In recent years, vision-related problems have acquired a greater relevance due to the ageing of the world’s population, which leads to an increase in visual problems, such as cataracts, glaucoma, age-related macular degeneration and diabetic retinopathy, occurring more frequently among over-60s [1,2]^[1][2]. Many visual diseases are associated with neurodegenerative disorders [3,4]^[3][4]. Young people over the age of 18 also suffer from visual problems, which increase especially with the growing use of electronic devices ^[5]. The rising number of people with vision impairment leads to a greater interest in dedicated care and treatments. This situation increases the costs in the global economy destined for the care of these disorders ^[6]. In addition, ocular therapy is a serious challenge because of the difficulty in targeting a drug to the appropriate ocular tissues.

In this landscape, technological research is actively involved, with the aim of developing innovative systems for targeted drug delivery ^[7]. The eye is a very complex structure, both anatomically and physiologically, and the treatment of pathologies affecting this organ is therefore not simple [8,9,10]^[8][9][10]. This is related to the various aspects that limit the transportation of drugs to the target site: anatomical barriers, physiological processes, mechanisms and metabolic aspects [11,12]^[11][12]. Reaching the target becomes more complicated if therapy is addressed to the posterior segment of the eye [13,14,15,16]^{[13][14][15][16]}. For this purpose, the major administration route remains intravitreal injection, which is invasive and produces undesirable effects such as pain and discomfort, inducing patient noncompliance [17,18]^[17][18]. The preferred route of administration would undoubtedly be the topical one, but conventionally it is used to treat diseases of the anterior eye. In fact, it is estimated that only a very small percentage of the drug instilled in the eye surface reaches the anterior chamber (around 5%) and even less in the posterior segment [19,20,21]^[19][20][21].

Nanotechnology represents a field of recent interest to overcome these issues. One potential strategy for improving drug delivery to the different eye tissues uses nanocarriers with specific size and surface properties, designed to ensure successful achievement of the drug to the target tissue, as well as the potential for a controlled release of the loaded drug, reducing the frequency of treatment and improving the retention time on the corneal surface [22,23,24]^[22][23][24]. Currently, the most widely studied nanosystems are used in the treatment of anterior eye diseases such as cataracts ^[25], glaucoma ^[26], dry eye syndrome ^[27], keratitis ^[28], conjunctivitis ^[29] and uveitis ^[30], but also posterior eye diseases such as retinitis ^[31], macular degeneration ^[32], endophthalmitis ^[33] and ocular tumors ^[34].

One possible strategy is to follow the nanosystem movements using a fluorescent probe. Fluorescence is a simple and non-invasive way to track the drug through the eye tissues, and it is also widely used in diagnostics to visualize diseased tissues, lesions and pathological markers. The development of personalized medicine and the need for early intervention in the diagnosis and treatment of specific diseases have promoted the birth and development of a new discipline: theranostics [37]^[35]. It can be defined as the combination of diagnostics with a specific therapeutic treatment. In vitro diagnostics and prognostics, in vivo molecular imaging, molecular therapeutics, image-guided therapy, biosensors, nanobiosensors and bioelectronics, system biology and translational medicine and point-of-care are some recent application examples.

2. Fluorescent Nanosystems in Ocular Application

Among the most investigated fluorescent nanosystems, there are lipid-based nanocarriers—such as nanostructured lipid carriers (NLCs) and solid lipid nanoparticles (SLNs), polymeric nanoparticles and nanocapsules, hybrid nanoparticles, cubosomes, emulsomes, nanoemulsions, niosomes, liposomes, films, nanomicelles and hydrogels. Fluorescence is introduced through the methods commonly used to prepare nanosystems [97,98]^[36][37]. The fluorescent nanosystems are essentially divided into (i) probe-loaded, in which the dye or probe is encapsulated into the system mostly during the formulation processes, and (ii) labeled/grafted, in which the probe is covalently bound to the surface of the nanosystem (often linked to some matrix component, such as polymers or lipids), always forming an adduct. As above cited, the tissues that compose the eye are many and with different properties. The difficulty for a nanosystem to reach the target tissue is high; thus, the profile of drug delivery is not always predictable. When the target is located in deeper ocular tissues, it is even more difficult to predict the ideal pathways followed by the carriers in vivo and through the ocular barriers. Tracking the drug after topical administration is important for several factors. Firstly, it allows for assessment of the effective achievement of the target site in order to accomplish the desired therapeutic action. Another factor to consider is the non-productive distribution of the drug in non-desired tissues, which could lead to the possible occurrence of side effects in addition to reducing the effective drug concentration. Furthermore, studying the pathways followed by the nanosystems is necessary to avoid issues related to barriers, tight junctions and physiological phenomena (tear flow and blinking), which could impair the routes. Size, surface charge and morphology of the nanocarriers have a great influence on their biodistribution, clearance and cellular uptake [99,100,101,102]^{[38][39][40][41]}. Before performing biodistribution studies, it is important to characterize the system and to proceed with in vitro and in vivo assays. For instance, mean size measurement, zeta potential, mucoadhesion studies and morphological analyses are, of course, also required to make the system as conformable as possible to a correct drug release. Tracking of nanosystems can be carried out in two ways, invasive and non-invasive; bioimaging using fluorescent molecules is a non-invasive method [103,104]^[42][43].

2.1.1. Fluorescent Lipid-Based Nanosystems

Lipid systems are of great interest for drug delivery in ocular tissues; their biocompatible and biodegradable composition makes them technologically safe, while their lipidic nature and structural characteristics allow them to pass through the corneal layers and achieve an efficient drug dosage even in the deepest tissues of the eye. The distribution of these systems occurs mainly in lipophilic layers, with minimal involvement of the stroma, since it has hydrophilic nature, and the lipid systems are difficult to distribute there. This was confirmed in the work of Namprem et al., in which confocal scanning microfluorometer (CSMF) analysis confirmed poor penetration of NLC labeled with Nile Red in hydrophilic compartments such as the stroma compared to corneal layers [105]^[44]. Due to eye barriers and obstacles to ocular administration, understanding the path taken by the designed nanosystem is necessary, especially if it is targeted to the back of the eye. The main route through which lipid systems reach the deeper tissues is the transcorneal one. There is growing evidence that successful drug delivery by functionalized nanocarriers depends largely on their efficient intra/paracellular transport, a process that is not fully understood yet. Therefore, the development of new imaging and diagnostic techniques is very important, particularly in a complex biological system such as the eye. Due to its lipophilic nature, one of the most used dyes for the preparation of fluorescent-lipid nanosystems is Nile Red (NR) Cubosomes labeled with Nile Red were prepared in the work of El Gendy et al. to assess the role of nine different lipids as penetration enhancers. The type of lipid used in the preparation plays an important role in tissue distribution. Among the prepared lipid systems, fluorescence analysis showed that the combination of oleic acid, Captex^® 8000 and Capmul^® MCM improved the penetration of the systems into the mucosa by increasing diffusivity due to both surfactant properties and the ability to disrupt the organization of the lipid bilayer [106]^[45]. Once again, Nile Red was used in the work of Kapadia et al. in order to visualize drug-loaded emulsomes. For the physico-chemical characterization and subsequent analyses, the nanosystems were loaded with triamcinolone acetonide, while for the studies of precorneal retention and ocular distribution, the fluorescent dye was loaded instead of the drug. The restudyearch revealed that after topical administration, the pathways taken to reach the back of the eye were basically three: corneal (via the iris and aqueous humor), conjunctival and systemic. The drug may diffuse through the sclera by lateral diffusion, followed by penetration of Bruch’s membrane and retinal pigment epithelium (RPE). To a lesser extent, the drug may be absorbed into the systemic circulation either through the conjunctival vessels and the nasolacrimal duct, and gain systemic access to the retinal vessel [107]^[46]. Another lipophilic DiI dye (1,1-dioctadecyl-3,3,3 tetramethyl indocarbocyanine perchlorate) was used to label lipid nanocapsules (LNCs) fluorescently. An important finding was made in the study by Eldesouky et al., where, despite the lipophilic nature of the dye, better penetration was achieved by encapsulation in lipid systems compared to simple dispersion. Fluorescence analysis showed that, without the use of lipid nanocarriers, the dye is unable to cross the hydrophobic corneal layer [108]^[47]. Mucoadhesion plays a key role in the enhancement of bioavailability. Efforts are made to design systems that have the ability to improve retention on the ocular surface. In this respect, the use of chitosan to improve the delivery of drugs into the eye tissues for its properties as a mucoadhesive agent, controlled drug release and permeation enhancer is interesting ^[24]. It is used in conjugation with a drug, such as in the study of Dubashynskaya et al., to improve the intravitreal delivery of dexamethasone [109]^[48]. In the major cases, it was used as a coating of nanocarriers to promote intraocular penetration, as reported by which designed modified NLCs with three different types of chitosan: chitosan acetyl-L-cysteine (CS-NAC), chitosan oligosaccharides (COS) and carboxymethyl chitosan (CMCS). The distribution profile was evaluated by loading the hydrophobic dye C6 into the NLCs. It was revealed through CLSM analysis that only NLCs modified with COS and CS-NAC were able to pass the cornea through the opening of tight junctions between epithelial cells [110]^[49]. Rhodamine-labeled NLCs were used to assess the corneal retention of such lipid nanocarriers, modified with a complex containing boronic acid, which is able to bind with high affinity the sialic acids of mucin. The NLCs were loaded with dexamethasone and designed for the treatment of dry eye syndrome. Fluorescence marking revealed the increased retention time due to the mucoadhesive property of the nanosystem, which also proved to be a potential not irritant treatment for dry eye syndrome [111]^[50]. Another key factor that improves retention time on the ocular surface is the positive charge of nanosystems interacting with the negative charges of mucin. The addition of octa-arginine (R8) to the nanoemulsions prepared by Liu et al. imparted a positive charge to the system with the aim of increasing eye retention. Once again, C6 was used to label lipid emulsions of disulfiram. In particular, the permeation of these systems under the influence of particle size and the presence of R8 was investigated and revealed that the addition of R8 and a size of ~50 nm improved the ocular delivery performance of nanosystems. In addition, the restudyearch showed that C6 passed through the corneal epithelium mainly by paracellular pathways, but there was also a fluorescent signal in the cytoplasm, indicating a transport also by transcellular pathways [112]^[51]. The internalization of lipid nanoparticles occurs mainly through an endocytosis mechanism. This is in fact the route taken by the mRNA-based solid lipid nanoparticles prepared by Gómez-Aguado et al. The SLN were developed in order to produce IL-10 to treat corneal inflammation and was loaded with Nile Red to assess cellular uptake in corneal epithelial cells (HCE-2 cells). This platform could also be used as a theranostic model as GFP (green fluorescent protein) is produced inside the cells, so the intensity of the fluorescence is indicative of the amount of protein produced. Since GFP, once produced, remains at the intracellular level, instillation on the ocular surface of mice of the samples permitted the identification of the corneal layers where transfection occurred. All the prepared mRNA-based SLN formulations showed higher fluorescence intensity than naked mRNA, demonstrating the enhancement of their targeting ability [113]^[52]. Fluorescein is one of the most widely used fluorescent dyes for drug tracking and visualization of ocular damage following treatment. Fluorescein was used by Jounaki et al. for tracking vancomycin loaded NLCs. The aim of the work is the idea that NLCs for topical use could be a valid substitute of intravitreal injection in the treatment of bacterial endophthalmitis caused especially by Staphylococcus. Both drug-loaded and fluorescein-loaded NLCs (0.2 mg/mL) were prepared by cold homogenization technique and were used to evaluate precorneal retention with an inverted fluorescent microscope. The increased fluorescence found in the corneal epithelium demonstrated that dye-loaded, stearylamine-coated NLCs were retained more in the ocular surface. Indeed, the cationic lipid stearylamine is trapped in the mucin layer and retained due to the interaction between the fillers, facilitating the penetration and delivery of the drug to the intraocular tissues [101]^[40]. In the work of Kakkar et al., fluorescein was also used in concentrations almost like the previous work (0.25 mg/mL) to track hybrid nanoparticles. Solid lipid nanoparticles were prepared and then coated with PEG in order to encapsulate the antimycotic fluconazole. Analysis to assess the penetration into the ocular internal layers revealed that fluorescence was observed in the vitreous humor, retina, sclera and choroid after instillation of a single drop of Fluconazole-SLNs into the rat eye. In addition, the ex vivo study showed that the system exhibited a 164.64% higher flux through the porcine cornea when compared to the commercial drops ZoconVR [114]^[53]. In addition to coating the nanosystems, fluorescein was used to label them binding it covalently to the material of the nanosystem. In the work of Puglia et al. [66]^[54] an adduct is prepared between fluorescein and stearic acid named ODAF (N-(30,60-dihydroxy-3-oxospiro[isobenzofuran-1(3H),90-[9H]xanthen]-5-yl]-octadecanamide). In this case, the dye was grafted (and not loaded) and the conjugation of the lipid with the dye leads to a fluorescent probe. Solvent-diffusion technique was used to prepare SLNs of about 120 nm. The in vivo distribution from 1 h to 16 h was evaluated in rabbits and the results showed that, after ocular instillation, ODAF SLNs were mostly located in the cornea (up to 2 h), whereas over a longer time (from the second hour to the eighth hour) the fluorescent signal gradually extended toward the back of the eye, confirming the ability of controlled delivery by the lipid nanosystems [66]^[54]. Considering that the influence of blinking and tearing on ocular drug absorption was rarely evaluated in studies, Pretor et al. evaluated absorption of two lipid-based formulations, a liposome and a SLN, in presence of these two physiological conditions. The SLNs were also labeled with a fluorescent phospholipid, thus constituting another example of a grafted nanosystem. From the restudyearch, using C6 as the fluorescent compound, it is evaluated that liposomes are shown to provide a greater absorption, despite the influence of blinking (shear stress of 0.1 Pa.) and tear flow. This interesting study was carried out by coupling the use of microfluidics with channels and cultured HCE-T cells as well as the use of a fluorescent dye to simulate the physiological mechanisms; it could be useful to add this kind of assay to the basic characterization of the nanosystem addressed to ocular targets [115]^[55]. In the rhodamine family, Rhodamine B is widely available and low-cost. The following two studies promote the use of this molecule for tracking nanosystems. The first is focused on the preparation of lipid systems (niosomes vesicles) and Eudragit nanoparticles for the treatment of eye fungal infections. Encapsulation of fluconazole within these systems resulted in being a good way to increase the bioavailability of the drug compared to free drugs. The systems obtained were innovative in terms of formulation as there is a triple step: the drug was first complexed using β-cyclodextrin, then encapsulated into niosomes, and the niosomes were finally incorporated into an in-situ gelling system made by Poloxamer, HPMC and chitosan. Niosomes were labeled with Rhodamine B and then were compared to labeled polymeric nanoparticles. The fluorescent signal of CLSM analysis increased in intensity when the NPs were incorporated into the hydrogel, whereas the signal of the pure dye was limited to the superficial epithelial layers, suggesting effective permeation of the nanosystems into the inner tissues [116]^[56]. Rhodamine B was also used to study the transport of curcumin as a model drug in multilamellar liposomes. These were coated with sodium alginate grafted acrylic acid conjugated with riboflavin. These multi-dye vesicles (rhodamine and curcumin), prepared using the lipid film hydration technique, have proven to be excellent carriers for drug delivery to the retina.

2.1.2. Polymer-Based Nanocarriers

Topical delivery of polymeric nanosystems is useful to improve corneal penetration and prolong the therapeutic response of several drugs. Nanocarriers need to be evaluated to find clinical application; specifically, their distribution in biological environment should be examined in order to understand the most appropriate strategy to address specific ocular pathologies. Plausible routes of topically instilled drug delivery for the treatment of ocular diseases involving the posterior segment include several pathways, including corneal, non-corneal and uveal routes. Successful nanocarrier development, therefore, involves fluorescent labeling useful for investigating mechanisms and biodistribution profiles of the designed systems. Polymeric nanostructures to be used as imaging diagnostic agents include various kinds of systems, such as nanoparticles, niosomes, film and nanomicelles and in-situ gel. The review of Swetledge et al. offers a detailed discussion on the biodistribution of polymer nanoparticles in major ocular tissues [118]^[57]. To improve retention time on the ocular surface, release profile and mucoadhesion performance, nanocarriers are often coated with polymers. Poly-lactide (PLA), polyglycolide (PGA), poly-lactide-co-glycolide (PLGA) and chitosan, Eudragit^®, but also different copolymers such as PLGA-PEG, poly-(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) constituted by hydroxybutyrate (HB) and hydroxyvalerate (HV) and chitosan modified copolymer are some of these. Among them, many polysaccharides are used as a useful coating for nanocarriers. Some of these, including chitosan, alginate sodium, hyaluronate sodium and cellulose derivatives, are approved for ophthalmic use by the FDA and are already present in the composition of ophthalmic products on the market [119]^[58]. Depending on the type of polymer, the most suitable fluorescent probe should be chosen. A study conducted by Zhukova et al. focused on understanding the interactions between probes, polymeric nanoparticles and the biological environment. Four dyes with different degrees of hydrophobicity were encapsulated (C6, rhodamine 123, DiI) or covalently bound to the polymer (amine Cyanin 5.5, Cy5.5) in order to label PLGA nanoparticles. To increase the accuracy of the interpretation of in vivo biodistribution data, dual-labeled nanoparticles were administered, using C6 as the encapsulated label and Cy5.5 as the grafted label. Neuroimaging results showed that the signal of the nanoparticles bounded with Cy5.5 was detected in retinal vessels, whereas the signal of the encapsulated C6 was found outside of blood vessels and in tissue background. The extra vasal distribution of C6 could falsify the data interpretation, leading to erroneous assumptions that the nanoparticles could efficiently cross the blood-retinal barrier. Assessing the affinity of the dye to the polymer and the lipophilic structures could be useful in scaling up these issues. Although C6 has not proved to be an ideal label, it aided in explaining the phenomenon whereby drugs are delivered to tissues through encapsulation in nanocarriers without involving any nanoparticle penetration [120]^[59]. Similar results were obtained by Zhang et al. tracking in vivo the distribution of PLGA-NPs in the retinal blood circulation after intravenous injection. NPs were labeled with lipophilic perchlorate carbocyanins (DiI) or hydrophilic rhodamine 123 (Rho123). DiI fluorescent signal was detected for a long time (>90 min) in retinal vessels, in contrast with Rho123 whose fluorescence was short (>15 min), due to diffusion from particles and elimination from the blood circulation. To avoid artefacts, dual-labeled nanoparticles were also injected intravenously in rats. Colocalization of fluorescent markers was performed by conjugating the polymer with Cy5.5 and loading the systems with probes (DiI/Rho 123). Cy5.5 signal was detected for both cargoes in retinal vessels for more than 90 min; however, colocalization was observed only for lipophilic DiI dye, which was more closely related to the hydrophobic polymer matrix. These findings further confirm that the affinity of the dye for the polymer and cell membranes played a key role in biodistribution kinetics [121]^[60]. The hydrophilic properties of rhodamine B make it a suitable fluorescent candidate for polymers of a hydrophilic nature such as chitosan, whose mucoadhesive qualities have been exploited by X et al. for the design of topical films for the treatment of glaucoma. Corneal permeation studies demonstrated the mucoadhesive efficacy of polymeric films in transporting rhodamine B molecules through the cornea with a high permeation rate [122]^[61]. A water-insoluble derivative of the rhodamine family is rhodamine B isothiocyanate, which has affinity for hydrophobic polymers. This dye was used as a label for nanoparticles consisting of hydrophobic PHBV polymer to obtain information regarding the depth and rate of penetration after topical administration. Confocal analysis showed improved penetration deepness of encapsulated marker compared to the free one, used as a control [123]^[62]. Recently, hydrophobic C6 was doubly used as a model drug and a fluorescent marker to track surface-modified PLGA-NPs with chitosan, glycol chitosan and polysorbate 80 in retinal tissues. Tracking of NPs after topical instillation was performed by fluorescence microscopy, revealing intense staining throughout the whole eyeball, anterior segment including cornea and conjunctiva, lens, iris/ciliary body and retina, with a peak at 30 min after administration and the disappearance of the signal after 60 min. Ocular tissue autofluorescence was distinct around the outer segments of the photoreceptor. Based on the average size of the NPs (<200 nm), the specific pathway of the NPs to the retina did not exclude any of the plausible routes of delivery to the posterior segment (corneal, noncorneal or uveal pathways) [124]^[63]. C6 was also used to label polymeric nanomicelles designed for the topical treatment of fungal keratitis. The nanomicelles consisted of a chitosan oligosaccharide-vitamin E copolymer conjugated to phenylboronic acid (PBA-CS-VE) to enhance corneal retention. C6 delivery through a monolayer of HCE-T cells and 3D cell spheroids demonstrated strong corneal penetration ability. Several characteristics of the polymer were able to influence nanomicelle uptake, but the key role in the process of cellular endocytosis was attributed to the high-affinity interaction between the PBA portion and sialic acid on the surface of the cell membrane [125]^[64]. Another study using C6 as a fluorescent probe was reported by Sai et al., aiming to evaluate the corneal transportation of an in-situ gelling system based on mixed micelles. This formulation designed for curcumin was composed of micelles, consisting of 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-2000] (PEG-DSPE) and poly(oxyethylene) esters of 12-hydroxystearic acid (Solutol HS 15), incorporated in a gellan gum gel. Incubation of human corneal epithelial cells (HCEC) with the fluorescently labeled systems showed time-dependent and improved absorption for the encapsulated dye, compared to free C6. Transcorneal penetration was investigated in vivo by CLSM and results suggested that curcumin was able to penetrate more effectively when incorporated into the gelled systems, probably due to the increased retention time conferred by the gellan gum, which was five-fold higher than the mixed micelles alone [126]^[65]. A pilot study with C6 was performed to evaluate the feasibility of the approach in assessing the biodistribution of PLGA-PEG nanoparticles suspended in hydrogels. The preliminary study showed an important limitation due to the high green autofluorescence of the examined ocular tissues. To deal with the drawbacks highlighted by the pilot study, PLGA nanoparticles in the full study were labeled with Cy-5, a far-red fluorophore that did not overlap with the natural autofluorescence of the ocular tissues.

2.1.3. Metallic-Based and Inorganic-Based Nanosystems

Inorganic nanodevices became of great interest in ocular delivery due to their unique properties such as low cost, easy preparation methods, small size, tuneable porosity, high surface-volume and robust stability. Fluorescent labeling has been applied to these delivery systems to assess their ability to cross ocular barriers and provide therapeutic efficacy [128]^[66]. Corneal barrier functions were investigated by Mun et al. using two types of silica nanoparticles (thiolate and PEGylated) fluorescently labeled with 5-(iodoacetamido)-fluorescein (5-IAF). Permeation studies were performed in vitro on intact or β-cyclodextrin pretreated bovine corneas. To provide experimental parameters close to in vivo conditions and to avoid artifacts such as the potential risk of corneal swelling when using Franz diffusion cells, the “whole-eye” method was used. 5-IAF-loaded thiolate silica nanoparticles, PEG-grafted silica nanoparticles (5-IAF-PEG), sodium fluorescein and fluorescein isothiocyanate dextran solutions were tested. It resulted that fluorescein salt (376 Da) did not uniformly penetrate the cornea; however, the dye was detected in the stroma. Larger molecules such as FITC-dextran (400 Da) and 5-IAF-PEG formed a layer on the corneal surface with no permeation of the epithelial membrane. Β-cyclodextrin pre-treatment disrupted the integrity of the cornea by providing homogeneous permeation of the low-molecular-weight dye, although it did not improve the penetration of larger molecules. Concerning NPs, no permeation was reported regardless of surface modification, particle size and pre-treatment with β-cyclodextrin, thus suggesting that the tight junctions of the corneal epithelium acted as the main barrier to permeation. The absence of penetration and confinement on the corneal surface was observed for thiolated NPs because of the formation of disulfide bonds between the NPs thiol groups and the cysteine domains of the mucus glycoprotein layer. The interaction between mucin and -SH thiol groups remained a limiting permeation factor even after the removal of the epithelial layer. NPs PEGylation was able to mask thiol groups, allowing passage into the stroma [129]^[67]. Baran-Rachwalska et al. designed a novel platform consisting of hybrid silicon-lipid nanoparticles, aiming to deliver siRNA to the cornea by topical administration. A fluorescent oligonucleotide duplex, siRNA transfection indicator (siGLO), was employed as a tracking probe to assess in vitro cellular uptake on a human corneal epithelial cell line (HCE-S) and in vivo corneal penetration on wild-type mice. Red fluorescence of the oligonucleotide marker allowed detection of nanoparticles in all layers of the cornea 3 h after instillation, in contrast to the control siGLO. The tracking of biodegradable nanosystems in corneal tissues was confirmed by the reduction of protein expression in the corneal epithelium, making them ideal candidates for therapeutic oligonucleotide delivery [130]^[68]. Biodegradable mesoporous silica nanoparticles (MSNs) loaded with carboplatin were designed by Qu et al. for the treatment of retinoblastoma. Carboplatin, being an anticancer drug, causes severe side effects; therefore, it is necessary to focus the action strictly on the target site. For this purpose, MSNs were surface modified by conjugation with an ideal target, epithelial cell adhesion molecule (EpCAM), in order to increase specificity as well as therapeutic efficacy. To assess the targeting efficacy of the designed systems, the reseauthorchers evaluated the cellular uptake of untargeted and targeted MSNs in retinoblastoma Y79 tumor cells. Rhodamine B and Lysotracker Green were used as fluorescent probes to track cellular and subcellular uptake of the vectors. Increased cellular uptake for targeted MSNs was attributed to EpCAM-specific receptor-mediated cellular internalization. Lysosomal localization of MSNs confirmed that the nanosystems followed the endocytosis pathway for drug delivery [131]^[69]. A hexa-histidine with metal ions nanosystem was designed to deliver Avastin in the treatment of corneal neovascularization (CNV). Pre-corneal retention time and ability to cross ocular barriers were studied on a rat CNV model induced by alkaline burns by FITC labeling the systems. Avastin encapsulated in the vectors showed a longer precorneal adhesion time compared to the free drug. These innovative systems have emerged as a promising platform for ocular topical delivery of protein drugs [132]^[70]. An interesting zirconium-porphyrin metal-organic framework (NPMOF) has been designed for drug tracking and delivery. The bright fluorescence self-emitted by the metal-organic framework qualifies the carriers to be applied for imaging. NPMOF was used as a skeleton for the delivery of methylprednisolone, a very efficacious corticosteroid in the treatment of retinal degenerative diseases. Adult zebrafish with photoreceptor degeneration induced by high-intensity light exposure were used to test in vivo distribution and therapeutic efficacy. Red fluorescence signals were detected in choroid, retina, photoreceptors and retinal pigment epithelium for up to 7 days. Recovery of visual function by rapid regeneration of photoreceptors and proliferation of Müller’s glia and retinal regeneration were reached after a single intravitreal injection. NPMOF vectors represent a novel delivery system for the treatment of diseases affecting the posterior eye segment [133]^[71].

2.1.4. Protein-Based Nanosystems

Protein-based nanosystems have attracted considerable interest in recent years and are designed for drug delivery, diagnostics and bioimaging. These highly bio-compatible systems, which have been extensively studied in the biomedical field, owe their properties to the protein they are composed of. Among the proteins used in their preparation, there are antibodies, enzymes, animal and plant proteins, collagen, plasma proteins, gelatin and proteins derived from virus capsids [134]^[72]. Fluorescent proteins are usually used to monitor protein-protein interactions, protein localization and gene expression. However, without any carrier, the fluorescent efficiency of a single protein is relatively low. The use of fluorescent protein-labeled nanomaterials improves loading due to increased surface area and allows the development of fluorescent nanosystems useful in bioimaging and biosensing. In the study carried out by Yang et al., nanoparticles were prepared from regenerated silk fibroin. This protein, which is the most abundant in silk, is considered to have high biocompatibility and degradability properties. In the biomedical field, it has been used for drug delivery in small nanosystems, biological drug delivery, gene therapy, wound healing and bone regeneration. The formulation is targeted for intravitreal injection with the aim of increasing the bioavailability of the drug in the retina. Fluorescein isothiocyanate labeled bovine serum albumin (FITC-BSA) has been encapsulated as a model drug. In vitro cytotoxicity studies were conducted on ARPE-19 cells, showing that these nanosystems were very compatible. In addition, in vivo comparison of the biodistribution in posterior ocular tissues in rabbits revealed increased retention in the retina due to encapsulation in the nanosystem rather than with a solution of model drug [135,136]^[73][74]. Albumin is widely used in the preparation of ocular nanosystems [137]^[75].

2.2. Diagnostics

Labeling nanoparticles with fluorescent probes was demonstrated to be a useful approach to improve the effectiveness of some diagnostic tests aimed to detect ocular pathologies. In fact, some eye diseases require a prompt diagnosis in order to contain possible damages related to the ongoing pathways involved. Age-related macular degeneration (AMD) is the main cause of vision loss for over-65-year-olds [37]^[35]; this pathology has often been analyzed to improve diagnostic techniques since it has several predisposing factors, and early detection is crucial to avoid degeneration toward blindness [140]^[76]. AMD has an unclear etiology, although oxidative stress is considered one of the main risk factors [141]^[77]; as a matter of fact, clinical studies demonstrated the importance of supplementation with antioxidants in order to slow down the progression of AMD [142,143]^[78][79]. Physiological antioxidant patterns involve metallothioneins (MT), low molecular mass proteins characterized by the presence of cysteine sulfur ligands, which are able to scavenge free radicals, thus protecting cells and tissues. The retina is particularly subject to oxidative stress due to visible and UV light exposure; moreover, age progression involves a reduction of MT expression, predisposing to AMD [144]^[80]. For this reason, bioimaging these proteins in ocular tissues could be an important tool useful to highlight the tendency to develop AMD. For this purpose, fluorescent gold nanoclusters involving Cu and Zn and bioconjugated with specific primary antibodies were developed by Cruz-Alonso and coworkers [145]^[81]. Laser ablation (LA)-inductively coupled plasma (ICP)-mass spectrometry (MS) technique was used to identify ⁶³Cu⁺ and ⁶⁴Zn⁺ in the retina of post-mortem donors since MT bind both Cu and Zn [146]^[82]. This method showed results comparable with conventional immunohistochemistry for MT proteins, with amplification of signals related to the presence of nanoclusters, which allowed the obtainment of higher resolution bioimages. An in vivo model of human “wet” AMD is laser-induced choroidal neovascularization (mouse LCNV) mouse, in which the inflammatory biomarker vascular cell adhesion molecule-1 (VCAM-1) is highly expressed. Gold nanoparticles functionalized with anti-sense DNA complementary to VCAM-1 mRNA were developed by Uddin et al., who aimed to detect this molecule, thus assessing the occurrence of oxidative stress [147]^[83]. The fluorescence in-situ hybridization (FISH) technique was used to perform photothermal-optical coherence tomography (PT-OCT) involving a fluorescent probe (Alexafluor-647) bonded to 3′ end of anti-sense DNA in order to highlight its interaction with the target mRNA. The conjugation of anti-sense DNA to gold nanoparticles proved to protect from the degradation performed by DNase while enhancing the uptake, probably through endocytosis, as suggested by transmission electron microscopic (TEM) images of retinal cells; moreover, it was verified that no inference in the fluorescence was produced due to low pH, which is characteristic of inflamed tissues. Compared to the control group, in vivo systemic injection in mice confirmed the enhancement in the fluorescent signal for anti-sense DNA coupled with nanoparticles, which mostly depended on VCAM-1 mRNA hybridization, thus demonstrating the potentiality of the developed platform as a tool to obtain direct images of endogenous mRNA in a tissue. In some cases, this pathology requires transplantation of photoreceptor precursors (PRPs) in the subretinal space, which was successfully performed, guaranteeing a certain vision restoration [148]^[84]. For a certain period, monitoring of the efficiency of the transplantations needs to be performed. As confirmed by Chemla and coworkers [149]^[85], gold nanoparticles could be transplanted together with photoreceptor precursors cells labeled with a fluorescent probe (Alexa 594) in order to ameliorate the efficiency of computed tomography (CT) and optical coherence tomography (OCT) in assessing the success of the transplant. The nanoparticles were firstly characterized in order to assess their safety, thus demonstrating no toxicity toward the transplanted cells and no occurrence of inflammation in the retina and vitreous. Furthermore, this platform demonstrated to enhance X-ray signal detected by CT and related to cell survival without interference from the particles secreted from the cells [150]^[86]; moreover, they were also able to increase optical signal for OCT by up to 1.4-fold and track cells migration toward layers deeper than the injection site. These results confirm the efficiency of such a platform in the monitoring of transplantation but also suggest a potential use for ameliorating existent molecular imaging in cell therapy and diagnostic. Another important diagnostic test is fundus fluorescein angiography (FFA), which allows highlighting vascular leakages in retinal and choroidal pathologies [151]^[87]. This clinical tool is useful to diagnose several ocular diseases: age-related macular degeneration, which is characterized by hemorrhaging and exudation in the retina [140]^[76]; diabetic retinopathy, which involves retinal damages related to microvascular modification which are clinically not revealable in the early stages [152]^[88]; diabetic macular edema, whose pathophysiology implicates modifications of choroidal and retinal vasculature due to BRB impairment [153]^[89]. Furthermore, the aforementioned diseases are characterized by alterations of ocular vessels, and share the consequent compromission of visual activity, if not quickly detected and treated. Fluorescein sodium (FS) is injected intravenously to perform this analysis, diffusing in the blood vessels, thus allowing uresearchers to observe them through a confocal scanning laser ophthalmoscopy system. Despite it being considered relatively safe, nausea and vomiting frequently occur, while severe effects such as anaphylaxis are rare. The main drawbacks are the diffusion of FS into normal tissues and cellular absorption, with long retention, which were overcome using nanoparticles. Cai et al. coworkers developed a high molecular weight polyethyleneimine (PEI) nanoparticles which demonstrated to successfully couple fluorescein [151]^[87]; moreover, in vitro studies showed good cytocompatibility, no significant difference in apoptosis rates considering various concentration tested, no genotoxicity, and no morphological changes or significant difference in endothelial tube formation. Cellular uptake assays, carried on with different concentrations of free FS and FS-NP, confirmed similar rapid uptake by cells, with a concentration-dependent and time-dependent fluorescence of main retinal vessels and microvessels. Furthermore, free FS was longer retained in cells when compared to FS-NP, as highlighted by in vivo fluorescence studies, suggesting a potential decrease in FS toxicity. These results confirm the potentiality of this platform as a diagnostic tool to detect retinal vessels; moreover, PEI enhances fluorescein metabolism, thus reducing its toxicity. Other polymeric nanoparticles developed as a potential diagnostic tool are composed of copolymerized glycerol mono methacrylate (GMMA), glycidyl methacrylate (GME) and ethylene glycol dimethacrylate (EGDMA), which were functionalized with Vancomycin, Polymyxin B or Amphotericin B, in order to detect the presence of Gram-positive bacteria, Gram-negative bacteria and fungi through a specific bond with the respective antibiotic or antimycotic [154]^[90]. The occurrence of such bonds was differently highlighted using fluorescent Vancomycin, and probes such as fluorescein isothiocyanate (FITC) and Calcofluor White.

2.3. Nanotheranostics

The recent development of systems that integrate the treatment of diseases with their diagnostics is referred to as theranostics. When the system is in a nanoscale range, it is called nanotheranostics.