electrodes. Through these unique properties of nanocarbon, it is possible to detect biomolecules with slower electron transfer rates, such as nicotinamide adenine dinucleotide phosphate and pyrimidine bases, allowing direct detection of single-nucleotide polymorphisms and, consequently, the oxidization of all DNA bases individually, enabling the identification of DNA bases and their derivatives (cytosine and its derivative 5-methylcytosine, for example).93,94 These nanocarbon films have been used to characterize DNA methylation directly from a 6mer oligonucleotide without any treatment,93 and 24mer oligomers containing methylation sites for RB (RB1) gene fragments and the CpG (cytosinephosphoguanosine) repeat sequences (60mers) could be detected using nanocarbon film electrodes.83 Moreover, electrochemical experiments using the nanocarbon film electrode provided quantitative detection of DNA methylation ratios solely by measuring methylated 5¶-CpG repetition oligonucleotides (60mers) with different methylation ratios.

CONCLUSIONS The pursuit of personalized medicine approaches for combating ocular diseases may be possible only through the development of nanotechnology platforms that include molecular-level engineering. Nanoparticle engineering is a common thread for numerous drug delivery and imaging paradigms; covered herein we attempt not only to show that unmodified NPs are like ceramic and Au used for direct disease therapy and imaging, but also to detail interesting and representative biomimetic strategies that are also used for specific diseases. Finally, through combining MEMS and NEMS strategies, interesting manufacturing and sensor development can be accomplished for early detection and, in some cases, treatment of ocular diseases. REFERENCES 1. Daniel MC, Astruc D. Gold nanoparticles: assembly, supramolecular chemistry, quantum-sizeYrelated properties, and applications toward biology, catalysis, and nanotechnology. Chem Rev. 2004;104:293Y346.

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