3.2.2. AgNPs

Numerous in vitro and in vivo studies demonstrated the toxicity of AgNPs that perturbs several cellular pathways [4]. The most accepted mechanism through which they induce side effects, despite still under investigation, seems to be related to Ag ions released from NPs surface [68–70]. In terms of production process, AgNPs can be synthetized by different chemical routes such as chemical-based reduction [71], sol-gel, or green synthesis [72,73].

Jun et al. [74] used zebrafish embryos as model organism to test their delivery at an ocular level. They investigated the toxicity of AgNPs at concentration of 0.4 mg/<sup>L</sup> and observed down- expressions of different lens crystalline genes by unknown mechanisms that either induced apoptosis or blocked nuclear DNA or RNA export.

Sriram et al. [75] demonstrated the cytotoxicity and Reactive Oxygen Species (ROS)-induced apoptosis after exposure of AgNPs with two different size (22.4 and 42.5 nm) in bovine retinal endothelial cells (BRECs) finding size-dependent toxicity. As a matter of fact, smaller NPs induced adverse effects on cells. Kim and colleagues [76] exposed New Zealand white rabbits (according to OECD test guidelines) to AgNPs in order to understand the possible eye irritation effect. Oedema and conjunctival redness have been observed after 1 h of AgNPs incubation, but the cornea, iris, or conjunctiva resulted unaffected after 24, 48, and 72 h of NPs exposure. In similar experiments, the instillation of AgNPs (5000 ppm) were tested on another in vivo model, the guinea pigs, observing a transient conjunctivae irritation during 24 h of early observation [77].

### **4. Noble-Metal NPs as Therapy and Diagnostic for Retinal Disease**

AuNPs scatter or absorb light at specific wavelengths as a function of their physico-chemical properties. This characteristic is particularly suitable for bioimaging and to treat some diseases like cancer by NIR-triggered photothermal therapy (PTT), in which light is converted to heat in the tumor

site where NPs accumulate [78]. In the NIR (650–900 nm), the absorption coe fficients of haemoglobin and water are very low, and for this reason, the penetration of NIR wavelength in tissues is very high, allowing NPs stimulation without causing damage [79]. Song et al. [80] reported application of gold nanodisks (GNDs) on human retinal microvascular endothelial cells (HRMEC) and mouse oxygen-induced retinopathy model. The GNDs were optically suitable for nanomedicine applications due to their ability to produce detectable signals regardless of polarization or direction of light source and to scatter in NIR region (830 nm). In addition, GNDs were internalized less e fficiently in cells with respect to AuNPs or Au rods (AuNRs), thus minimizing the ROS production and other several adverse effects. The authors used di fferently charged anisotropic NPs (nanorods or nanodisks) and compared them with AuNPs. After simulating the role of di fferent sizes to select the best diameter (160 nm), they showed that GNDs were able to bind vascular endothelial growth factor (VEGF) inhibiting in vitro angiogenesis with high performance and abolished the VEGF-induced migration of endothelial cells. When these nanostructures were applied to mice eyes, a strong signal in optical coherence tomography (OCT) was detected with a consequent reduction of retinal degeneration (Figure 3). The OCT system involves the use of light energy that, once absorbed by tissues, causes a thermos-elastic expansion generating ultrasonic waves detected by a transducer.

**Figure 3.** Schematic representation of gold nanodisks (GNDs) e ffects in retinal degeneration and Optical Coherence Tomography (OCT) imaging. The GNDs were particularly suitable for their optical properties connected to the shape: GNDs absorbed in the Near Infrared Region (NIR). The size and surface charge also influenced the eye di ffusion. In OCT images, GNDs exhibited a strong signal compared to the dual window (DW) processing method, which is used to detect modulation of OCT signals due to scattering or absorption. Reprinted from [80] Copyright (2017), with permission from Elsevier.

Recent therapies to treat some diseases strictly connected to retinal degeneration consider the transplantation of photoreceptor precursors (PRPs) as e fficient support improving visual acuity [81]. The analysis of cell survivor in the host retina is a key factor in this kind of treatment. Chemla et al. [82] used AuNPs in order to improve the tracking of transplanted PRPs. Cells were labelled with photothermic AuNPs and transplanted in the vitreous and sub-retinal space of Long-Evans pigmented rats (4–8 weeks old) by gauge needle.

By using coherence tomography, computed tomography and fluorescence fundus imaging, NPs have been tracked for 1 month without recording adverse e ffects. Real-time tracking is essential for evaluating the e fficiency of retinal cell replacement therapies. These studies confirmed the possibility to use AuNPs to improve retinal cell therapy in humans. AuNPs-based photothermal therapy has been also used to treat eye tumors in vitro and in vivo. Some eye cancers provoke retinal detachment leading to a decreased visual eye activity.

In this framework, Kim et al. [83] tested anticancer properties of Doxorubicin (Dox)-conjugated Fucoidan (Fu)-encapsulated AuNPs (Dox-Fu@AuNPs) for the treatment of VX2 squamous carcinoma cells and xenograft tumors in New Zealand White rabbits. After intra-tumoral injection of Dox-Fu@AuNPs in the eye, a complete suppression of the tumor has been observed by dual-chemotherapy and PTT treatment. No damage to retina has been also detected. These composite nanostructures enabled the improvement of the OCT imaging technique (thanks to the selective light absorption of AuNPs) [84].

The quality of photoacoustic-based imaging was also improved thanks to selective light absorption of AuNPs. In this way, a double e ffect in terms of therapy and diagnostic was achieved.

As reported above, OCT is largely used for imaging in ocular clinical research due to the possibility to take structural information based on the reflectivity of tissue. In this respect, Gordon and colleagues [85] confirmed that Au nanorods (with a peak absorption of 750 nm and diameter of 10–35 nm) injected in the retina of Wild-type C57BL/6 mice are able to enhance the contrast, thus significantly improving the quality of a scanned lesion. Additionally, intravitreal injection of Au nanorods functionalized by anti-VEGF antibody triggered a reduction in the extent of anatomic laser damage and lesion-associated photothermal signal density in mice treated in laser induced choroidal neovascularization (LCNV) model and injected with ICAM2-targeted GNR. AuNPs (3–5 nm) were also used as inhibitor of VEGF on choroid-retina endothelial cells (RF/6A) [86]. A similar e ffect was showed using AgNPs, which have been found to reduce the VEGF activation and induced cell proliferation and migration in bovine retinal endothelial cells [87]. In addition, AgNPs were shown to inhibit the angiogenesis, typical of metastasising cells and of retinal neovascularization development. Ag is widely used because it has the grea<sup>t</sup> advantage of being a cost-e ffective material. For this reason, it is a good candidate for the replacement of the current therapies used for the treatment of various retinal angiogenic disorders. In this respect, Sheikpranbabu and co-workers [88] investigated the effect of green AgNPs, obtained from *Bacillus licheniformis*, on VEGF-and Interleukin 1 β (IL-1β) expression—that induced retinal endothelial cell permeability. AgNPs were able to stop VEGF-and IL-1β-induced permeability in retinal endothelial cells from porcine retina.

### **5. Bio-Inspired NPs for Ocular Drug Delivery**

The use of bio-inspired materials mimicking natural components for applications in living organisms has widely spread recently [89]. In fact, inorganic NPs have the serious drawback of not being degradable and are also di fficult to ge<sup>t</sup> excreted from the body [90]. Bio-inspired nanomaterials, including liposomes, niosomes, and chitosan/alginate NPs have been then explored as valid alternatives to metal NPs for treatment of several diseases [91]. In addition, these 'soft' NPs have been applied for drug delivery in the posterior segmen<sup>t</sup> of eye, including retinal photoreceptors.

### *5.1. Liposomes as Drug Delivery Carrier*

Liposomes consist of a lipid bilayer, resembling a cellular membrane, that surrounds an aqueous core. These structures are widely used in pre-clinical studies to improve drug delivery, for example through Blood Brain Barrier (BBB) [92]. The BBB and BRB have the same functional properties [93,94]: so liposomes improve drug delivery to the retina and its photoreceptors due to their high half-life, permitting the long term drug absorption [95]. Liposomes also o ffer the ability to significantly improve pharmacokinetics and pharmacodynamics of any loaded drug compared to its free-circulating counterpart. They are also considered as highly biocompatible and biodegradable as well up to some extent both in vitro and in vivo. For this reason, several anticancer drugs such as doxorubicin, daunorubicin, and epirubicin were encapsulated in liposomes core with results demonstrating a significant decrease of many severe adverse e ffects (nausea, weakness, vomiting) with respect to the same concentration of free (non-encapsulated) drug. In this context, Zhang et al. [96] subcutaneously injected tacrolimus-encapsulated liposomes in Lewis rats with autoimmune uveoretinitis (EAU) by subcutaneous injection. Confocal analysis over rhodamine-conjugated liposomes (containing also the

tacrolimus) revealed that such vesicles remained in ocular fluids even after 14 days. They found a reduced intraocular inflammation and decrease of EAU development without observing significant adverse e ffects.

Liposomes encapsulated with the anti-inflammatory and anti-angiogenic drugs berberine hydrochloride and chrysophanol were applied in myoblasts and in ischemic animal models. These drugs have the ability to contrast oxidative stress by enhancing the activities of antioxidant enzymes such as superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) [97,98]. Berberine hydrochloride and chrysophanol have been used in ocular liposome-mediated delivery to overcome their low stability. In this respect, a recent study [99] showed a liposome system that can entrap the two drugs at same times using the third polyamidoamine dendrimer (PAMAM G3.0) as a delivery carrier for age-related macular degeneration (AMD) therapy in the ocular posterior chamber.

PAMAM G3.0-coated compound liposomes were used to evaluated HCECs (Human Corneal Epithelial Cells) internalization, trans-corneal permeability in New Zealand white rabbits (weighing 2.0–2.5 kg) and male Sprague–Dawley rats (weighing 210–250 g). Pharmacokinetics were evaluated together with in vitro assessments of anti-reactive oxygen species (ROS) e fficacy. The PAMAM G3.0-coated compound liposomes enhanced bio-adhesion on rabbit corneal epithelium after topical administration and exhibited grea<sup>t</sup> permeability in HCECs cells. The possibility to deliver a protective agen<sup>t</sup> against photo-oxidative retinal damage in a light-damaged animal model was also investigated through the use of dendrimers (Figure 4A). Results showed that dendrimers encapsulated with chrysophanol–berberine hydrochloride induced the greatest protection of retinal function in light-exposed rats compared to unloaded dendrimers and free drug. In addition, no inflammation and damage of cornea, iris, and conjunctiva was observed after drug-loaded dendrimers instillation for 14 days. Upon exploring the fundus of the eye (interior surface of eye opposite the lens including the retina, optic disc, macula, fovea, and posterior pole) [100], modifications in the vessel structure and distribution have not been observed after di fferent treatments. (Figure 4B).

**Figure 4.** (**A**) Protection evaluation against photo-oxidative retinal damage in a light-damaged animal model by the use of di fferent formulation: chrysophanol–berberine hydrochloride suspension (CBs), compound liposomes (CBLs), and PAMAM coated compound liposomes (P-CBLs). Retina was stained with hematoxilyn/eosin (scale bare = 20 μm). (**B**) Fundus retinography after incubation with the di fferent formulations. Adapted from [99] under the terms of the Creative Commons Attribution 4.0 International License.

Liposomes o ffer also the grea<sup>t</sup> ability of an easy surface functionalization with a plethora of active macromolecules and targeting moieties that bind in a selective manner some specific receptors constitutive of di fferent cell lines [101,102].

The surface modification of liposomes has also been widely studied to improve their specificity of targeting and to deliver them in di fferent eye compartments. It has been reported [103] that the cationic PEGylated liposomes having an average zeta potentials below +20 mV were able to e fficiently penetrate the murine retina. In contrast, liposome having surface zeta potential values above +20 mV were retained on vitreous section. This did not change whether PEGylated or non-PEGylated liposomes were used. Another critical factor for a liposome-based therapy is the phospholipids composition characterizing them. For example, edaravone-loaded egg L-phosphatidylcholine liposomes prevent retinal ganglion cells (RGCs) from undesired apoptotic processes [104]. This drug is a free radical scavenger used for acute brain infarction and amyotrophic lateral sclerosis treatment [105]. Surprisingly, minor RGC protection was observed with edaravone-loaded distearoyl phosphatidylcholine (DSPC) liposomes. This evidence sugges<sup>t</sup> a link between the viscoelastic properties of the lipid bilayer and the final biological e ffect. In this specific case, the higher fluidity was a key factor to improve the ocular therapy. Another study [106] also tested the use of liposomes for the treatment of di fferent kinds of hereditary degeneration of the retina. The degeneration of retina a ffects rod photoreceptors important for capturing light under low illumination conditions. Because of the pathology development, the patients a ffected by retina degeneration become aware of defects only when lesions are in an advanced stage. A key factor for this disease seems to be rods-specific Pde6b gene mutation in the transduction pathway that triggers an excessive accumulation of cGMP (that have two e ffectors: cyclic nucleotide gated ion channels (CNGCs) and cGMP-dependent protein kinase (PKG) [107]. Consequently, rod cell death was induced and in some cases the secondary loss of cones was observed [108]. The authors identified a liposomal cGMP analogue that was able to overcome the blood–retinal barrier (BRB) while strongly preserving retinal function and reducing rod photoreceptor loss in three di fferent in vivo models for retina degeneration [106,109]. The vesicle structure of liposomes was suitable to confine proteins and peptides into aqueous core overcoming potential agglomeration and protein misfolding events. However, the surface functionalization with peptides is the most common method used for retina therapy. For example, liposomes functionalized with tanticoagulant annexin A5 have been demonstrated to have enhanced uptake levels through corneal epithelial barriers [110].

This vector has been injected topically to deliver 127 ng/g of bevacizumab to posterior segmen<sup>t</sup> of both rats and rabbit eye. Annexin A5-functionalized liposomes loaded with bevacizumab were also rapidly delivered to retina of the two animal models. Annexin A5 unilamellar liposomes made by di fferent polymers (2-dipalmitoylsn-glycero-3-phosphocholine,1,2-dioleoyl-sn-glycero-3-phospho-l-serine and cholesterol) have been also used to target transforming growth factor (TGF)-ß1 into the vitreous of rabbits [111]. It has been found that a higher concentration of molecule with respect to the experimental group treated with the unloaded drug only. The choice for human recombinant TGF-β1 relied on the drug's ability to prevent retinal damage elicited by Aβ oligomers.

### *5.2. Niosomes for Drug and Gene Delivery*

Gene therapies is an approach where diseases are cured by modifying the expression of specific genes regulating pathology. This happens through the administration of nucleic acids aiming to replace abnormal gene sequence [112]. Liposomes and lipoplexes have been taken into consideration as suitable non-viral vectors platform for gene editing. Non-viral gene delivery approaches were widely applied in clinical trials since 2004 to attempt replacing the use of viral vectors that possess high risk of inducing oncogenesis, immunogenicity, mutagenicity phenomena [113].

Among non-viral vectors, niosomes have been recently tested as a potential candidate for gene delivery. Niosomes are vesicular systems comprising of bilayer made up of nonionic surfactants and they can internalize several types of bio-macromolecules such as nucleic acids and drugs. The most commonly used surfactant are Sorbitan fatty acid esters, Spans (Span 20, 40, 60, 65, 80, and 85), Tweens (20, 40, 60, and 80) [114] and brij (30, 35, 52, 58, 72, and 76) [115].

In a recent study [116], niosomes made by cationic lipid 1,2-di-octadecenyl-3-trimethylammonium propane, Squalene, and Tween 20 have been combined with GFP-encoding genetic materials consisting on minicircle (MC-GFP, 2.3 kb), its parental plasmid (pGFP, 3.5 kb), and a larger plasmid (pEGFP, 5.5 kb). Their e fficacy in retinal disease treatment has been investigated. These noisome-DNA complexes, named as nioplexes, demonstrated good transfection ability in human ARPE19 retinal pigment epithelium cells (in vitro) and in embrionary rat retinal primary cells (in vivo). Fluorescence showed GFP positive signal emission after transfection with the nioplexes carrying plasmidic DNA. In addition, such transfected cells showed physiological glial cell morphology confirming good cell viability in all tested cases. (Figure 5). The successive injections of nioplexes in rat retinas confirmed the higher capacity of cationic niosomes vectoring minicircle DNA to deliver genetic material into retina cells. Therefore, nioplexes based on cationic niosomes vectoring minicircle DNA are demonstrated as powerful agents in retinal diseases therapies. Cationic formulations made by a combination of Tween-60:DOTMA:lycopene have been used to transfect ARPE-19 cells with pCMS-EGFP plasmid with a transfection e fficiency close to 35% [117]. Also in case, in vivo experiments in rat eye showed the e fficiency of nioplexes to transfect outer segments of the retina, Niosomes constituted by Span 40 or Span 60 and cholesterol were produced to encapsulate and topically deliver in vivo acetazolamide. The intraocular pressure (IOP) after drug delivery system application was reduced in rabbits. In particular, multilamellar acetazolamide niosomes developed with Span 60 and cholesterol (7:4 molar ratio) were more e fficient in reducing IOP, and at same time, no irritation and retinal damage were observed [118]. Gugleva et al. [119] prepared niosomes by di fferent surfactants to deliver doxycycline hyclate in rats eye. Doxycycline hyclate is an antibiotic used for the treatment of eye infections. It was demonstrated to inhibit e ffect on matrix metalloprotease 9 (MMP-9) activity and mitogen activated protein kinase in experimental murine dry eye [120]. Niosomes were synthesized using several surfactants (Span 20, Span 60, Span 80, Tween 60) and cholesterol in di fferent molar ratios and tested on male albino rabbits. The experiments clearly showed that the prepared niosomal formulations were high tolerated and did not induce any irritations.

**Figure 5.** Fluorescence immonostaining by GFP expression in embrionary rat retinal primary cells. The green signal was referred to the transfection event of DST20 nioplexes with MC-GFP, (**B**) pGFP 3.5 kb, or ( **C**) pEGFP 5.5 kb. ( **D**–**F**) Positive controls incubated with Lipofectamine ™ 2000. Nuclei were stained with Hoechst 33,342 (blue) and neuronal dendrites with MAP2 (red). Scale bars: 20 μm. Reprinted from [116] Copyright (2019), with permission from Elsevier.
