**2. Drug Ocular Administrations**

The delivery of drugs in the eyes presents many difficulties. As in the case of the brain, it presents multiple physical boundaries: corneal and conjunctival epithelium, blood-aqueous barriers (BAB), and blood-retinal barriers (BRB). These barriers finely control the transit of molecules and fluids across eye and, in most cases, the drug is unable to enrich the deeper layers. The BRB consists of two different barriers: (i) the outer BRB characterized by endothelial cells lining the choroidal vasculature (fenestrated blood vessels) and by tight-junction-coupled RPE cells, and (ii) inner BRB. The latter is made by endothelial cells in conjunction with pericytes, astrocytes, and Müller glial cells. The inner BRB shows a lot of similarities with blood–brain barrier (BBB) with the exception of Müller glia [18]. In general, the most common drug administration is through eye drops and suspensions' that target only the anterior segmen<sup>t</sup> of eye, mainly due to the patient compliance. With such a method, less than 5% of topically applied dose is delivered to deeper ocular tissues, such as in vitreous cavity [19]. The chemical nature of bioactive molecules also influences the uptake. For example, BRB is selectively permeable to lipophilic molecules in the presence of specific eye drugs transporters. In this way, the formulation of new therapies should take in consideration this limitation [20]. Ocular transporters are divided in two groups: the solute carrier (SLC) family and the ATP-binding cassette (ABC) family. The electrochemical gradient was used by SLC transporters to induce the uptake of molecules through cell membrane, whereas ABC transporters employ ATP [21]. However, physiological eye phenomena, such as tear turnover, reflex blinking, ocular static, or lacrimal drainage reduce the action of the drug and consequently the eye drop administration fails to treat

retina disease [22]. As a matter of fact, in unstimulated conditions, total aqueous tear volume is 7 μL and the normal tear turn-over is about 1.2 μL/min [23]. Thus, the precorneal drug half-life is about 3 minutes [24]. Therefore, treatment of posterior segmen<sup>t</sup> injuries still remains a challenge. For all the reasons explained above, medical research seeks alternative routes such as intravitreal injections. This method consists of direct administration into vitreus via *pars plana* using a needle. In this way, a high concentration of drug is guaranteed at the retina level, but the molecule half-life depends on its molecular weight. In fact, proteins and peptides characterized by high molecular weight (ranging from 40 kDa to 70 kDa) and steric hindrance showed longer retention [25,26]. However, a lot of complications (hemorrhages, retinal detachment, cataracts) can manifest after injection [27]. An alternative method is periocular route (Figure 2) that was demonstrated to be an effective route to direct drugs to the posterior eye segmen<sup>t</sup> consisting of subconjunctival, sub-tenon, retrobulbar, peribulbar, and posterior juxtascleral [28]. Administration of drug by sub-tenon injection was found to be more suitable as demonstrated by Ghate et al. [29], where sodium fluorescein was used in rabbits' eyes by subocular administration. The study concluded that injection of drug via sub-tenon resulted in highest vitreous concentration of sodium fluorescein (two sodium fluorescein (NaF) concentrations, 2.5 mg in 0.1 mL (c1) and 2.5 mg in 0.5 mL (c2)) compared to the other routes. However, also in this case, a lot of adverse effects have been observed, such as strabismus, hyphema, and intraocular pressure [30].

**Figure 2.** Common drug administration routes through the eye. Topical administration (**1**,**2**), subconjunctival injection (periocular route) (**3**), subretinal injection (**4**), and intravitreal injection (**5**). Adapted from reference [31].

### **3. Physico-Chemical Properties of Nanomaterials**

A "Nanomaterial" is defined as a "material with any external dimension in the nanoscale or having internal structure or surface structure in the nanoscale" (ISO, 2010) [32]. Similarly, a definition of "nanoparticle" is a "nano-object with all three external dimensions in the nanoscale" where nanoscale was defined as size ranging from 1 nm to 100 nm (ISO, 2008) [33]. NPs can be obtained by many synthetic

routes, starting from chemical elements such as carbon, metals, metal oxide, biological molecules, and polymers [34]. The biological e ffects are strongly a ffected by physico-chemical properties of NPs such as surface charge, size, shape, and solubility. In addition, they exhibit greater surface area per unit mass compared to bulk materials [35]. Because of the above mentioned properties and of the material they are made, NPs are ideal tools to treat retinal disease as active components of the therapy without the help of drugs [36,37]. In addition, NPs with a smaller size (<20 nm) are demonstrated to have the ability to cross eye barriers including cornea, conjunctiva, and BRB [38,39]. NPs for ocular therapy include inorganic NPs (metal oxide and noble metal NPs), as well as soft-biopolymer-based NPs. Metal NPs are more suitable as active therapy tools (for their intrinsic properties), and soft-NPs are more efficient in encapsulating drugs and macromolecules due to their ability to form aqueous-suspended vesicles having a hollow lumen. All these nanomaterials are di fferent in charge, shape, and size, but they are able to be internalized by cells to treat retinal disease [40]. Finally, recent applications of soft nanorobot (with size range in the molecular scale) have been studied. These structures have the capability to deliver active biomolecules in ocular sections due to their ability to make changes in a controlled and predictable manner to the environment following external stimuli [41].

### *3.1. Noble Metal NPs: The Case of Gold (Au) and Silver (Ag)*

Noble metals, especially AuNPs and AgNPs, are characterized by unique optical properties. For this reason they are used for a variety of applications owing to collective oscillations of conduction electrons coupled with incident light [42–45]. This phenomenon, known as Localized Surface Plasmon Resonance (LSPR) is strongly influenced by NPs' shape and the metal they are formed. The surface plasmon resonance bands of metal NPs can be tuned from visible to Near Infrared Region (NIR), which is a typical wavelength to penetrate and analyze biological tissues [46]. Even if AuNPs are the most studied in nanomedicine field due to their good chemical stability and well-controlled size/surface functionalization and biocompatibility, AgNPs have the advantage of possessing an antibacterial and antiangiogenesis e ffect [47,48].

### *3.2. Au and AgNPs: Safety Studies in In Vitro and In Vivo Retinal Models*
