*5.3. Hydrogels-Based Nanocarriers*

Hydrogels are three-dimensional, cross-linked networks of natural or synthetic water-soluble polymers that are suitable for applications like tissue engineering and controlled/sustained drugs delivery [121,122]. The aqueous environment is particularly advantageous to confine soluble active molecules, oligonucleotides, and proteins. For this reason, they are powerful tools in retinal treatment disease [123]. In addition, smart or environment-responsive hydrogels were synthetized to improve drug delivery e fficacy [124].

### 5.3.1. Natural Hydrogels: Chitosan NPs (CHNPs)

Chitosan is a natural hydrogel cationic polysaccharide of co-polymers glucosamine and N-acetyl glucosamine [125] obtained by alkaline deacetylation of chitin that is constitutive of crustacean shells of shrimps, lobster, and crab. It presents a positive charge that can be exploited to form polyelectrolyte vesicular complexes able to act as delivery systems. The positive charged chitosan is also ideal to bind a negatively charged corneal surface, to improve precorneal retention time and to decreases clearance [126]. In addition to chitosan, also the biocompatible alginate is extensively employed in biomedical applications. Alginate can also be a useful biopolymers for designing responsive hydrogels [127]. Because of its characteristics, alginate is also used for ophthalmic applications often in combination with chitosan [128]. For example, chitosan coated sodium alginate NPs [129] loaded with Fluorouracil (5-FU) have been successfully tested for eye delivery demonstrating a high 5-FU release in aqueous humor compared to free drug in rabbit eye. In another approach, chitosan NPs were encapsulated with levofloxacin for the treatment of ocular infection [130]. The formulation of NPs were optimized to enhance corneal retention without inducing eye irritation. These nanovectors demonstrated strong antibacterial agents against *Pseudomonas aeruginosa* and *Stafiloccocus aureus*.

Andrei and co-workers [131] designed biocompatible chitosan and alginate-based micro/NPs with a specific morphology aiming to deliver cefuroxim, a second-generation cephalosporin antibiotic. The authors performed an in vivo assessment to evaluate the biodistribution following an intravitreal administration of fluorescein-chitosan NPs on wistar rats. They observed an e fficient NPs distribution in lens and retina. Similarly, chitosan-dextran NPs have been employed to deliver the antibiotic moxifloxacin that has been retained for a long time in vivo thanks to the mucoadhesive properties of NPs [132].

In recent work, humanin, a neuronal peptide that mediated inflammatory responses, was used in the treatment of age-related macular degeneration (AMD) [133]. This peptide was a suppressor of the IL-6 cytokine receptors and protects neuronal cells from necrosis [134]. The authors studied the e ffect on the inflammatory response reduction using chitosan NPs as vector vesicle (with a size of 346 ± 52 nm) in which humanin (a micropeptide encoded in the mitochondrial genome) derivative AGA-(CR8) HNG17 (AGA-HNG) were encapsulated. The e ffects of NPs were assessed in retinal pigment epithelial cells. The percentage release profile of free protein after five days was 60%, after which it enriched a plateau whereas the nanostructured exhibited a slow and substantial release with 80% of protein released after fifteen days. Cheng et al. [135] developed a thermosensitive hydrogel containing latanoprost and curcumin-loaded NPs (CUR-NPs). Curcumin was selected because of its antioxidative and antiinflammatory properties that made it suitable also for treatment of eye pathologies such as chronic anterior uveitis and light-induced retinal degeneration [136]. Latanoprost was chosen because it acted on IOP restore due to uveoscleral outflow increase [137]. In glaucoma disease, IOP is generated by loss of aqueous humor drainage through the uveoscleral or trabecular outflow pathway that can be damaged by oxidative stress at trabecular meshwork level [138]. In this condition, Latanoprost and CUR-NPs were demonstrated to decrease the oxidative stress in trabecular meshwork (TM) cells that resulted in decreased levels of apoptosis, inflammation-related gene expression, and mitochondrial damage [135]. In another work [139], neuroprotective and neuro regenerative agen<sup>t</sup> Erythropoietin (EPO) was encapsulated in chitosan/hyaluronic acid (HA) nanoparticulate (size ≤300 nm). Di fferent formulations have been tested and delivered to porcine corneas, scleras, and conjunctivas by ex vivo permeation, finding out a good combination between targeting e fficiency and biocompatibility.

Pandit et al. [140] encapsulated high levels of bevacizumab (an anti-angiogenesis drug) within chitosan-coated PLGA NPs (size 222.28 ± 7.45) and demonstrated its e fficient deliver to the retina followed by e fficient release. As matter of fact, CS-coated NPs showed better permeability of the bevacizumab across sclera as compared to the free drug solution. Another category of carrier is made by glycol chitosan (GCS) NPs [141]. These biocompatible NPs, demonstrated to load high amount of plasmid DNA, have been successfully delivered to retinal pigment epithelium of adult wild-type albino mice. More importantly, the encapsulated pDNA was intact and maintained a proper conformation.
