Nanotechnology in retinal diseases: From disease diagnosis to therapeutic applications

Abstract

Nanotechnology has demonstrated tremendous promise in the realm of ocular illnesses, with applications for disease detection and therapeutic interventions. The nanoscale features of nanoparticles enable their precise interactions with retinal tissues, allowing for more efficient and effective treatments. Because biological organs are compatible with diverse nanomaterials, such as nanoparticles, nanowires, nanoscaffolds, and hybrid nanostructures, their usage in biomedical applications, particularly in retinal illnesses, has increased. The use of nanotechnology in medicine is advancing rapidly, and recent advances in nanomedicine-based diagnosis and therapy techniques may provide considerable benefits in addressing the primary causes of blindness related to retinal illnesses. The current state, prospects, and challenges of nanotechnology in monitoring nanostructures or cells in the eye and their application to regenerative ophthalmology have been discussed and thoroughly reviewed. In this review, we build on our previously published review article in 2021, where we discussed the impact of nano-biomaterials in retinal regeneration. However, in this review, we extended our focus to incorporate and discuss the application of nano-biomaterials on all retinal diseases, with a highlight on nanomedicine-based diagnostic and therapeutic research studies.

I. INTRODUCTION

The retina is an intricate and multi-layered neural tissue positioned at the posterior aspect of the eye.^1,2 It serves as a crucial component in the process of vision by sensing light and translating this sensory input into electrical signals to be interpreted by the brain.³ Any abnormality within the retina can lead to varying degrees of vision loss.^4–6 The worldwide prevalence of retinal diseases varies, encompassing a range from 5.35% to 21.02%, particularly among individuals aged 40 years and above.⁷ In developed countries, the retinal diseases are the leading causes of irreversible blindness, with notable conditions, such as age-related macular degeneration (AMD), ischemic retinopathy, diabetic retinopathy (DR), inherited retinal disorders (IRDs), and proliferative vitreoretinopathy (PVR).^8–10 Traditionally, medications, pharmacological agents, and surgical interventions exhibit limited efficacy, biocompatibility, and significant health risks.^11,12 Due to the blood–retinal barrier (BRB), oral and intravenous routes are rarely employed in ophthalmic formulations.¹³ The BRB consists of the inner BRB (iBRB) of the retinal arteries and the outer BRB (oBRB) of the retinal pigment epithelium. Both have nonleaky tight junctions, which limit blood components' ability to enter intraocular chambers.¹⁴ This explains why oral or intravenous medications rarely reach therapeutic concentrations in intraocular tissues (Fig. 1). However, to address these challenges, nanotechnology has opened new avenues in the field of retinal sciences in the past decade.

FIG. 1.

The blood retinal barrier regulates the flow of molecules, ions, and water in and out of the retina. Blood-retinal barrier has two parts: the inner barrier, which consists of Müller cells, pericytes, and endothelial cells, and the outer barrier, which consists of tight junctions between pigmental epithelial cells that control paracellular trafficking. These two barriers regulate both visual function and retinal homeostasis. The figure is created with Biorender.com.

The complexity of retinal diseases, and their limited treatment options, have prompted researchers to explore the potential of nanoscale materials and devices in transforming ocular healthcare. In our previous review article on the applications of nano-biomaterials on retinal regeneration,¹⁵ we focused only on the studies involving the role and applications of nano-biomaterials on retinal regeneration. This review article focuses on the role of nanoparticles, nanowires, hybrid nanostructures, and nanoscaffolds in all retinal diseases, emphasizing the nano-biomaterials-based diagnostic and therapeutic research studies. Based on the structural and morphological characterizations, nanomaterials are classified as nanoparticles, nanowires, hybrid nanostructures, and nanoscaffolds (Fig. 2).^16–18 Each nanomaterial retains unique characteristics that integrate with other materials to imbibe individual advantages. The potential of nanomaterials has been explored for imaging, tissue engineering, diagnosis, drug delivery, biosensors, and therapeutics in several forms of retinal degeneration.^19–21 The variety of nanomaterials employed provides the flexibility to control the shape, size, and surface characteristics, offering the potential for their application as advanced therapeutics for retinal diseases.^22–24 Nanoscale diagnostic tools offer the potential for highly sensitive and specific detection of biomarkers associated with retinal pathologies, enabling clinicians to diagnose conditions at earlier stages.²⁵ With these advancements, the Food and Drug Administration (FDA) has approved approximately 250 nanomaterial-based products that hold great promise for enhancing therapeutic efficiency and monitoring tissue regeneration processes.^26–28

FIG. 2.

This graphic depicts multifunctional nanostructures: nanoparticle (NP), nanowire (NW), nanoscaffolds, and hybrid nanostructures, which have a range of uses in biomedical research. The surface of these nanostructures are modified with drugs (conjugated or incorporated to the surface), a PEGylated lipid bilayer (to increase solubility and lower immune response), targeting groups (to improve distribution, efficacy, and preference of nanostructures), and imaging agents (e.g., reporter molecules and fluorescent dyes). The figure is created with Biorender.com.

Based on this background, the present review aims to explore the latest advances in nanomedicine and the importance of nanoparticles, nanowires, nanoscaffolds, and hybrid nanostructures in the management of retinal disorders, which are summarized in Table I. By delving into the transformative potential of nanotechnology in ocular healthcare, we aim to offer new perspectives on how nanomedicine can revolutionize retinal healthcare.

TABLE I.

Relevant studies of several nanostructures used for retinal diseases.

Nanostructure	Nanomaterial	Inference	Reference
Nanoparticles	Nanoceria particles	Mitigate the adverse impact of reactive oxygen species and reduce retinal neovascularization	Tisi et al.29
	Superparamagnetic iron oxide nanoparticles	Magnetization of rat mesenchymal stem cells	Yanai et al.30
	Gold	Passing through blood-retinal barrier	Kim et al.31
		Inhibition of retinal neovascularization	Shen et al.32
		Inhibits choroidal neovascularization	Singh et al.33
	Platinum nanozymes	Reduces photoreceptor degeneration	Cupini et al.34
	Hyaluronic acid-coated gold nanoparticles	Distribution of drugs	Laradji et al.35
	Silver	Regulates microglia activity and retinal inflammation	Palacka et al.36
		Effect on VEGF- and IL-1beta-induced vascular permeability in porcine retinal endothelial cells	Sheikpranbabu et al.37
		Inhibition of angiogenesis in bovine retinal endothelial cells	Gurunathan et al.38
Nanowires	Gallium phosphide and silicon	Long-term effects on postnatal retinal cells	Piret et al.39
		Short and long-term effects on mouse retinal cells	Piret et al.40
	Poly(ε-Caprolactone) (PCL)	Suitability for subretinal implantation	Christiansen et al.41
Hybrid nanostructures	Melatonin-loaded lipid-polymer hybrid nanoparticles (melLPHNs)	Antioxidant and neuroprotective properties	Romeo et al.42
	Gold nanorods	Gold nanorods augment photoacoustic microscopy and optical coherence tomography	Nguyen et al.43
	Photoreceptor-binding upconversion nanoparticles	Creation of miniature near-infrared (NIR) light transducers	Ma et al.44
Nanoscaffold	Fluocinolone acetonide (FA) treated nanofibrous scaffold, netrin-1-conjugated PEDOT	Growth and differentiation of retinal pigmented epithelial cells	Egbowon et al.45
		Facilitate axon guidance and functional recovery of retinal ganglion cells	She et al.46
	Gelatin/chitosan nanofibrous scaffolds	Implantation in the subretinal space	Noorani et al.47
	Biodegradable scaffold	iPSC-based therapies	Sharma et al.48

II. NANOPARTICLES FOR RETINAL DISEASE

Nanoparticles are characterized by their minuscule size, typically ranging between 1 and 100 nanometers.⁴⁹ Nanoparticles are classified as metallic and nonmetallic. Metallic nanoparticles include gold, silver, platinum, iron, cobalt, nickel, cerium oxide, and yttrium oxide while nonmetallic nanoparticles include carbon, silica, and calcium phosphate. Among them, gold nanoparticles are recommended for treating retinal diseases.^10,50,51 The small size of nanoparticles offers several unique physical and chemical properties in the field of medicine and pharmaceutical delivery to treat various forms of retinal disorders.^52,53 Early signs of retinal degeneration can be detected through targeted imaging. This can be achieved by binding nanoparticles to the pathological markers associated with retinal diseases.^54,55 Furthermore, nanoparticles' chemical and physical characteristics enable them to absorb or scatter light at specific frequencies or wavelengths that have applications in bioimaging and photothermic therapy.^56,57 Gold-based nanoshells absorb visible and near-infrared parts of the electromagnetic spectrum and can transform them into heat, resulting in photothermal ablation of sick cells. It has been demonstrated that inert gold nanoshells can be readily functionalized with various molecules to bind cellular targets selectively.^56,57 This noninvasive monitoring and early detection are vital for timely interventions that may halt the progression of retinal degeneration. Furthermore, nanoparticles have the promising property of delivering therapeutic agents directly to the affected retinal cells.⁵⁸ Nanoparticles-based drug delivery systems can improve the bioavailability and targeted release of drugs, reducing side effects and enhancing the overall efficacy of treatments.⁵⁹ Figure 3 illustrates the routes of transportation that nanoparticles employ to cross the blood–retina barrier. Nanoparticles can also be engineered to carry genes or RNA molecules for gene therapy approaches.^60,61 Despite these promising applications, challenges include the need for thorough safety assessments and addressing potential long-term effects of nanoparticle use in the eyes.

FIG. 3.

Approaches of nanoparticles transportation across the blood–retinal barrier (BRB). (a) Tight junctions (TJs) loosen up due to a surfactant in NPs or BRB disturbances caused by pathological circumstances, allowing NPs to flow through the BRB. (b) NPs enter the retina via receptor-mediated transcytosis. (b) The NPs engage with their appropriate endothelial cell surface receptors, causing plasma membrane incursions, vesicle formation, and crossing of NP to the other side of the membrane. (c) The NPs can cross the BRB through carrier-mediated transport. (d) Adsorptive transcytosis allows chitosan or polysaccharides coated NPs to pass through the BRB. The figure is created with Biorender.com.

A. In vitro studies on nanoparticle implantation and imaging

Several in vitro studies have been performed to assess the health hazards of nanoparticles. For instance, Chen et al. used rare earth metals as nanoparticles to prevent retinal degeneration. The authors assessed intracellular levels of toxic reactive oxygen intermediates (ROIs) in rodents' retinal cell cultures after using nanoceria particles (vacancy-engineered mixed-valence-state cerium oxide nanoparticles). The study's findings revealed that nanoceria particles inhibited the progression of ROI-induced cell death involved in retinitis pigmentosa, macular degeneration, and other retinal diseases.⁶² Later, the efficacy of silver nanoparticles on angiogenesis was assessed in bovine retinal endothelial cells, such as PEDF. The authors observed that silver nanoparticles hinder vascular endothelial growth factor (VEGF)-induced cell migration, proliferation, tube formation, and sprouting by inhibiting and targeting the activation of PI3K/Akt pathways.³⁸ Yanai et al. used another metallic nanoparticle, superparamagnetic iron oxide nanoparticles (SPIONs), to magnetize rat mesenchymal stem cells (MSCs) and targeted these magnetized cells for its delivery to the upper hemisphere of the rodent's retina. These studies also reveal that SPIONs did not hamper the viability and differentiation capabilities of the cells.³⁰

B. In vivo studies on nanoparticle implantation and imaging

The efficacy of gold nanoparticles passing through the blood–retinal barrier (BRB) was determined by intravenously administering gold nanoparticles to C57BL/6 mice.³¹ The authors found that 20 nm of gold nanoparticles could pass through the BRB without showing any structural abnormality, retinal toxicity, and cell death in the retinal layers. However, 100 nm of gold nanoparticles could not be detected in the retina, suggesting that the small size of the nanoparticles offers more benefits in drug delivery.³¹ In another study, Kim et al. investigated the effects of gold nanoparticles on retinal neovascularization inhibition. For the same, the animal model of retinopathy of prematurity (ROP) was intravitreally injected with gold nanoparticles. The authors found that gold nanoparticles significantly inhibited retinal neovascularization without affecting cellular viability or retinal toxicity in retinal microvascular endothelial cells.⁶³ In 2014, Giannaccini et al. used magnetic nanoparticles for ocular drug delivery to target the retinal pigmented epithelium (RPE) layer. They intraocularly injected nanoparticles into Xenopus embryos and found that they were retained in RPE for several days.⁶⁴ Mitra et al. tested the efficacy of Y₂O₃ nanoparticles in a murine light-stress model to ameliorate degeneration. The study's results suggested that Y₂O₃ nanoparticles are nontoxic and well-tolerated.⁶⁵ The effect of topically applied hyaluronic acid-coated gold nanoparticles and uncoated gold nanoparticles as drug delivery vehicles to the mouse eye was compared. Retinal sections of eyes treated with hyaluronic acid-coated gold nanoparticles revealed a higher distribution of drugs in the posterior section of the eye and unaltered visual function. The findings suggest that nanoparticles can be safely administered and serve as noninvasive drug carriers to the retina.³⁵ According to a recent publication, peptide-conjugated lipid nanoparticles successfully transfer mRNA to rats' and nonhuman primates' neural retinas, increasing the potential applications of lipid nanoparticle-mRNA therapy for hereditary blindness.⁶⁶ Figure 4 illustrates the transfer and translatability of GFP mRNA in different neural retinal cells via peptide-conjugated lipid nanoparticles.

FIG. 4.

Peptide-conjugated lipid nanoparticles (LNPs) mediate GFP expression in the neural retina of nonhuman primates (NHP) after subretinal administration. (a) Fundus autofluorescence image after 48 h of peptide conjugated LNP subretinal administration (n = 1). (b) Confocal images of NHP retinal cross sections stained with DAPI (blue) and anti-GFP antibody (red). The H&E image depicts the retinal morphology in locations with GFP expression. (c)–(f) Confocal photographs of retinal cross sections stained with cell-specific and anti-GFP antibodies. Cone arrestin for cones, rod arrestin for rods and s-cones, glutamine synthetase (Glts) for Müller glia, and RPE65 for RPE cells. (g) Confocal pictures of retinal cross sections stained with CD3 (T cells; green) and IBA-1 (microglia; red) to show the elicited immunological responses. Co-localization is represented by arrowheads. The scale bar represents 100 mM in all the image panels. Reproduced with permission from Herrera-Barrera et al., Sci Adv. 9, eadd4623 (2023). Copyright 2023 Authors, licensed under a Creative Commons Attribution Noncommercial License (CC BY-NC) License.

C. Therapeutic studies on nanoparticle implantation and imaging

The anti-angiogenic properties of silver nanoparticles on VEGF-induced bovine retinal endothelial cells (BRECs) were explored to assess their impact on the proliferation and migration of BRECs in the presence of VEGF. The authors reported that a 500 nM (IC50) concentration of silver nanoparticles could inhibit cell survival by inactivating the PI3K/Akt-dependent pathway.⁶⁷ Similarly, Sheikpranbabu et al. investigated the effect of silver nanoparticles on VEGF- and IL-1beta-induced vascular permeability in porcine retinal endothelial cells (PRECs). Results from the rhodamine isothiocyanate (RITC)-dextran flux assay indicated that silver-conjugated nanoparticles (Ag-NP) efficiently blocked the flux of dextran in VEGF- and IL-1beta-induced PRECs monolayer by targeting the Src kinase pathway.³⁷

In vitro and in vivo studies have used superparamagnetic iron oxide nanoparticles (SPIONs) to deliver stem cells to the retina. In the in vivo study, FluidMAG-D-labeled stem cells were intravitreally injected into a rodent model of retinal degeneration (S334ter-4 transgenic rat), and a gold-plated neodymium disk magnet was inserted within the orbit in a rodent group. Results showed increased retinal localization and stem cell delivery to the retina, suggesting that magnetic stem cell therapy has significant therapeutic benefits in the dystrophic retina.³⁰ The inhibitory effect of gold nanoparticles on VEGF-induced choroid-retinal endothelial (RF/6A) cells was tested, and it was discovered that gold nanoparticles did not alter cell viability or adherence. However, they efficiently inhibited VEGF-induced migration in RF/6A cells, with the inhibitory impact mediated by the Akt/eNOS pathway.⁶⁸

In summary, several studies demonstrate that nanoparticles can significantly enhance therapeutic and imaging modalities currently available in clinical settings. Recently, the discovery of fibrin-targeted nanoparticles functioning as T1 and T2 MRI contrast agents for identifying and diagnosing tiny blood clots in acute ischemic stroke has been reported.⁶⁹ In addition, the ability to target a specific retinal locus with intravenous injection of rat mesenchymal stem cells coupled with superparamagnetic iron oxide nanoparticles was also demonstrated.³⁰ Moreover, treatment with magnetic mesenchymal stem cells increases the amount of anti-inflammatory molecules, indicating that mesenchymal stem cell therapy may be used for therapeutic purpose.³⁰ According to a study by Maya-Vetencourt et al. subretinal implantation of poly [3-hexylthiophene (P3HT) nanoparticles recovered vision in a rat model of retinitis pigmentosa.⁷⁰ Layer-by-layer (LbL) microcapsules packed with nerve growth factor (NGF) induce hippocampus neurons' cellular growth and structure.⁷¹ The LbL-microcapsules were proposed as a potential vehicle for delivering NGF to particular subpopulations of brain neurons.⁷¹ Therefore, studies suggest that nanoparticles offer a platform for the development of new techniques for diagnosing and treating retinal diseases.

III. NANOWIRES FOR RETINAL DISEASE

Nanowires are minute single-crystal structures with a diameter of 10 nm. Nanowires are composed of a variety of materials, such as cobalt, copper, silicon, and gold.^72,73 They exhibit both electrical and optical properties relevant to maintaining retinal health. Due to their structural and morphological similarity with photoreceptors, the optical properties enable the creation of high-resolution imaging devices that can provide detailed insight into the structure and function of the retinal tissue.^74,75 The enhanced imaging capability can contribute significantly to early disease detection and monitoring, facilitating personalized treatment strategies.^15,76 Nanowires can be engineered for highly sensitive and specific detection of biomarkers associated with retinal diseases. Their small size allows for precise interactions at the molecular level, enabling early and accurate identification of pathological changes in the retina.^72,77,78 For example, modifying an indium (III) oxide (In₂O₃) nanowire with a fibronectin-based binding agent allows detection of the SARS virus N-protein.⁷² Nanowires modified with polyethylene glycol (PEG) can identify and quantify biomarkers in physiological fluids and can be used to create wearable tear-borne biomarkers.⁷⁸ Furthermore, nanowires can deliver targeted treatments directly to affected retinal cells. This focused approach reduces adverse effects while increasing the therapeutic efficacy of therapies.⁷⁹ Nanowire-based therapies may offer new avenues for restoring or preserving vision in individuals affected by these debilitating conditions.^10,80

A. Effect of nanowires on retinal cells development and function

In two studies, arrays of gallium phosphide nanowires and silicon wires were used to prepare long-term cultures of postnatal retinal cells, such as rod and cone photoreceptors, bipolar cells, and ganglion cells. The cells survived 18 days in vitro with gallium phosphide nanowires and silicon nanowires. The use of gallium phosphide nanowires resulted in the absence of neuronal overgrowth, expressed synaptic vesicle marker (synaptophysin), neurite elongation with long nanowire (4 μm long), and regulation of cell behavior, indicating that gallium phosphide nanowires are the next generation of implantable devices.³⁹ On the other hand, silicon nanowires were used in both short- and long-term cultures of murine retinal cells. Silicon nanowires caused phenotypic changes, such as the absence of neurites and under-expressed retinal cell markers, altered cell behavior, increased porosity, and neurotoxicity.⁴⁰ Semiconductor nanowires have been developed to manipulate cell behavior. Using fluorescence microscopy, we evaluated the amount of laminin adsorbed on gallium phosphide nanowires, demonstrating that up to four times as much laminin was absorbed per surface area. Notably, laminin binding to nanowires is attributable to geometric factors rather than electrostatic interactions. This highlights the advantageous characteristics of nanowire substrates, particularly in promoting cellular growth.⁸¹

B. Studies focused on the use of nanowires in animal models

In 2012, Christiansen et al. conducted an in vivo study involving the implantation of Poly(ε-Caprolactone) (PCL) short nanowires and other scaffolds into the subretinal space of 28 porcine eyes. The utilization of PCL nanowires proved effective without causing inflammation or tissue disruption. The PCL short nanowire demonstrated an optimal level of stiffness for successful surgical delivery to the subretinal space, suggesting its potential suitability for subretinal implantation.⁴¹ Tang et al. developed gold nanoparticle-decorated titania nanowire arrays as artificial photoreceptors to restore light responsiveness with complex spatiotemporal properties in retinal degenerative disorders.⁸² The innovative arrays were tested on blind mice with degenerated photoreceptors. Nanowire arrays successfully restored blue, green, and near-UV light sensitivity in retinal ganglion cells with high spatial resolution of over 100 μm. Additionally, subretinal implantation of nanowire arrays resulted in the functioning of neurons in the primary visual cortex, exhibiting light-responsive behavior (Fig. 5). This suggests a potential recovery of light sensitivity and improvement in the pupillary light reflex, indicating a promising avenue for behavioral recovery in individuals with retinal degenerative diseases.⁸²

FIG. 5.

The subretinal implantation of nanowire (NW) array restores light sensitivity in blind mice after 4–8 weeks. (a) and (c) The infrared images show pupil contraction in the right and left eyes of a blind mice with and without NW implants, as well as in the left eyes of wild-type mice right before and during near UV light stimulation (a) or before and during green light stimulus (c). White arrows show the UV LED or green LED reflections on the cornea of the eye. (b) and (d) The bar graph depicts pupil constrictions of experimental mice's eyes at various LED irradiances, with UV LED wavelength peaking at 395 nm (b) and with green LED peaking at 580 nm (d). The data are reported as mean ± standard error of measurement (SEM). Two-tail paired Student's t-test was used to compare the differences between groups. P-value < 0.05 for blind vs blind + NW groups. Each circle represents the number of animals utilized in the study. Panels (a) and (c) have scale bars of 500 μm. Reproduced with permission from Tang et al., Nat Commun. 9, 786 (2018). Copyright 2018 Authors, licensed under a CC BY (Creative Commons Attribution) license.

C. Nanowire based therapeutic studies

Nanowires are employed to study the long-term effects of electrodynamic characteristics on cellular processes, such as adhesion, proliferation, differentiation, and morphogenesis.⁸³ Consequently, scientists have created nanowires with improved cell adhesion, biocompatibility, and electroconductivity.^84,85 In a study, mouse cortical stem cells were grown on vertical nanowire substrates to analyze gene expression through RNA microarrays. The findings indicated a significant upregulation of genes associated with cell morphology, cell adhesion, and cell metabolism on the nanowire substrate. Cells cultured on the nanowire substrate had a rounded shape with a mature appearance, characterized by multiple extended processes firmly adhered to the nanowire surfaces.⁸⁶

It was also demonstrated that a vertically oriented gold nanoparticle-decorated titania (Au-TiO₂) nanowire array-based artificial photoreceptor soaks up light and produces photovoltaic activity, triggering spiking processes in the interfaced neurons and restoring light responses in the degenerated retina.⁸² It has also been demonstrated that subretinal implantation of nanowire arrays in blind mice and monkeys restores visual acuity and can be utilized to treat visual deficiencies in people with photoreceptor degeneration.⁸⁷ A recent study demonstrated that optoelectronic synaptic transistors based on cesium lead bromoiodide (CsPbBrI2) nanowires effectively capture visual information that changes over time. These transistors can also be utilized to create artificial optoelectronic synapses that can be used to build dynamic machine vision systems.⁸⁸ Silicon and titanium nanowires coated with gold nanoparticles were effective at simulating photoreceptors due to their ability to accept and convert light signals into electrical signals.⁸⁹ They also have a greater surface-to-area ratio for sensing light and charge transfer. Silicon nanowires, in particular, help sense light and transport signals to the retina's internal layer to restore vision.

IV. HYBRID NANOSTRUCTURES FOR RETINAL DISEASE

Hybrid nanostructure is a combination of two or more nanomaterials that formulate a new structure with enhanced and superior properties.⁹⁰ Until, a broad range of nanostructures have been explored with hybrid approaches that mimic the biological, physiochemical, and electrical properties of the retina.^75,91 Hybrid nanostructures are not confined to compositional changes but structural characteristics including individual component distribution, exposed facet, spatial arrangement, components interface, and the crystal phase.⁹⁰ These properties of hybrid nanostructures led to promising results in terms of high drug loading capacity, good targeting features, greater biocompatibility, versatile surface activities, and minimal toxicity in retinal-related conditions.^90,91 For instance, the integration of biocompatible polymers with inorganic nanoparticles is suitable for intraocular drug delivery due to controlled drug release and improved biocompatibility.⁹²

Hybrid nanostructures have emerged as a theragnostic approach in managing retinal diseases. For AMD, lipid-based hybrid nanoparticles have been developed to encapsulate and deliver anti-VEGF drugs, which has resulted in prolonged drug retention and improved outcomes.⁹³ In the context of retinal imaging and diagnosis, quantum dots are ideal for fluorescent labeling and imaging. Quantum dots are semiconductor nanoparticles, combined with organic polymers or graphene oxide to formulate a quantum dot-based hybrid nanostructure, resulting in early detection and monitoring of retinal diseases.^94,95 Hybrid nanostructures comprising biocompatible scaffolds, stem cells, and growth factors can promote retinal regeneration and repair damaged retinal tissue.^75,96 Studies have shown that hybrid nanofibers aid in guiding the differentiation of stem cells into retinal cell types, thus leading to an advancement in cell replacement therapy in conditions like retinitis pigmentosa.^97,98 Various clinical trials have suggested that stem cell incorporation into the eye is technically possible and has no discernible long-term structural or functional damage.⁹⁷ Furthermore, several studies have shown that using nanomaterials (TiO₂ nanotube) as a 2D cell culture substrate can encourage the differentiation of stem cells into particular retinal cell lineages.⁹⁸

A. Studies on the use of hybrid nanostructures in retinal cells

In 2008, Redenti et al. developed a composite graft by cultivating mouse retinal progenitor cells (mRPCs) on laminin-coated novel nanowire PCL scaffolds.⁹⁹ These scaffolds included smooth, short (2.5 μm), and long (27 μm) nanowires. The composite graft was co-cultured with the retina of C57BL/6 and rhodopsin knockout retinal explants. The study found that short and long PCL nanowires resulted in robust RPC proliferation, PCL-mediated RPC migration, enhanced expression of mature bipolar (protein kinase C) and photoreceptor (recoverin) markers, and downregulation of early progenitor markers. This suggests that the implantation of the composite graft in the subretinal space may repair diseased retinal tissue by inducing the differentiation of cells.⁹⁹

In the realm of neural prostheses applications for improving the stability of microelectrodes, Zhou et al. conducted in vitro studies using multiwall carbon nanotube (MWCNT)-doped poly(3,4-ethylenedioxythiophene) (PEDOT) composite films layered onto a platinum microelectrode. The results of cell assays demonstrated that PEDOT/MWCNT films promoted adherence and neurite outgrowth in cultured rat pheochromocytoma cells.¹⁰⁰

Furthermore, in the field of neural electrodes, a study involved the fabrication of single-walled carbon nanotube (SWNT)/polypyrrole (PPy) composite films with strong adhesive force and controlled pore size. These films were cultured with rat pheochromocytoma (PC12) cells to enhance the performance of neural electrodes. Lab findings showed that the SWNT/PPy composite film, being porous, was not harmful and encouraged neuronal development, suggesting it could be helpful in long-term implantable neural electrodes for both stimulation and recording.¹⁰¹

B. Hybrid nanostructure implantation and imaging in animal models

Studies have shown that gold nanorods (AuNRs) coated with either poly(styrene sulfonate) (PSS-AuNRs) or anti-CD90.2 antibodies (Ab-AuNRs) can be utilized for ocular optical coherence tomography (OCT) imaging as a contrast agent. Intravitreal injection of these nanorods in mice aimed to enhance contrast in OCT images. However, the use of AuNRs led to ocular inflammation, limiting their potential as a contrast agent in OCT.¹⁰² Similarly, Christiansen et al. (2012) conducted a study using different modifications of poly(ε-Caprolactone) (PCL) scaffolds (smooth, short, and electrospun) in the subretinal space of 28 porcine eyes. After 6 weeks, all three PCL scaffolds were well-tolerated in pig eyes without causing inflammation or significant tissue disruption. However, multifocal electroretinography (mfERG) results showed membrane-induced photoreceptor damage and reductions in P1 amplitude with the smooth PCL scaffold.⁴¹

In a study by Zhou et al., platinum wires coated with PEDOT/MWCNT films were utilized in comprehensive in vitro and in vivo experiments. The in vivo experiments involving implantation into the rat cortex revealed a diminished tissue response after 6 weeks, as demonstrated by glial fibrillary acidic protein and neuronal nuclei staining. This observation suggests that galvanostatically polymerized PEDOT/MWCNT films can potentially enhance the durability of stimulation microelectrodes. Later, ocular injectable photoreceptor-binding upconversion nanoparticles (pbUCNPs) were designed and utilized to bind to retinal photoreceptors, culminating in the development of small near-infrared (NIR) light transducers for NIR light image perception. The findings showed that mice equipped with these pbUCNPs nanoantennae could perceive NIR light, ambient-daylight compatible light patterns, and detailed NIR shape patterns.⁴⁴

C. Studies on hybrid nanostructures for therapy and diagnostic imaging

To date, hybrid nanostructures have shown promising contributions in ocular diagnostic and screening tests.^103,104 However, the use of nanostructures for therapeutic interventions is still limited. In therapeutics, nanomaterials are used to deliver drugs to the retina and gene therapy through topical delivery, subconjunctival administration, and intravitreal or subretinal injections, but these therapies have been linked to ocular complications like inflammation, retinal detachment, increased vitreous hemorrhage, and intraocular pressure.^10,105 A study revealed the development of melatonin-loaded lipid-polymer hybrid nanoparticles (mel-LPHNs), a new and safe hybrid platform that can be used to treat retinal diseases topically.⁴² The biological assessment of mel-LPHNs in an in vitro model of diabetic retinopathy revealed superior neuroprotective and antioxidant properties compared to an equivalent quantity of melatonin in aqueous solution.⁴²

Several studies have shown that polymeric hybrid nanoparticles can be used for gene delivery, vaccine delivery, and bioimaging.¹⁰⁶ It was also demonstrated that bevacizumab-PLGA formulations preserve bevacizumab's active antiangiogenic properties and can prevent choroidal neovascularization for an extended period.¹⁰⁷ A study also presented an approach for noninvasive treatment of choroidal neovascularization with hybrid cell-membrane-cloaked biomimetic nanoparticles. The retinal blood cell-retinal endotheliocyte (RBC-REC) membrane was put on top of the PLGA nanoparticle to make the hybrid nanoparticle. The hybrid membrane-coated nanoparticles were able to target choroidal neovascularization and also attenuated the growth of new blood vessels. Adding RBC membranes to nanoparticles made it much harder for macrophages to eat the final nanoagents, which made it easier for them to build up in choroidal neovascular regions.¹⁰⁸

V. NANOSCAFFOLDS FOR RETINAL DISEASE

Nanoscaffolds refer to a nanostructured framework comprising natural or synthetic polymers. It provides a three-dimensional (3D) platform for cell growth, cellular interaction, performing biochemical functions, cell signaling, and tissue regeneration.^109,110 It supports cellular growth by influencing cell adhesion, migration, proliferation, differentiation, and colonization.^109,110 The nanoscale fabrication of scaffolds simulates the structural features and pattern of the extracellular matrix. Thus, the colonized cells on nanoscaffolds are biocompatible, immunocompatible, and biofunctional.^109,110

Ocular tissue engineering approaches use scaffold-based extracellular matrix in the retinal pigment to mimic in vivo structures and natural mechanical properties. Recently, the concept of using polymers for retinal transplantation has emerged, and an ideal polymer for various applications in tissue engineering characterized by being biodegradable, porous, and approximately 50 μm thick, with the correct Young's modulus. Several polymers that meet these criteria and have shown promising results include poly(lactic acid) (PLA), poly(lactic-co-glycolic acid) (PLGA), poly(glycerol-sebacate) (PGS), and polycaprolactone (PCL).¹¹¹ In the specific context of ocular applications, gelatin has been widely used in tissue engineering and regenerative medicine for many years. Gelatin has excellent biocompatibility, ease of processing, and cost-effectiveness. Over the past decade, extensive research has been conducted to assess the use of gelatin-based materials in various ocular applications, including bio-adhesives, bio-artificial grafts, and cell-sheet carriers.¹¹² The versatility and favorable properties of gelatin make it a valuable candidate for developing innovative solutions in the field of ophthalmology. Gelatin scaffolds have been prepared by cross-linking gelatin with natural or synthetic biopolymers.^113,114

A. Research on the application of nanoscaffolds in retinal cells

Various groups are investigating strategies to cure damaged retinal pigment epithelium by implanting different types of cells on scaffolds made of degradable, non-degradable, natural, and artificial substrates.¹⁰⁹ Ultrathin nanofibrous membranes are prepared using various materials to mimic the inner collagenous layer of Bruch's membrane. In a study by Warnke et al., an ultrathin 3D nanofibrous membrane was created using collagen type I and PLGA.¹¹⁵ On the other hand, Xiang et al. fabricated an ultrathin porous nanofibrous membrane with a thickness ranging from 3 to 5 μm.¹¹⁶ The materials used in this membrane included antheraea pernyi silk fibroin (RWSF), PCL, and gelatin. When human retinal pigmented epithelial cells were cultured on these 3D nanofibrous membranes, the results showed the formation of an oriented monolayer with polygonal cell shapes. Additionally, the cells exhibited tight junctions between them, had abundant sheet-like microvilli on their apical surface, and showed an upregulation of RPE65 protein expression. This suggests that these nanofibrous membranes have the potential to support the growth and differentiation of retinal pigmented epithelial cells.^47,116 RWSF/PCL/Gt nanofibrous membranes also exhibited similar characteristics along with the secretion of polarized PEDF, higher cell growth, excellent biocompatibility, and lack of inflammation.⁴⁷ These studies suggest that ultrathin membrane implantation in the subretinal space in AMD patients ensures good outcomes.^115,117 A nanopatterned porous PCL film scaffold also mimics the functions of healthy Bruch's membrane due to its biocompatibility with the subretinal space in patients with AMD. Fetal human retinal pigmented epithelial cells cultured on porous PCL exhibit improved maturity and function markers, including cell density, pigmentation, growth factor secretion, barrier function, and cell behavior.¹¹⁸ Recently, gelatin/chitosan nanofibrous scaffolds were implanted in the subretinal space to rectify damaged retinal pigmented epithelial cells. SEM results revealed proper adhesion of cultured retinal pigment epithelium cells on gelatin/chitosan scaffolds along with upregulated RPE65 and cytokeratin 8/18 markers.⁴⁷

Distorted vision arising from diseased photoreceptor cells and inner retinal cells can be addressed through the use of biodegradable elastomeric membranes made of poly(glycerol-sebacic acid) (PGS). To enhance their efficacy, the surface of these PGS membranes is subject to chemical modification with peptides incorporating an arginine-glycine-aspartic acid (RGD) extracellular matrix ligand sequence. In addition to this, nanofiber meshes containing laminin and PCL are applied as a coating, promoting the attachment of photoreceptor layers. In vitro findings demonstrated successful cell adhesion in isolated embryonic retinal tissue, an increased number of recoverin and rhodopsin labeled photoreceptors, and the absence of ganglion cells, rod bipolar cells, and amacrine cells. This suggests an improvement in graft-host neuronal connections and holds promise for correcting distorted vision.¹¹⁹

B. Research on nanoscaffolds implantation and imaging in animals

The ultrathin nanofibrous membranes made of RWSF, PCL, and gelatin (RWSF/PCL/Gt) demonstrate promising uses and effectively implant membranes in rabbit eyes.¹¹⁶ Histological staining revealed well-tolerated RWSF/PCL/Gt membranes in the intraocular space without any evidence of inflammation in the sclera or retina. This suggests that these membranes could serve as a viable cell scaffold option for transplantation.¹¹⁶

In another study, retinal pigment epithelial cells (RPECs) generated from human embryonic stem cells (hESC) were subretinally transplanted into immunosuppressed rabbits and Royal College of Surgeons (RCS) rats using ultrathin and porous polyimide (PI) membranes. Electroretinography and optical coherence tomography results demonstrated rescue after hESC-RPE injection in rats and successful placement of the PI in rabbits. However, pigmentation on the hESC-RPE-PI was lost over time. While the porous ultrathin PI membrane shows promise as a scaffold, the challenge lies in addressing insufficient immunosuppression for reducing xenograft-induced inflammation.¹²⁰

Another area of interest involves degradable scaffolds to enhance retinal pigment epithelium cell transplantation technology. For instance, a fibrin hydrogel implant was delivered to the sub-retinal space of a pig eye. After 8 weeks postoperatively, standard ophthalmic imaging techniques and histologic analysis revealed the fibrin scaffold's degradation, the neurosensory retina's reattachment, and a regular retinal pigment epithelial cell–photoreceptor interface.¹²¹

C. Preclinical and translational research on nanoscaffolds

Implantation of retinal pigment epithelium (RPE) is considered a potential therapy for various ocular diseases. A functional RPE monolayer can be manufactured by reproducing the natural environment or through a tissue engineering approach. The retinal pigment epithelium is cultured on a nanofibrous biological or synthetic scaffold for better cell growth, transplantability, and cell replacement therapy.¹²² Several research investigations have employed stem cell-derived RPE-based therapy in individuals with AMD. A study used a biodegradable PLGA nano-scaffold to deliver clinical-grade AMD-patient-derived RPE patches in rats and laser-induced porcine RPE-injury AMD models.¹¹⁵ As illustrated in Fig. 6, the iPSC-RPE patches were safe, and the AMD-iRPE patch was fully functional after being integrated into Bruch's membrane. The biodegradable scaffold approach improved the incorporation and function of RPE-patch in rats and the laser-induced porcine RPE-injury model, suggesting the promising role of autologous iPSC-based therapies in retinal disorders.⁴⁸ Another study using a pig model of injury-induced choroidal neovascularization showed that it supports RPE cells and replaces Bruch's membrane with an amniotic membrane.¹²³

FIG. 6.

Incorporation and evaluation of the iRPE patch from AMD patients in laser-induced swine retinal degeneration model for functional assessment. (a)–(c) Optical coherence tomography (OCT) images of healthy retina and PLGA scaffold or AMD-iRPE patch transplanted retina. The transplanted scaffold is visible above an area of laser induced RPE ablation, as indicated by the horizontal line in (b) and (c). (d)–(f) The retinal sections were immunostained with STEM121 [green, arrowhead in (f) shows AMD-iRPE intgeration], PNA [white, arrowhead in (e) indicates retinal tubulations], and RPE65 (red). (g) The immunostaining of retina for cone opsins (white), and STEM121 (green) in pig eye transplanted with human iRPE. (h) and (i), the immunostaining of healthy pig retinas and iRPE patch transplanted retinas with rhodopsin (green) and RPE65 (red) or STEM121 (red). The white arrow indicates the presence of phagocytosed photoreceptors in panels (h) and (i). Z-sections reveal the location of photoreceptor outer segments. Reproduced with permission from Sharma et al., Sci. Transl. Med. 11, eaat5580 (2019). Copyright 2019 the AASC (American Association for the Advancement of Science).

Numerous clinical investigations have demonstrated that introducing retinal pigment epithelial cells using nanoscaffolds led to a well-differentiated monolayer integration that improved photoreceptor survival and rescue in the subretinal area. Furthermore, nanoscaffold-mediated RPE delivery exhibits superior resilience to oxidative damage.¹²⁴ In a first-in-human, Phase 1/2a trial, 16 patients with advanced non-neovascular age-related macular degeneration were implanted with a parylene scaffold (CPCB-RPE1) to cure severe vision loss. A 94% success rate was recorded, with no unanticipated major adverse events associated with the implant or surgery.¹²⁵ Furthermore, RPE and Parylene-C implants have been employed in a number of preclinical and translational investigations to treat visual loss.^126,127 In recent years, gelatin, chitosan, collagen, and hyaluronic acid have been used to prepare natural nanofiber scaffolds. These natural nanoscaffolds have been found to stimulate healthy RPE growth and the release of restorative factors, along with superior cellular adhesion than synthetic scaffolds.

VI. CHALLENGES AND FUTURE PERSPECTIVES

Nanomaterials play a pivotal role in revolutionizing the diagnosis and treatment of retinal diseases due to their versatility in drug delivery, imaging, and regenerative medicine applications.⁸⁰ However, challenges associated with their application in retinal regeneration and repair include regulatory approval, scalability of synthesis methods, preventing immune responses, and long-term safety, stability, and biocompatibility.¹²⁸ Therefore, nanotechnology seeks more advancements to develop nanomaterials with better physicochemical properties and retention ability. Nanomaterials are being engineered for better biocompatibility to ensure that they do not cause inflammation after implantation. The delivery methods of nanomaterials in the retina are invasive and may trigger inflammation, infection, retinal detachment, or vision loss.^48,129,130 Another challenging aspect is controlled drug delivery. The retina is a sensitive site for precisely delivering drugs, and nanomaterials are designed to ensure targeted delivery to specific retinal cells or layers, such as photoreceptors or retinal pigment epithelium cells, for the effective and controlled release of drugs over extended periods without causing any toxicity.¹³¹ Nanomaterials should be engineered to endure the eye's hostile environment, which includes light, temperature variations, and mechanical stress. Finally, the most challenging step is regulatory approval. Nano-based therapies may alter an individual's immunity and immune system. Thus, nanomaterials-based medicines necessitate thorough testing, which can be costly and time-consuming.¹³²

A fundamental question in nanoparticle discussion is whether nanoparticles are toxic. Extensive research is being conducted to assess the toxicity and long-term effects of these nanostructures on ocular tissues.^133,134 Various studies have shown that nanoparticles may trigger immunogenicity and immunotoxicity.^133,134 Lin et al. studied the toxicity of PCL, PLGA, and PEGylated PLGA (PEG-PLGA) utilizing retinal pigmental epithelial cell line and the human retinal endothelial progenitor cell models.¹³⁵ It was observed that PLGA and PCL both showed toxic effects, whereas PEG-PLGA does not have harmful effects. The traditional investigations of nanotoxicity rely on trials using in vivo animal models or in vitro cell culture. The ability and strength of these models to forecast human nanotoxicity are topics of continuous discussion in the scientific community.^136–138 Since laboratory animals have far shorter lifespans than humans, it is challenging to simulate chronic long-term exposure in such simplified models, limiting nanoparticle risk assessment's applicability. However, data on long-term, low-dose nanoparticle exposure may offer insightful new information about the long-term harmful consequences of nanoparticles. Assessing and comprehending the mechanisms of nanotoxicity in medically and biologically relevant models requires a great deal of effort and attention. It might be necessary to create new toxicity evaluation models that use machine learning techniques, high-throughput screening techniques, and three-dimensional microfluidic-based tissue chips and organoids to simulate human physiology more accurately.^139–143 These sophisticated tissue models and single-cell analytical techniques like single-cell elemental measurement, single-cell RNA sequencing, and enhanced optical imaging may offer potent instruments for evaluating nanotoxicity at the individual cell level.^144–148

Future research should focus on optimizing the design of nanostructures for specific retinal disease targets and exploring innovative strategies for precise drug delivery and tissue regeneration. Additionally, personalized approaches, such as tailored nano materials-based treatments to individual patients, may be beneficial and effective treatment strategies.¹⁴⁹ Nanomaterials can also facilitate the delivery of gene-editing tools or viral vectors for gene therapy in retinal diseases with a genetic component, like retinitis pigmentosa. Integrating nanotechnology with stem cells could be a futuristic approach for retinal rejuvenation.^97,150 Hence, more research studies and clinical trials are needed to understand the state-of-the-art future systemic strategy of nano-biomaterials in retinal disease.

AUTHOR DECLARATIONS

Conflict of Interest

The authors have no conflicts to disclose.

Author Contributions

Geetika Kaur: Writing – original draft (equal); Writing – review & editing (equal). Shivantika Bisen: Writing – original draft (supporting); Writing – review & editing (supporting). Nikhlesh K. Singh: Conceptualization (lead); Funding acquisition (lead); Methodology (lead); Resources (lead); Supervision (lead); Validation (lead); Visualization (lead); Writing – original draft (equal); Writing – review & editing (equal).

DATA AVAILABILITY

Data sharing is not applicable to this article as no new data were created or analyzed in this study.