# Corneal endothelial dysfunction treatments: Recent advances in non-invasive treatment strategies

Nataliia Gnyliukh, Rabah Boukherroub, Sabine Szunerits

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Nataliia Gnyliukh, Rabah Boukherroub, Sabine Szunerits. Corneal endothelial dysfunction treatments: Recent advances in non-invasive treatment strategies. Nano Today, 2025, 63, pp.102740. 10.1016/j.nantod.2025.102740. hal-05039974

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Submitted on 18 Apr 2025

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Distributed under a Creative Commons Attribution 4.0 International License
Review

Corneal endothelial dysfunction treatments: Recent advances in
non-invasive treatment strategies

Nataliia Gnyliukh<sup>a, *</sup> <sup>ID</sup>, Rabah Boukherroub<sup>b</sup>, Sabine Szunerits<sup>a,b,**</sup> <sup>ID</sup>

ARTICLE INFO

Keywords:

Corneal endothelium

Topical therapeutics

Non-invasive treatment

Human corneal endothelial disorders

Nanoparticles

A B S T R A C T

The corneal endothelium plays an essential role in the maintenance of a healthy vision. However, in contrast to
epithelial cells with high proliferation rates and a turnover lifespan of six to seven days that allows maintenance
of constant cell density and high control of cell adhesion, human corneal endothelial cells (HCECs) do not
normally proliferate. Therefore, loss of HCECs density over time leads to corneal transparency loss, and the
development of corneal edema, bullous keratopathy and other eye diseases. Developments for repairing HCECs
are continuously searched for and are evolving around possible topical drug applications as well as emerging
strategies such as CRISPR-Cas9 gene editing, antisense oligonucleotides (ASOs) and nanotechnological concepts.
Here, the state-of-the-art treatment strategies for corneal endothelial diseases (e.g. Fuch's endothelial corneal
dystrophy (FECD), corneal edema, bullous keratopathy) will be outlined with a special focus on where topical
applications have shown positive therapeutic outcomes.

The human eye: anatomy and barriers to drug delivery

The eye is a highly complex organ that perceives more than 90 % of
surrounding information [1]. The outermost part of the eye, the cornea
(Fig. 1A), is vital for protecting the eye from bacterial/viral infections
and maintaining its structural integrity by shedding. This protective
layer is also essential for creating a focused image via focusing light onto
the retina in the posterior part of the eye [2]. The list of abbreviations
used in this review is given in Table 1.

Corneal epithelium

The outermost layer of the cornea is composed of five to six corneal
epithelial cell layers, covered with a thin tear film layer, with an
approximate thickness of 50 µm (Fig. 1A). The two most anterior cell
layers are flat and contain tight junctions (TJs) of 1–2 nm in size that
regulate the passage of small molecules and ions through the para-
cellular pathway [2]. Beneath the flat corneal epithelial cell layer are
multi-layered wing cells. Composed of collagen IV, laminin, and fibro-
nectin, this membrane is important in maintaining the corneal

epithelium.

TJs are essential for preventing the free flow of fluids from tears, penetration of pathogens, and maintaining the structural integrity of the cornea [3]. They are composed of integral transmembrane proteins, which are connected to actin filaments through adaptor proteins like Zonula occludens-1 (ZO-1) [4]. ZO-1 forms a continuous seal between adjacent corneal epithelial cells, thereby regulating the paracellular pathway for molecules that can pass. This restriction majorly prevents topically applied drug absorption. The search for approaches that can disrupt reversibly these TJs and lead to time-limited increased permeability is a viable approach for overcoming these limitations for drug passage.

The epithelium layer is directly followed by Bowman's membrane (Fig. 1A). This acellular layer consists of randomly oriented primarily type I collagen fibers with a thickness of 8–12 µm and functions as a protective barrier to the subepithelial nerve plexus, contributing to epithelial innervation and sensory recovery [5]. Recent studies have shown that, although Bowman's membrane does not contribute directly to corneal functioning, it contributes to its wound healing process, especially to stromal healing [5,6].

<footnote><sup>a</sup> Laboratory for Life Sciences and Technology (LiST), Faculty of Medicine and Dentistry, Danube Private University (DPU), Viktor-Kaplan-Straße 2, Geb. E, EG Nord, Wiener Neustadt 2700, Austria</footnote>

<footnote><sup>b</sup> Uni. Lille, CNRS, Univ. Polytechnique, Hauts-de-France, UMR 8520 - IEMN, Lille F 59000, France</footnote>

<footnote>* Corresponding author.</footnote>

<footnote>** Corresponding author at: Laboratory for Life Sciences and Technology (LiST), Faculty of Medicine and Dentistry, Danube Private University (DPU), Viktor-Kaplan-Straße 2, Geb. E, EG Nord, Wiener Neustadt 2700, Austria.</footnote>

<footnote>E-mail addresses: nataliia.gnyliukh@dp-uni.ac.at (N. Gnyliukh), sabine.szunerits@univ-lille.fr (S. Szunerits).</footnote>
The corneal stroma, a predominant part of the corneal structure, follows the Bowman's membrane. This transparent 450–600 µm thick layer consists of predominantly type I collagen fibrils enmeshed in a proteoglycan matrix [2]. The parallel assembly of the collagen fibrils and proteoglycans is critical for the transparency and refraction properties of the stroma. Additionally, the stroma contains keratocytes (stromal cells) that contribute to its structural integrity and function. Between the stroma and final posterior corneal endothelial layer lies the 7-µm thick Descemet's basement membrane that consists mainly of type IV collagen, nidogens and perlecan [7,8]. This basement membrane plays a crucial role in corneal homeostasis and transparency by anchoring the endothelium to the stroma and controlling hydration and nutrient intake of the cornea.

## Corneal endothelium

The corneal endothelium consists of a single layer of hexagonal-shaped cells, and plays an important role in the maintenance of a healthy vision (Fig. 1A) [184]. The stability of this endothelial cell layer is crucial for the regulation of corneal hydration and maintenance of its transparency. Unlike human corneal epithelial cells, which have high proliferation rates and turnover, allowing the maintenance of constant cell density and high control of cell adhesion, human corneal endothelial cells (HCECs) do not normally undergo mitosis in vivo [2]. HCECs are arrested in the G₁-phase of the cell cycle through the regulation of specific signaling pathways that are involved in cell cycle progression. Cell-to-cell contact-dependent inhibition has been shown to play a central role in the HCECs proliferation inhibition through an increase in

p27Kip1 protein levels [9]. P27Kip1 is one of several cyclin-dependent kinase inhibitors that, through inhibition of cyclin E/CDK2 complex, are controlling the cell transition from G₁ to S phase. However, when cell-to-cell contact is compromised, corneal endothelial cells (CECs) do not typically proliferate in vivo, indicating the existence of more crucial regulatory mechanisms. Other factors involved in the control of CECs proliferations are β-catenin and transforming growth factor β2 (TGF-β2). HCECs possess TGF-β receptors that transduce the inhibitory signal from aqueous humor to endothelial cells and down-regulate CDK4 and consequently block the transition into S-phase [10].

At birth, the density of HCECs is around 3.000–4000 cells mm⁻² with the number slowly decreasing at a rate of about 0.3–0.6 %/year to 2.500 cells mm⁻² during adulthood. This loss of HCECs is compensated through changes in the remaining cells, leading to migration, enlargement, and increased heterogeneity to maintain tight cellular connection and cell layer integrity [11]. When the HCECs density drops below the critical value of 400–500 cells mm⁻², several changes occur in these cells including a decline in the Na⁺/K⁺-ATPase pump function, HCECs enlargement or development of an irregular shape, increase in corneal thickness and loss of cellularity [2]. These changes lead to a decrease in corneal transparency, the development of corneal edema, bullous keratopathy, and, as a result, partial or full vision impairment.

It has been additionally demonstrated that alterations in ZO-1 expression can impact the barrier function of the corneal endothelium, leading to changes in endothelial cell density and, as a result, corneal thickness and transparency reduction [10]. Importantly, the mechanics of G₁-phase arrest does not imply a “terminal” differentiation, where cells irreversibly lose the ability to proliferate [10]. During the G₁-phase

<figure><img src="image_2.png" /><figcaption>**Fig. 1.** Focus on the Cornea. (A) Image of eye structure. (B) Various invasive and non-invasive drug delivery approaches to the eye, with a list of their advantages and disadvantages for each type of drug delivery system. (Created with BioRender.com).</figcaption></figure>

<figure><img src="image_3.png" /></figure>
<table><caption>**Table 1**
List of abbreviations.</caption><thead><tr><th>Abbreviation</th><th>Definition</th><th>Abbreviation</th><th>Definition</th></tr></thead><tbody><tr><td>AA</td><td>Ascorbic acid</td><td>LASIK</td><td>Laser-assisted in situ keratomileusis</td></tr><tr><td>AAV</td><td>Adeno-associated virus</td><td>LCA</td><td>Leber congenital amaurosis</td></tr><tr><td>ALT</td><td>Argon laser trabeculoplasty</td><td>LIMK</td><td>LIM-domain-containing protein kinases</td></tr><tr><td>AMG</td><td>Amniotic membrane grafting</td><td>mAChR</td><td>Muscarinic acetylcholine receptor</td></tr><tr><td>ARE</td><td>Antioxidant responsive element</td><td>MBS</td><td>Myosin-binding subunit</td></tr><tr><td>ASOs</td><td>Antisense oligonucleotides</td><td>miRNA</td><td>Micro RNA</td></tr><tr><td>ATF4</td><td>Activated transcription factor 4</td><td>MLC</td><td>Myosin light chain</td></tr><tr><td>BCECs</td><td>Aovine corneal endothelial cells</td><td>MYPT1</td><td>Myosin light chain 1</td></tr><tr><td>bpV(pic)</td><td>Aisperoxo (picolinato) oxovanadate (V)</td><td>NAD</td><td>Nicotinamide adenine dinucleotide</td></tr><tr><td>CCT</td><td>Central corneal thickness</td><td>NIC</td><td>Nicotinamide</td></tr><tr><td>cDNA</td><td>Coding DNA</td><td>Nrf2</td><td>Nuclear factor erythroid 2-related factor 2</td></tr><tr><td>CECs</td><td>Corneal endothelial cells</td><td>p53</td><td>Transformation-related protein 53</td></tr><tr><td>CHED</td><td>Congenital hereditary endothelial dystrophy</td><td>PCEDs</td><td>Persistent corneal epithelial defects</td></tr><tr><td>CHOP</td><td>Oxotremorine altered DNA damage-inducible 3</td><td>pDNA</td><td>Plasmid DNA</td></tr><tr><td>CPI-17</td><td>17 kDa protein kinase C-potentiated inhibitor</td><td>PK</td><td>Penetrating keratoplasty</td></tr><tr><td>DLEK</td><td>Deep lamellar endothelial keratoplasty</td><td>PPCD</td><td>Posterior polymorphous corneal dystrophy</td></tr><tr><td>DMEK</td><td>Descemet's membrane endothelial keratoplasty</td><td>PRK</td><td>Photorefractive keratectomy</td></tr><tr><td>DNA</td><td>Deoxyribonucleic acid</td><td>PTEN</td><td>Phosphatase and tensin homolog deleted on chromosome ten</td></tr><tr><td>DSAEK</td><td>Descemet stripping automated endothelial keratoplasty</td><td>PTK</td><td>Phototherapeutic keratectomy</td></tr><tr><td>DSEK</td><td>Descemet's stripping endothelial keratoplasty</td><td>RNA</td><td>Ribonucleic acid</td></tr><tr><td>DSO</td><td>Descemet stripping only</td><td>ROCK</td><td>Rho/Rho-associated protein kinase</td></tr><tr><td>EGF</td><td>Epidermal growth factor</td><td>ROS</td><td>Reactive oxygen species</td></tr><tr><td>EMT</td><td>Endothelial-to-mesenchymal transition</td><td>RPE</td><td>Retinal pigment epithelial</td></tr><tr><td>ERM</td><td>Ezrin radixin and myosin</td><td>SFN</td><td>Sulforaphane</td></tr><tr><td>FECD</td><td>Fuch's endothelial corneal dystrophy</td><td>siRNA</td><td>Small interfering RNA</td></tr><tr><td>FGF</td><td>Fibroblast growth factor</td><td>SLT</td><td>Selective laser trabeculoplasty</td></tr><tr><td>GAG</td><td>Glycosaminoglycan</td><td>SMILE</td><td>Small incision lenticule extraction</td></tr><tr><td>GRP78</td><td>Glucose-regulated protein 78 kDa</td><td>TGF-β</td><td>Transforming growth factor β</td></tr><tr><td>HCECs</td><td>Human corneal endothelial cells</td><td>UPR</td><td>Unfolded protein response</td></tr><tr><td>HSV</td><td>Herpes simplex virus</td><td>UVB</td><td>Ultraviolet B</td></tr><tr><td>IC</td><td>Intracameral</td><td>VEGF</td><td>Vascular endothelial growth factor</td></tr><tr><td>IOP</td><td>Intraocular pressure</td><td>ZO-1</td><td>Zonula occludens-1</td></tr><tr><td>Keap1</td><td>Kelch-like ECH-associated protein 1</td><td></td><td></td></tr></tbody></table>

arrest, cells can usually undergo repair processes, respond to growth factors, or address cellular stress before resuming the cell cycle. This reversibility is crucial for maintaining cellular homeostasis, ensuring proper cell growth, and responding to environmental cues. Interestingly, it has been shown that, in the rabbit model, HCECs can proliferate and the activation of β-catenin played an important role [10]. This knowledge indicates possible therapeutic targets that can influence endothelium regeneration *in vivo*.

The anatomy and physiology of the eye both largely prevent the uptake of drugs via simple eye drop applications. The lipophilic nature of the corneal epithelium and the presence of TJs make this most outer layer the primary barrier to be overcome for any drug [12]. As this layer is naturally water-repelling it offers great resistance to the passing of hydrophilic drugs, with lipophilic drugs penetrating easily into the corneal epithelium [13]. However, the corneal epithelium is in direct contact with a mucin layer produced by conjunctival goblet cells. Once the mucous layer has formed across the surface, the cornea becomes hydrophilic, providing eye nutrition and lubrication [14]. The corneal epithelium is integrated with the corneal stroma, the thickest layer of the cornea, with stromal-epithelial interactions being key determinants of corneal function. It consists of a highly hydrated collagen network, offering minimal resistance to hydrophilic drugs, but behaves as a rate-limiting barrier for deeper ocular permeation for hydrophobic and lipid-soluble drugs. The posterior corneal surface that transports water from the stroma to the anterior chamber is the corneal endothelial layer [12]. It thus regulates hydration for optimal transparency and vision.

Currently, conditions like dry eye, glaucoma, conjunctivitis and inflammatory eye issues are treated using several types of eye drop medicine [15-18] (Table 2A). While these topically applied drugs (Fig. 1B) have demonstrated good therapeutic action in cornea disorders of the epithelium, the drugs often do not travel deeper than the aqueous humor and, therefore, do not reach the endothelium or other intraocular tissues.

Indeed, no therapeutic effective drug doses reach the posterior eye segment using topical drug application (Fig. 1B), as TJs in the corneal epithelium majorly restrict drug absorption through the corneal stroma and corneal endothelium. Passing through TJs is fundamental for drugs to reach the aqueous humor and diffuse further to the corneal endothelial layer. This becomes even more relevant for the treatment of endothelial corneal diseases (Table 2B), such as Fuch's endothelial corneal dystrophy (FECD), corneal edema, bullous keratopathy, persistent epithelial defects (PEDs), corneal ulcers, or corneal endothelial keratitis to cite the most frequent ones. These different corneal endothelial dysfunctions can lead to severe pain, vision blurriness, and partial or full visual loss [19].

FECD, corneal edema and Bullous keratopathy are some of the most common corneal endothelial disorders caused by ageing, genetic factors, diabetes, untreated keratitis, cataract and glaucoma surgeries [20-22]. They result in an accelerated loss of corneal endothelial cells, which leads to a decrease in the activity of CECs and causes an increase in the hydration state of the corneal stroma. Accumulation of extracellular fluid in the cornea is a frequent cause of corneal edema symptoms (e.g. blurred vision, glare, sensitivity to light) due to corneal swelling and the development of folds/breaks in the Descemet's membrane. Bullous keratopathy can develop as a result of progressive FECD and corneal edema. Due to corneal swelling, blister-like bullae form and rupture, causing decreased visual acuity, loss of contrast, glare, and photophobia. Raptured bulla can be easily invaded by pathogens and can lead to corneal ulcer disease. Some other common corneal endothelial disorders, like congenital hereditary endothelial dystrophy (CHED) and posterior polymorphous corneal dystrophy (PPCD), which can cause partial or full vision loss if left untreated, are genetic-based.

Up to this day, the only approved treatment strategy for corneal endothelial dysfunctions is transplantation of either whole corneal tissue or selective endothelial transplantation. (Table 2B). The procedure involves replacing damaged corneal tissue with healthy donor tissue.
<caption>**Table 2**</caption>

Representative topical formulations for the treatment of eye: (A) Treatments linked to the epithelial cell layers of the human eye. (B) Treatments related to the endothelial cell layers of the human eye. * Composition of artificial tear products: hyaluronic acid, dextran, viscosity-enhancing agents such as carbomer 940, polyvinyl alcohol, polyvinylpyrrolidone and polyethylene glycol, hydroxypropyl guar (HP-guar), carboxymethyl cellulose (CMC), sodium hyaluronatehydroxypropyl methyl-cellulose (HPMC hypromellose), which aid lubrication [42]. ** Composition of lubricating ointments: dextran, polyvinyl alcohol, polyethylene glycol 400 (PEG 400), carboxymethylcellulose, hypromellose, polysorbate, povidone, glycerin, or propylene glycol [43].

<table><thead><tr><th>Disease</th><th>Disease etiology</th><th>Topical Drug</th><th>Mechanism of drug function</th><th>Drug side effects</th><th>References</th></tr></thead><tbody><tr><td>Exposure keratitis</td><td>Corneal damage caused by dryness due to inadequate eyelid closure, resulting in loss or insufficiency of the tear film</td><td>Artificial tear drops* (0.2-1 %)<br>Lubricating ointment** (0.4-0.3 %)</td><td>Corneal lubrication and hydration, reduction of dryness and discomfort<br>Corneal lubrication and hydration, especially during sleep<br>Reduction of eye inflammation</td><td>Allergic reactions, like itching or hives, swelling, eye pain<br>Blurred vision, burning, stinging, irritation</td><td>[44,45]</td></tr><tr><td>Bacterial keratitis</td><td>Inflammation of the cornea caused by a bacterial infection</td><td>Tobramycin (14 mg mL<sup>-1</sup>), cefazolin (50 mg mL<sup>-1</sup>), or vancomycin (50 mg mL<sup>-1</sup>)</td><td>Antiviral inhibitor of herpes virus DNA polymerase<br>Antiviral inhibitor of herpes virus DNA polymerase</td><td>Irritation, itching, blurred vision, eye pain, hives, swelling</td><td>[46,47]</td></tr><tr><td>Herpetic keratitis</td><td>Corneal infection caused by herpes simplex (HSV) virus</td><td>Acyclovir ointment (3 %)<br>Ganciclovir gel (0.15 %)</td><td>Stimulates corneal innervation, tear secretion, and epithelial cell growth due to differentiation and maintenance of neurons</td><td>Hives, sensitivity to light, itching, swelling<br>Blurred vision</td><td>[48,49]</td></tr><tr><td>Neurotrophic keratitis</td><td>Loss of corneal sensitivity and corneal damage due to the damage to the trigeminal nerve</td><td>Cenegermin (0.002 %)</td><td>Biological nutrients present in natural tears, such as growth factors, proteins, and vitamins, promote corneal healing process</td><td>Pain, ocular hyperemia, inflammation, increased lacrimation, irritation, redness, or swelling of the eye, headache</td><td>[50]</td></tr><tr><td rowspan="5">Persistent epithelial defects (PEDs) and corneal ulcers</td><td rowspan="5">Failure of re-epithelialization due to trauma, dry eyes, exposure keratopathy, limbal stem cell deficiency, and neurotrophic corneal disease<br>Corneal ulcer is a severe form of PED where the defect extends into the corneal stroma</td><td>Autologous serum (20 %)</td><td>Promotion of cell proliferation, migration, and differentiation, enhances the expression of growth factors</td><td>Risk of infection</td><td>[51,52]</td></tr><tr><td>Insulin (1-50 IU mL<sup>-1</sup>)</td><td>Increase of cell adhesion, migration, and differentiation, enhancement of growth factors expression</td><td>Enhanced formation of retinal acellular degenerated capillaries</td><td></td></tr><tr><td>Fibronectin (1 mg mL<sup>-1</sup>)</td><td>Stimulation of cell migration and proliferation, reduction of oxidative stress, enhancement of growth factor expression</td><td>No reported significant side effects</td><td></td></tr><tr><td>Epidermal Growth Factor (EGF) (0.05 mg mL<sup>-1</sup>)</td><td>Promotion of cell proliferation, migration, and differentiation, enhancement of growth factor expression</td><td>Dry eye, proptosis, and swelling of the extraocular muscles.</td><td></td></tr><tr><td>Insulin-Like Growth Factor-1 (IGF-1) (0.3 mg kg<sup>-1</sup>)</td><td>Promotion of cell proliferation, migration, and differentiation, enhanced expression of growth factors</td><td>Hypersensitivity reactions</td><td></td></tr><tr><td rowspan="3">Meesmann's dystrophy</td><td rowspan="3">Development of intraepithelial microcysts, which cause chronic eye dryness, photophobia, lacrimation, and visual loss. It is caused by the mutations in the KRT3 or KRT12 genes encoding cornea-specific keratin cause</td><td>Artificial eye drops (0.5 %)</td><td>Lubrication of the cornea for reduction of symptoms</td><td>Short-time burning, itching, and stinging</td><td>[53]</td></tr><tr><td>Sodium chloride (5 %)</td><td>Maintain ocular surface hydration and promote epithelial healing by increasing adherence of the epithelium to the underlying stroma</td><td>Short-time burning and stinging</td><td></td></tr><tr><td>Artificial eye drops (0.2-1 %)<br>Lubricating ointment (0.4-0.3 %)</td><td>Lubrication and hydration of cornea, reduction of dryness and discomfort<br>Prolonged lubrication and hydration to the cornea, especially during sleep</td><td>Itching, hives, swelling of the face, lips, or tongue, eye pain<br>Blurred vision, burning, stinging, or irritation</td><td></td></tr><tr><td>Band keratopathy</td><td>Precipitation of calcium salts, mainly hydroxyapatite, on the underside of the corneal epithelium, which causes chronic ocular inflammation,</td><td>Ethylenediaminetetraacetic acid (EDTA) (1.5-3.75 %)</td><td>Maintains calcium ion chelation and dissolution of calcium deposits in the cornea</td><td>Temporary blurring of vision and brief stinging, irritation, allergic reaction</td><td>[54]</td></tr></tbody></table>

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<table><caption>**Table 2 (continued)**</caption><thead><tr><th colspan="6">(A)</th></tr><tr><th>Disease</th><th>Disease etiology</th><th>Topical Drug</th><th>Mechanism of drug function</th><th>Drug side effects</th><th>References</th></tr></thead><tbody><tr><td rowspan="5">Salzmann's nodular degeneration</td><td rowspan="5">Growth of blue-greyish fibrous nodules in the cornea caused by trauma, keratitis, corneal surgery, trachoma, or use of contact lenses</td><td>Sodium hyaluronate (0.2%)</td><td>Viscoelastic agent used with EDTA eye drops to reduce postoperative pain and discomfort by lubrication and moisturizing the cornea and induction of cell adhesion</td><td>Temporary blurred vision</td><td rowspan="5">[55-57]</td></tr><tr><td>Prednisolone acetate (1%)</td><td>Topical steroid reduces inflammation, pain and discomfort</td><td>Burning, stinging</td></tr><tr><td>Dexametasone (0.1%)</td><td>Reduction of swelling, redness and irritation</td><td>Irritation, burning, stinging or itching, blurred or cloudy vision, eye pain</td></tr><tr><td>Cyclosporine (0.05%)</td><td>Increase of tear production and eye lubrication</td><td>Burning, pain, itching, pain upon instillation of drops, redness of the sclera or inside of the eyelids</td></tr><tr><td>Lifitegrast (5%)</td><td>Reduction of eye inflammation</td><td>Irritation, discomfort or blurred vision</td></tr><tr><td></td><td>Perfluorohexyloctane (100%)</td><td>Increase tear film breakup time and corneal hydration</td><td>Blurred vision</td><td></td></tr><tr><th colspan="6">(B)</th></tr><tr><td></td><td><strong>Disease</strong></td><td><strong>Disease etiology</strong></td><td><strong>Available treatment</strong></td><td></td><td></td></tr><tr><td>Fuchs endothelial corneal dystrophy (FECD)</td><td>Ageing, genetic factors (mutations in (alpha-2 chain of type VIII collagen), ZEB1 (Zinc finger E-box-binding homeobox) and others), diabetes, glaucoma, untreated keratitis, cataract and glaucoma surgeries</td><td>Disorder caused by loss of corneal endothelial cells. CECs are not able to mediate the recurred dehydration state of corneal stroma. Cell loss causes thickening of Descemet's membrane and deposition of extracellular matrix in stroma. This leads to cornea swelling (corneal edema) and loss of corneal transparency, resulting in vision loss</td><td>Invasive: Descemet's stripping endothelial keratoplasty (DSEK)<br/>Non-invasive: no approved treatment available</td><td>[20,22,58]</td><td></td></tr><tr><td>Corneal edema</td><td>Infection, inflammation, injury, following eye surgery, or eye diseases like dry eye, FECD, PPCD or glaucoma</td><td>The process of corneal swelling results from the accumulation of extracellular matrix in corneal stroma. It leads to corneal transparency and vision loss</td><td>Invasive: Descemet's stripping endothelial keratoplasty (DSEK)<br/>Non-invasive: hypertonic saline drops, hypertonic saline ointment (in case of high ocular pressure)</td><td>[59]</td><td></td></tr><tr><td>Congenital hereditary endothelial dystrophy (CHED)</td><td>Mutations in the SLC4A11 (sodium borate transporter) gene</td><td>Rare autosomal recessive genetic corneal disorder. It is characterized by bilateral corneal opacification due to stromal edema and Descemet membrane thickening. Non-treated, CHED can cause blurred or complete loss of vision</td><td>Invasive: penetrating keratoplasty (PK), endothelial keratoplasty (EK)<br/>Non-invasive: no approved treatment available</td><td>[60]</td><td></td></tr><tr><td>Bullous keratopathy</td><td>Surgery, trauma, or pre-existing diseases like FECD, glaucoma and diabetes</td><td>It is characterized by chronic corneal edema, leading to the formation of liquid-filled subepithelial bullae. Bullous keratopathy can cause pain, blurred vision, photophobia, tearing, foreign body sensation, epithelial erosion, and corneal opacity, ultimately leading to blindness</td><td>Invasive: Descemet Stripping Endothelial Keratoplasty (DSEK), Descemet Stripping Automated Endothelial Keratoplasty (DSAEK), Descemet Membrane Endothelial Keratoplasty (DMEK), Amniotic Membrane Grafting (AMG), Phototherapeutic keratectomy (PTK)<br/>Non-invasive: no approved treatment available</td><td>[61]</td><td></td></tr><tr><td>Posterior polymorphous corneal dystrophy (PPCD)</td><td>Mutation in COL8A2 (alpha-2 chain of type VIII collagen), ZEB1 (Zinc finger E-box-binding homeobox), OVOL2 (Ovo like zinc finger 2), and GRHL2 (Grainyhead like transcription factor 2) genes</td><td>Rare autosomal dominantly inherited corneal dystrophy characterized mainly in early childhood or adolescence, caused by genetic mutation. It is characterized by the presence of nodular, vesicular, and blister-like lesions, and deep stromal "railroad tracks", leading to clouding of the stroma and blurred vision</td><td>Invasive: Laser-assisted in situ keratomileusis (LASIK), photorefractive keratectomy (PRK), and small incision lenticule extraction (SMILE); Deep lamellar endothelial keratoplasty (DLEK), Descemet stripping endothelial keratoplasty (DSEK), or Descemet stripping automated endothelial keratoplasty (DSAEK)<br/>Non-invasive: no approved treatment available</td><td>[62-64]</td><td></td></tr><tr><td>Iridocorneal endothelial (ICE) syndrome</td><td>Viral infection (HSV, EBV)</td><td>Rare eye conditions that affect the corneal endothelium and iris; it more frequently affects young adults and middle-aged females. Although the exact mechanism of ICE syndrome is unknown, it is linked to increased proliferation and migration of corneal endothelial cells, which can lead to secondary angle-closure glaucoma, corneal edema, and atrophy of the iris.</td><td>Invasive: Descemet's stripping endothelial keratoplasty (DSEK)<br/>Non-invasive: no approved treatment available</td><td>[65]</td><td></td></tr></tbody></table>

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<table><caption>**Table 2 (continued)**</caption><thead><tr><th>(A)<br>Disease</th><th>Disease etiology</th><th>Topical Drug</th><th>Mechanism of drug function</th><th>Drug side effects</th><th>References</th></tr></thead><tbody><tr><td>Post-traumatic fibrous downgrowth</td><td>Penetrating keratoplasty, cataract surgery, trauma, or filtering surgery</td><td>It is caused by the invasion of connective tissue elements into the eye. It is characterized by a retrocorneal membrane that may be vascularized and is predominantly fibrous instead of cellular. It can lead to blindness if left untreated.</td><td></td><td>Invasive: corneoscleral grafting, laser photocoagulation, intracameral injections (IC) of bevacizumab<br>Non-invasive: intraocular pressure (IOP)-lowering eye drops, topical steroids</td><td>[58]</td></tr><tr><td>Glaucoma</td><td>High intraocular pressure (IOP)</td><td>It is a group of eye conditions that can cause corneal endothelial cell loss, which can lead to corneal decompensation; however, the mechanism is not well understood.</td><td></td><td>Invasive: Laser trabeculoplasty (ALT or SLT), Trabeculectomy, viscoscanalostomy, deep sclerectomy, glaucoma drainage device implantation<br>Non-invasive: intraocular pressure (IOP)-lowering eye drops (most popular - Latanoprost, Timolol, Brimonidine)</td><td>[66]</td></tr><tr><td>Photokeratitis</td><td>Exposure to ultraviolet B radiation (290-320 nm) from the sun or artificial sources.</td><td>In UVB-induced corneal damage, the endothelium can show widespread intracellular edema and some decomposition of cells with higher exposure levels</td><td></td><td>Invasive: photocoagulation<br>Non-invasive: artificial tears, fluorescein</td><td>[67]</td></tr></tbody></table>

Although it remains the most popular way of treatment, the corneal transplantation procedure carries several risks for the patient, such as the elevated risk of tissue rejection, glaucoma, cataract, corneal edema, and detachment of the retina. The process of wound healing and visual

rehabilitation after such operations can be slow and regular check-ups are needed for the maintenance of a healthy transparent cornea. Another drawback of corneal transplantation is the need for healthy donor tissue. Due to the extreme shortage of donors, only 1 in

<figure><img src="image_4.png" /><figcaption>**Fig. 2.** Current topical remedies for corneal endothelial dysfunctions. Strategy pathway of ongoing studies using corneal endothelial cells (CECs) *in vitro* and animal models with corneal endothelial dysfunction *in vivo* as well as current clinical studies testing the efficiency of novel topical treatments for corneal endothelial diseases (image of corneal endothelial cells, reprint with permission from Wisely et al. [184] (Created with BioRender.com).</figcaption></figure>
approximately 70 patients will receive a new cornea. Replacing damaged parts of the cornea rather than the whole structure with techniques like Descemet's Stripping Endothelial Keratoplasty (DSEK) and Descemet's Membrane Endothelial Keratoplasty (DMEK), offers quicker visual recovery and reduced risk of graft rejection [19]. However, these procedures are still highly invasive, need a long recovery time, and require donor tissue.

Cell therapy is currently undergoing extensive clinical trials as an alternative to transplanting. Due to the ability of CECs to proliferate *in vitro*, scientists have created a sustainable source of cells for transplantation, reducing the reliance on donor tissue [23–26]. Different model organisms, including rabbits, chimps and humans, were used, and long-term corneal regeneration could be validated when using *in vitro* cultivated HCECs therapies [27]. Long-term human studies recently revealed the successful restoration of endothelial tissue *in vivo* after HCECs injection. No major side effects have been seen until now, making this technique a strong candidate to replace corneal transplantation [28, 29]. While highly promising, these cell-based therapies remain invasive.

*Are there current remedies for corneal endothelial dysfunctions, such as topical treatments for different corneal epithelial defects?*

While there are still no clinically approved topical treatments for diseases related to endothelium dysfunction (Table 2B), new strategies are constantly being explored. The current research advances in pre-clinical and clinical testing of the various therapeutic pathways that aim at corneal endothelial dysfunction treatments in a non-invasive manner are the focal point of this review article (Fig. 2).

To overcome ocular drug absorption limitations, due to inherent cellular barriers and loss mechanisms associated with the physiology of the eye, the development of effective ophthalmic drug delivery concepts requires multidirectional considerations. Nanotechnology-based delivery systems are considered promising to treat corneal endothelial dysfunction and to improve the efficiency of topically administered drugs [30]. The hope is that these nanostructures can overcome ocular barriers to deliver the drug to the target site in the eye. The possibility to enhance the solubility of poorly soluble drugs via encapsulation or the formation of biphasic formulations improves the possibility for being topically delivered as eye drops. The high surface-to-volume ratio of NPs together with the variable chemistry of these particles is believed to prolong the ocular retention time with enhanced trans-corneal permeation [13,31]. Nanostructures, such as micelles, liposomes, dendrimers, niosomes as well as nanoparticles (NPs), have been investigated as effective options [32–34]. Polymeric NPs, synthesised from natural or synthetic polymers, can encapsulate hydrophilic and hydrophobic drugs, resulting in improved drug solubility and bioavailability. Metallic NP such as gold or carbon-based ones allow targeted treatment and are often considered to be junction modulators [35]. Different penetration modes need to be considered as with other junctions ranging from paracellular transport to transcellular pathways via carrier-mediated transport, passive diffusion, efflux pumps as well as receptor-mediated transcytosis or adsorptive-mediated transcytosis [36,37].

This review will discuss the potential of chemical therapeutics targeting HCEC proliferation and survival via inhibitors/modulators for different molecular pathways. Gene therapeutic approaches (gene editing, gene addition, ribonucleic acid (RNA) therapies) and nano-technological routes, notably those based on nanoparticles, will also be discussed. These therapies aim to increase cell proliferation and adhesion and limit the rate of cell death of HCECs. Cell proliferation is a very complex and tightly regulated process that depends on various intra- and extra-cellular signals. Its regulation is greatly studied because its malfunctioning is involved in the development of neurodegenerative and hematologic disorders, and cancer [38,39]. Since CEC are arrested in the G₁-phase of the cell cycle, they do not proliferate under normal conditions *in vivo* [27]. This property causes most primary and secondary corneal endothelial disorders when HCEC density critically drops, and the remaining cells cannot maintain tight cell-to-cell

connections and perform their pumping function, resulting in the development of corneal edema. During corneal endothelial damage, remaining endothelial cells often undergo endothelial-to-mesenchymal transition (EMT), which can result in endothelial scarring and blindness [40,41]. In this review, the state of the art of pharmacological alternatives as well as genetic therapeutic strategies together with recent nanotechnological approaches tested *in vitro* and *in vivo* animal models will be presented in more detail. This review will focus on the use of topical applications rather than intraocular and other invasive drug delivery methods. A concluding section will be devoted to possible future developments in the field based on our current knowledge.

## Chemical remedies for endothelial dysfunctions of the eye

*Topical applied inhibitors and modulators for the Rho/Rho-associated protein kinase (ROCK) pathway*

Various drugs have been investigated for their potential to enhance cell proliferation capacity and adhesion levels by modulating the Rho/ Rho-associated protein kinase (ROCK) pathway, one of the critical molecular regulators of cell proliferation, adhesion, motility, differentiation and apoptosis [68,69] (Fig. 3). Inhibition of ROCK activity by various ROCK inhibitors has been shown to decrease cell apoptosis levels, and induce HCECs culture adhesion levels, cell migration, and cell proliferation [68,69]. Indeed, ROCK is activated by extracellular signals through the activity of Rho GTPase, such as RhoA, Rac and Cdc42 [70–73]. It mediates phosphorylation of various substrates, such as the myosin-binding subunit (MBS) of myosin light chain 1 (MYPT1), myosin light chain (MLC), LIM-domain-containing protein kinases 1 and 2 (LIMK1/2), 17 kDa protein kinase C-potentiated inhibitor (CPI-17), α-adducin, and ezrin radixin and myosin (ERM) proteins [74]. Phosphorylation of these proteins leads to myosin rearrangement, actomyosin contraction and membrane blebbing, and results in cell apoptosis. In HCECs, there are two ROCK isoforms, ROCK1 and ROCK2, with their roles being considered similar [72]. It was seen that mice with ROCK1 delete have impaired eye closure, while ROCK2 deficiency leads to intrauterine growth retardation [75].

Several ROCK inhibitors (Table 3) [76] are approved in Japan, USA and/or Europe for the treatment of glaucoma and ocular hypertension [77–79]. For example, ripasudil was tested for the treatment of endothelial cells in the case of Fuch’s endothelial corneal dystrophy (FECD) and corneal wound healing on HCECs culture and rabbit *in vivo* models.

*Ripasudil*

4-Fluoro-5-[(2S)-2-methyl-1,4-diazepan-1-yl]sulfonyl]isoquinoline, known under the brand name ripasudil or K-115, is a type 1 ATP competitive kinase inhibitor that blocks the transfer of phosphate from ATP to a substrate [80]. Ripasudil targets kinase domains of ROCK1 with an $IC_{50}$ = 0.051 µmol L$^{-1}$ and ROCK2 [77,78] (Table 2). The feasibility of ripasudil eye drops to treat corneal endothelial injury was shown by Okumura et al. [81]. Treating HCEC cells with ripasudil (1 and 10 µM) for 48 h revealed increased cell proliferation compared to the control (Fig. 4A). Testing ripasudil eye droplets in a rabbit partial corneal endothelial wound model using slit-lamp microscopy revealed that wounds created by trans corneal freeing have hazy corneas after 48 h, while rabbits treated with ripasudil eye droplets showed decreased haze in their corneas (Fig. 4A). In addition, using Alizarin red staining revealed that the wound area was smaller in ripasudil eye droplets treated cases (Fig. 4A).

The protective effect on HCECs against induced apoptosis, by increasing the mitochondrial glycolic capacity of treated cells, has in addition been documented by Goldstein et al. [82]. Subjects treated with ripasudil showed restored corneal clarity compared to the untreated control conditions, where the wounded cornea remained hazy. In clinical studies, ripasudil has been applied as an adjuvant during the Descemet stripping only (DSO) corneal surgery as a post-operational
<figure><img src="image_5.png" /><figcaption>**Fig. 3.** Schematic representation of targeted signalling pathways crosstalk in corneal endothelial cells and chemical molecules that can act on them: HCECs apoptosis, proliferation, migration and cell cycle progression as currently understood from the literature presented in Chapter 2. Pharmacological activators (green): TTHX1114 (topical application), Oxotremorine, Mefenamic acid (MA), and Sulforaphane (SFN). Pharmacological inhibitors (red): Ripasudil (topical application), Netarsudil (topical application), Y-27632 (topical application), Ascorbic acid (AA) (topical application), SB431542, LY2109761, bpV(pic), and CHIR99021 (Created with BioRender.com).</figcaption></figure>

topical treatment, showing a positive effect in clearing the corneal edema [83,84]. Another study used topical ripasudil on patients with low HCECs density after cataract surgery [85]. The study reported a significant reduction of HCECs loss rates in treated subjects, indicating the potential of the ROCK inhibitor to maintain HCECs density, especially in cases with very low cell numbers. Ripasudil has also been reported to be beneficial for the treatment of high intraocular pressure (IOP) and bullous keratopathy in a patient with cytomegalovirus corneal endotheliitis [86] with only mild side effects, such as mild to moderate conjunctival hyperemia, ocular irritation, abnormal sensation in the eye, and conjunctival haemorrhage [85,87].

### Netarsudil

Another ROCK inhibitor that originally was developed for the treatment of open-angle glaucoma and ocular hypertension is (4-((1S)-1-(aminomethyl)-2-(isoquinolin-6-ylamino)-2-oxoethyl)phenyl)methyl 2,4-dimethylbenzoate, known under the commercial name of netarsudil (AR-13324) [88]. Similar to ripasudil, netarsudil interacts with the kinase domain and prevents substrate phosphorylation [89] (Table 3). Netarsudil is present in eye drops as pro-drug, netarsudil dimesilate, and is cleaved by esterases in the cornea to create the active form for the reduction of ocular side effects and increase of corneal penetration [89]. Studies in silico provided evidence for high docking of netarsudil to ROCK, and in vitro HCECs culture and ex vivo cultured wounded corneas showed a higher rate of cell adhesion and proliferation when treated with netarsudil [76]. A clinical trial investigated netarsudil for the topical treatment of corneal edema caused by FECD [90]. The treatment led to a significant reduction in corneal thickness and improvement of corneal clarity, resulting in overall improved patient-reported visual

disability, with mild or moderate side effects. In a case study from Li et al. of a patient with corneal endothelial decompensation caused by corneal endotheliitis, a disappearance of corneal edema after 7 months of treatment with 0.02 % netarsudil bidaily was observed (Fig. 4B) [91]. Another case study of a patient with corneal edema, developed after laser peripheral iridotomy, revealed that the initial application of netarsudil had a low effect due to the complete loss of HCECs. However, after the performance of Descemet's membrane endothelial keratoplasty, the application of netarsudil resulted in the stabilisation of vision and thinning of the cornea [92]. Assessment of the influence of netarsudil on the corneal endothelial cell morphology and central CCT in patients with ocular hypertension or open-angle glaucoma showed no severe side effects [93] (Fig. 4B). The development of honeycomb-like patterns of epithelial cysts after discontinuation of the treatment in patients with corneal edema treated with netarsudil was reported by LoBue et al. [94], although it has been described as rather a rare side effect.

### Y-27632

An experimental ROCK inhibitor (+)-(R)-trans-4-(1-aminoethyl)-N-(4-pyridyl)cyclohexanecarboxamide dihydrochloride, Y-27632, derived from 4-aminopyridine, has a similar mechanism of action to the previously discussed ripasudil and netarsudil [95] (Table 3). Y-27632 is currently undergoing clinical trials in Japan, holding great potential for treating corneal endothelial disorders. Comparison of Y-27632 to other ROCK inhibitors in vitro demonstrated its increased selectivity towards ROCK proteins compared to isoquinoline derivatives, with 20–30 times higher affinity for ROCK kinases than for other Rho effector kinases [95]. Studies on the in vivo transcorneal freezing rabbit model showed
<table><caption>**Table 3**

Therapeutic formulations with potential treatment value for different corneal endothelial disorders. Formulations that were topically applied are marked in red.</caption><thead><tr><th>Drug</th><th>Name</th><th>MW/[g mol<sup>-1</sup>]</th><th>Chemical structure</th><th>Action</th><th>IC<sub>50</sub>/ [µmol L<sup>-1</sup>]</th><th>Target</th><th>Mechanism of action</th></tr></thead><tbody><tr><td>Ripasudil</td><td>4-fluoro-5-[(2S)-2-methyl-1,4-diazepan-1-yl]sulfonyl]isoquinoline</td><td>323.39</td><td></td><td>ROCK inhibitor</td><td>ROCK1: 0.051<br>ROCK 2: 0.019</td><td>Rho-associated protein kinases<br>ROCK1 and ROCK2</td><td>Binding of isoquinoline unit to the ROCK kinase domain and preventing substrate phosphorylation</td></tr><tr><td>Netarsudil</td><td>4-[(1S)-1-(aminomethyl)-2-(isoquinolin-6-ylamino)-2-oxoethyl]phenyl]methyl 2,4-dimethylbenzoate</td><td>526.46 (prodrug)<br>453.54 (active form)</td><td></td><td>ROCK inhibitor</td><td>ROCK1 I: 0.079<br>ROCK2: 0.016</td><td>Rho-associated protein kinases<br>ROCK1 and ROCK2</td><td>Binding of isoquinoline unit to the ROCK kinase domain and preventing substrate phosphorylation</td></tr><tr><td>Y-27632</td><td>(+)-(R)-trans-4-(1-aminoethyl)-N-(4-pyridyl)cyclohexanecarboxamide dihydrochloride</td><td>247.34</td><td></td><td>ROCK inhibitor</td><td>ROCK1 &amp;ROCK2: 0.41</td><td>Rho-associated protein kinases<br>ROCK1 and ROCK2</td><td>Binding of pyridine carboxamide unit to the ROCK kinase domain and preventing substrate phosphorylation</td></tr><tr><td>bpV(pic)</td><td>Dipotassium bisperoxo (5-hydroxypyridine-2-carboxyl) oxovanadate (V)</td><td>367.27</td><td></td><td>PTEN inhibitor</td><td>0.031</td><td>Phosphatase and tensin homolog (PTEN)</td><td>Binds to the CX5R motif of PTEN and inhibits its activity</td></tr><tr><td>SB431542</td><td>4-[4-(2H-1,3-Benzodioxol-5-yl)-5-(pyridin-2-yl)-1H-imidazol-2-yl]benzamide</td><td>384.39</td><td></td><td>TGF-β receptor inhibitor</td><td>0.094</td><td>TGF-β receptor complex</td><td>Binds to the ATP binding domains, inhibiting substrate phosphorylation</td></tr><tr><td>LY2109761</td><td>4-(2-((4-(2-Pyridin-2-yl)-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl)quinolin-7-yl)oxy)ethyl)morpholine</td><td>441.52</td><td></td><td>TGF-β receptor inhibitor</td><td>0.069</td><td>TGF-β receptor complex</td><td>Binds to the ATP binding domains, inhibiting substrate phosphorylation</td></tr></tbody></table>

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<table><caption>**Table 3 (continued)**</caption><thead><tr><th>Drug</th><th>Name</th><th>MW/[g mol<sup>-1</sup>]</th><th>Chemical structure</th><th>Action</th><th>IC<sub>50</sub>/ [µmol L<sup>-1</sup>]</th><th>Target</th><th>Mechanism of action</th></tr></thead><tbody><tr><td>Nicotinamide</td><td>Pyridin-3-carboxamide</td><td>122.12</td><td></td><td>Vitamin B3 derivative</td><td>14.3</td><td>Cofactor of nicotinamide adenine dinucleotide (NADH/NAD+) and nicotinamide adenine dinucleotide phosphate (NADP+)</td><td>Inhibition of EMT through inhibition of TGF-β1 signalling pathway</td></tr><tr><td>CHIR99021</td><td>6-[(2-[(4-(2,4-Dichlorophenyl)-5-(4-methyl-1H-imidazol-2-yl)pyrimidin-2-yl)amino]ethyl)amino]nicotinonitrile</td><td>465.34</td><td></td><td>GSK-3 inhibitor</td><td>0.04</td><td>Glycogen synthesis kinase 3 (GSK-3)</td><td>Binds to the ATP binding domains, inhibiting substrate phosphorylation</td></tr><tr><td>Sulforaphane</td><td>1-isothiocyanato-4-methylsulfinylbutane</td><td>177.29</td><td></td><td>Nrf2 enhancer</td><td>5.9-14.5</td><td>Nuclear factor erythroid 2-related factor 2 (Nrf2)/Kelch-like ECH-associated protein 1 (Keap1) complex</td><td>SFN releases Nrf2 from Keap1 and activates the Nrf2-ARE pathway.</td></tr><tr><td>Ascorbic acid</td><td>(2 R)-2-[(1S)-1,2-dihydroxyethyl]-3,4-dihydroxy-2H-furan-5-one</td><td>176.12</td><td></td><td>Oxidative stress protector</td><td>6.1</td><td>PI3P /reactive oxygen species (ROS)</td><td>Reverses ROS-inhibited Akt phosphorylation</td></tr><tr><td>Oxotremorine</td><td>1-(4-Pyrrolidin-1-yl)but-2-yn-1-yl) pyrrolidin-2-one</td><td>322.19</td><td></td><td>Muscarinic acetylcholine receptor agonist</td><td>1.1</td><td>Muscarinic acetylcholine receptor</td><td>Alters expression of CHOP and GRP78. Upregulates expression of ATF4, protein carbonyl, and 8-OHdG</td></tr><tr><td>Mefenamic acid</td><td>2-[(2,3-Dimethylphenyl) amino]benzoic acid</td><td>241.29</td><td></td><td>Competitive inhibitor of human cyclooxygenase 1 (hCOX-1) and 2 (hCOX-2)</td><td>hCOX-1: 0.04 hCOX-2: 3</td><td>COX-1 and COX-2</td><td>Alters expression of GRP78, protein carbonyl and 8-OHdG</td></tr></tbody></table>

that a smaller wound area was observed after topical Y-27632 delivery compared to the control group [96]. Next, Y-27632 was tested as a topical treatment of the corneal-endothelial partially injured by transcorneal freezing model in rabbits [97]. Results showed increased proliferation activity of RCECs after Y-27632 treatment (Fig. 4C). It also revealed improved clearance of corneal edema after the 14-day treatment compared to the control (Fig. 4C). The first clinical trial involved 8 patients with corneal endothelial dysfunction, specifically corneal edema caused by early-stage FECD, and showed a positive influence of Y-27632 on the clearance of central corneal edema [98]. A clinical study of central corneal edema (CE) and diffused CE treatment with Y-27632 highlighted that, in the case of central CE, there was a significant improvement in corneal clearance after 3 months of topical treatment (Fig. 4C). However, no significant improvement was observed in case of treated diffused CE (Fig. 4C). A long-term clinical case involving the

usage of Y-27632 eye drops, after the removal of degenerated HCECs, underlined the maintenance of the clear corneal with stable cell density and corneal thickness throughout 12 years after the treatment [99]. This result proves the efficiency of the used treatment and Y-27632 safety judging by the absence of side effects. It can be concluded that various ROCK inhibitors can present a potential treatment for the early stages of corneal endothelial diseases.

### Activation of fibroblast growth factor (FGF) pathway with topical eye formulations

Epidermal growth factor (EGF) and fibroblast growth factor (FGF) have been tested on bovine CECs (BCECs) and human CECs (HCECs) cultures for stimulation of cell migration. The FGF signalling pathways control many various cell processes through several protein cascades
<figure><img src="image_6.png" /><figcaption>(B)</figcaption></figure>

<figure><img src="image_7.png" /><figcaption>(C)</figcaption></figure>

<figure><img src="image_8.png" /></figure>

(caption on next page)
**Fig. 4.** Topical application of ROCK inhibitors. (A) Effect of ripasudil eye droplets on HCECs proliferation and wound healing of a mechanically damaged corneal endothelial rabbit eye model. (a) Representative phase contrast images of control ripasudil (1 and 10 µM) treated HCECs. Scale bar: 50 µm. (b) Upper images: Slit-lamp microscopic images of rabbit eyes after corneal freezing non-treated and treated for 48 h with ripasudil (0.4% × 4/day or 0.8% × 2/day). Lower images: Visualisation of wound area via Alizarin red staining. Scale bar: 1 mm (reprint with permission from Okumura et al., Investigative Ophthalmology & Visual Science, March 2016, Vol.57, 1284-1292). (B) Effect of topical netarsudil on the corneal haziness and endothelial tissue morphology. Silt-lamp images of eye and endothelial cell layer with corneal endotheliitis at initial evaluation and after 4 and 7 months of topical administration of netarsudil 0.02% × 2/day (reprint with permission from Li et al., International Immunopharmacology 2024, Vol.136, 112195). (C) Treatment effect with Y-27632. (a) Fluorescence images of rabbit corneal endothelium cells treated for 48 h with control solution or Y-27632 (10 mM) using Ki67 staining. Cell borders and cell nuclei were visualised using Phalloidin and DAPI, respectively. (b) Slit-lamp microscopic images of corneal endothelial damage rabbit model after Y-27632 (10 mM) × 4/day treatment compared to non-treated eye (reprint with permission from Okumura et al., Investigative Ophthalmology & Visual Science, September 2015, Vol.56, 6067-6074.) (c) Fluorescence slit-lamp microscopic images of human eyes with central CE and diffused CE before treatment and after 6 months post Y-27632 (10 mM) × 6/day for 7 days treatment. (d) Quantification of central corneal thickness (CCT) in a span of 6 months in patients with central CE compared to diffuse CE treated with Y-27632 (10 mM) × 6/day for 7 days (reprint with permission from Okumura et al., Investigative Ophthalmology & Visual Science, April 2013, Vol.54, 2493-2502).

[100,101], and were found to be more effective than EGF due to specific and potent proliferation and mitogenic effects on the endothelial cell culture (Fig. 3). In the corneal epithelium, an increase in the FGF-1 mRNA levels after epithelial burning was noticed [102], with FGF-2 found to stimulate wound healing in corneal epithelium through Cdc42 activation, Rho inactivation, and the phosphatidylinositol 3-kinase pathway [103]. This links the FGF pathway to the previously discussed ROCK pathway and suggests the potential of FGF pathway manipulation for an increase in cell migration and proliferation. Treating scrapped corneal endothelial cells in cat eyes with purified FGF induced excellent cell proliferation. However, the use of the canonical FGF is limited due to its short half-life in a biological system (FGF-1 – 29 h, FGF-2 – 7.6 h) [104,105], and an engineered FGF-1 was created to overcome the stability issues, opening the way for eye treatment [106–108].

TTHX1114 (N-Met C16S, A66C/C117V FGF1) was first tested on *ex vivo* epidermal corneal tissue and showed strong proliferative CECs response and stimulation of epithelial regeneration [109]. The topical application of TTHX1114 for the treatment of HSV-induced corneal keratopathy in an *in vivo* mouse model led to a significant reduction of inflammation rates and improved corneal clearance compared with untreated samples [110]. This study also showed developed resistance and shorter duration of the disease after 5 days of treatment. Following these results, scientists tested the influence of TTHX1114 on the human cornea in *ex vivo* organ culture [111]. Specifically, the healing of the wounded corneal endothelium after DSO procedure and treatment of TTHX1114 was assessed. Results revealed significantly improved wound healing rates and less scarring upon drug treatment. Moreover, the treated corneal wounded area was completely covered in CECs monolayer with tight cell-cell junctions. Currently, the ongoing in-depth clinical trial is testing the effect of the topical application of TTHX1114 for the treatment of corneal endothelial disorders. One of the described side effects could cause a neovascularization condition in the cornea [112,113].

## Regulation of oxidative stress and apoptosis with ascorbic acid

Oxidative stress is known to result in corneal endothelial impairment and the development of FECD and CHED [114]. Reactive oxygen species (ROS) can accumulate in CECs [115,116] and the induction of endoplasmic reticulum (ER) stress, decrease in concentration of $Ca^{2+}$ ions in ER storage, and upregulation of the unfolded protein response pathway are key cellular responses to oxidative stress [117,118] (Fig. 3). ROS downregulate the PI3P/Akt pathway through inhibition of the $PI_3$-mediated phosphorylation of Akt and subsequent inhibition of cell proliferation and induction of autophagic cell death [119]. This imbalance between ROS generation and antioxidant capacity can lead to disruption of cellular redox status, causing oxidative stress and premature senescence in CECs. The balance between ROS-induced oxidative stress and apoptosis and the nuclear factor erythroid 2-related factor 2 (Nrf2)/antioxidant responsive element (ARE) pathway has a complex regulatory mechanism [116]. During FECD, oxidative stress leads to

oxidative deoxyribonucleic acid (DNA) damage, particularly mitochondrial DNA damage, morphological modification of CECs, and, as a result, apoptosis. Studies demonstrated that, during oxidative DNA damage, transformation-related protein 53 (p53) plays a critical role in regulating oxidative-stress-induced apoptosis also by inhibiting the antioxidant Nrf2-ARE pathway during FECD [114]. Briefly, during this disorder, there is a decline in the Nrf2 antioxidant path and a deficiency in its stabilizers [120]. Additionally, upon the induction of oxidative stress, FECD CECs lack the relocation of Nrf2 and increase in the p53-induced cell death. The application of antioxidants and an enhancement of the Nrf2 levels are currently considered in the modulation of corneal endothelial oxidative stress levels.

Ascorbic acid (AA) is a known antioxidant used in the treatment of various diseases linked to oxidative stress (Table 3). A previous investigation evidenced the protective influence of AA against stress-induced HCECs injury and apoptosis *in vitro*, and reduced damage of rabbit cornea *in vivo* after topical application of AA [121]. In a case study, AA treatment was performed on two patients with FECD and endotheliitis following cataract surgery [122,123]. The overall state of the corneal endothelium after the surgery was comparable to other previously approved procedures, inferring the positive impact of the AA on the state of HCECs. While encouraging, more systematic *in vivo* and clinical studies are required to determine the efficiency, dosage, and other corneal endothelial disorders that can be treated using topical AA.

## Perspectives for other potential topical treatments for corneal endothelial disorders

While the treatments described above have been actively tested for their function when applied topically, several other chemicals have a great potential for use against endothelial disorders (Table 3), but are administered in an invasive manner. Indeed, an interesting approach is to focus on the use of inhibitors of bisperoxo phosphatase and tensin homolog deleted on chromosome ten (PTEN) bpV(pic) (Table 3). In HCECs, PTEN is activated by ROCK activity shown to negatively regulate cell migration and cell cycle progression by inhibition of released HCECs from the $G_1$-arrest state [124–127] (Fig. 3). While the treatment of CECs monolayer *in vitro* demonstrated increased cell migration and accelerated wound healing, the intracameral (IC) injection of bpV(pic) in rat model cleared corneal edema and decreased corneal thickness [128]. In this respect, nicotinamide (NIC) helps corneal endothelial wound healing when injected intraocularly [129] (Table 2). It protects CECs from ultraviolet B light (UVB)-induced apoptosis, stimulates cell proliferation, and inhibits EMT through inhibition of TGF-$\beta$1 *in vitro* [130] (Fig. 3). Researchers also observed the reverse of CECs irregular to hexagonal shape and thinning of corneal thickness.

It is important to consider more closely the use of formulations of inhibitors for TGF-$\beta$ and GSK3 and enhancers of Nrf2 protein and muscarinic pathway, which are currently only tested *in vitro* or *ex vivo* for topical application. TGF-$\beta$, the primary signalling molecule in ROCK pathway, is essential in wound healing in the cornea and its excess can lead to the increase of extracellular matrix in the endothelium (Fig. 3)
[131]. TGF-β receptor inhibitors, SB431542 and LY2109761, had a positive influence on the HCECs density and hexagonal cell morphology *in vitro* [132,133] (Table 3). While SB431542 has been mainly used for the maintenance of the CECs culture *in vitro* and prevention of the transition to fibroblast phenotype [134], LY2109761 positively influenced the HCECs culture hexagonal phenotype and inhibited the EMT *in vitro* [135].

Another inhibition of EMT has been linked to the regulation of GSK3 activity, a serine-threonine protein kinase downstream of FGF receptor [136] (Fig. 3). Upon GSK3 inhibition by CHIR99021, the induction of CECs proliferation was observed *in vitro* [10]. Research also evidenced that a balanced combination treatment of CHIR99021 and TGFβ1 of HCECs culture *in vitro* induced cell proliferation and migration rather than EMT [136].

The Nrf2 protein regulates the antioxidant response to oxidative stress [137] (Fig. 3). Sulforaphane (SFN) is a known Nrf2 enhancer and activator of the Nrf2-ARE pathway [138] (Table 3). *In vitro* and *ex vivo* studies on CECs reported protective properties of SFN against oxidative stress through decrease of ROS production and reduction of apoptosis levels [139]. Moreover, when normal HCECs were pre-treated with SFN, the increased rate of nuclear translocation of Nrf2 was detected as well as the lower levels of induced ROS production.

Oxotremorine is a selective muscarinic acetylcholine receptor (mAChR) agonist and was identified as a novel protective compound against oxidative stress for CECs culture *in vitro* [140] (Fig. 3). In BCECs and HCECs culture *in vitro*, oxotremorine effectively reduced oxidative stress levels potentially via altered DNA damage-inducible 3 (CHOP) gene expression [140] (Table 3). Furthermore, the study also found that oxotremorine increased the expression of glucose-regulated protein 78 kDa (GRP78) and activated transcription factor 4 (ATF4), which are both involved in the unfolded protein response (UPR) and can help protect cells from oxidative stress.

Another compound studied along with oxotremorine is mefenamic acid (MA) [140] (Table 3). It belongs to a group of nonsteroidal anti-inflammatory drugs and its primary mechanism of action is the inhibition of human cyclooxygenase 1 and 2 (hCOX-1 and hCOX-2, respectively). Researchers tested the protective properties of mefenamic acid on the BCECs and HCECs cultures *in vitro* upon induction of oxidative stress [140]. Results showed similar protective effects of the cell culture against DNA damage through modulating the expression of stress response proteins like GRP78 and reducing oxidative damage markers like protein carbonyl and 8-OHdG.

In conclusion, a substantial amount of research has been performed to create drugs that can be applied topically for the treatment and potential prevention of various corneal diseases that are linked to the posterior endothelial cell layer. Interestingly, only 5 are applied topically so far, including engineered FGF-1 protein (Table 3). ROCK inhibitor drugs, like ripasudil and netarsudil, have been originally used for the topical treatment of glaucoma and ocular hypertension. Their potential against corneal endothelial disorders was discovered later; however, prior medical approval of their safety helped speed up the reach of the clinical trial stage. The main reason to deliver experimental drugs to the corneal endothelium via intraocular injection is the guarantee of their fast delivery to the target tissue layer at a desired functional concentration. Passage of the chemical molecules through the corneal epithelium has been one of the great challenges for developing effective topical treatments. The size and charge of the molecules play an important role in this passage, therefore bpV(pic) and NIC have a potential for the efficient penetration of corneal tissues. However, extensive studies on the eye organoids as well as *in vivo* studies are needed to determine the efficacy and safety of these drugs when applied topically. The same is necessary for the recently presented drugs that so far have been only tested *in vitro*. Development and testing of new potential topical drugs with high permeability and treatment efficiency is an important step towards the global goal of substituting most corneal transplantations. Moreover, apart from the chemical-based drugs, more

novel gene-based therapy approaches and various delivery molecules, like nanoliposomes and nanoparticles, present an exciting possibility for specific, long term and efficient treatment and will be discussed in the following section.

## Next generation of therapy approaches: from gene therapy to penetration enhancers in the form of carbon quantum dots therapy

### Gene therapies: from adenovirus-vectors-based approaches to nanoparticles

As shown in the previous section, the use of chemical formulations for the treatment or moderation of corneal disorder conditions remains the most used for the topical strategy. In the case of diseases caused by drug intolerance, inefficiency or genetic mutations, a full corneal transplantation or transplantation of the corneal endothelial cell layer remains the only option available. With cell administering remaining a highly invasive and complex procedure performed by a specialist with a lot of risk factors, gene manipulation might be a non-invasive alternative [141]. Gene therapy represents an exciting direction in modern medicine, bringing several advantages compared to traditional treatment strategies [142]. It offers targeted modification, modulation, or repairment of underlying genetic abnormalities, with long-lasting effects and, in some cases, the possibility of only one-time treatment. Gene therapy (e.g. gene addition, gene editing) is based on the delivery of various types of RNA (e.g. messenger RNA (mRNA), antisense RNA (asRNA)/antisense oligonucleotides (ASOs), small interfering (siRNA), microRNA (miRNA) and double-stranded DNA (dsRNA)) [143,144] or DNA and genome editing machinery, like CRISPR-Cas9, TALE nucleases, or zinc-finger nucleases [141,145,146].

The only approved gene therapy for eye diseases is Luxturna against Leber congenital amaurosis (LCA) [147] via subretinal injection. LCA, also known as biallelic RPE65-mediated inherited retinal disease, causes blindness in children [147]. Luxturna uses an adeno-associated virus (AAV) vector-based delivery system with a functional copy of coding DNA (cDNA) of RPE65 gene. This gene encodes a key enzyme of the visual cycle called retinoid isomerohydrolase that converts all-trans-retinyl esters to 11-cis-retinol during phototransduction [148]. 11-cis-retinol is crucial in the process of visual pigment regeneration in retinal pigment epithelium, specifically in the photoreceptor cells. The mutations in this gene induce faulty protein folding and loss of function, resulting in the retinal pigment epithelial (RPE) cells dystrophy and photoreceptor degeneration. Concerns are often voiced around the use of adenovirus-based vectors, including in gene editing like BMP7, Smad7, Decorin, and HLA-G, for the treatment of corneal scarring, neovascularization, and fibrosis [149,150]. While these examples highlighted the advancement for the treatment of corneal diseases linked to the outer corneal layers (e.g. epithelium and stroma) (Fig. 1), only a very limited number of studies were directed to gene therapy of the corneal endothelium and topical drug delivery [148].

ASOs are currently largely advanced for gene silencing, owing to their high specificity against target mRNA and the mechanism of following mRNA degradation [151] and many *in vivo* studies are directed at using ASOs towards inherited retina diseases [152]. Delivery of ASOs through various administration routes, including intravenous and IC injections to the cornea for treating corneal diseases was demonstrated by Chau et al. [153]. An ASO-mediated knockdown of *MALAT1* regardless of the mode of administration was observed by them independent of whether IV or IC injection was performed. Motivated by these findings, the topical delivery of ASO was additionally investigated (Fig. 5) [153]. No inhibition of *MALAT1* expression was observed, however.

Zarouchliot et al. developed a FECD subject-derived corneal endothelial cell model to investigate the impact of ASO treatment on corneal endothelial cells derived from FECD patients [154]. Transfecting with
<figure><img src="image_9.png" /><figcaption>**Fig. 5.** ASO-Mediated treatments of corneal endothelial cells. (a) Percentages of nuclei that contain 0 foci after treatment with the different ASOs. Mean ± SD are represented (n = 6). (b) Violin plots representing the distribution of the frequencies of each group of nuclei in cells treated with control and (CAG)₇ ASOs (n = 6). (c) Fluorescence images of ASO treatment on foci incidence. Sense RNA foci detection using Cy3-(CAG)₇ probe (red). Scale bars, 10 µm. (reprint with permission from Zarouchliot et al., The American Journal of Human Genetics April 2018, Volume 102, Issue 4, Pages 528–539).</figcaption></figure>

2'-O-Me-PS-(CAG)₇ ASOs induced reduction in CUG-specific RNA foci, a biomarker of CTG18.1-related pathology using a cell culture model. A significant reduction in foci number was observed, indicative of RNA (Fig. 5). *In vivo* studies in mice evidenced, in addition, the presence of ASOs in the corneal endothelium upon intravitreal injection. This study is an essential prerequisite for further investigation of topical application concepts.

Substantial effort has been directed in addition to establishing the use of non-viral-based gene therapy for the treatment of ocular diseases. Plasmid DNA (pDNA) is a non-viral delivery vector that uses engineered plasmid vectors containing regulatory elements for expression alongside the inserted therapeutic gene, in the form of a naked plasmid or packed into various types of nanoparticles, which can be another gene therapy option [155]. pDNA is relatively simple to construct at low production costs; it can accommodate large segments of genomic DNA, has high stability for transportation and long-term storage and owns an excellent safety profile, compared to viral vectors. One of the main limitations of plasmid-mediated gene therapy remains the poor transduction efficiency and therefore treatment efficacy. Researchers have demonstrated that injection of naked pDNA directly into the corneal stroma results in the efficient transfection of corneal keratocytes and epithelial cells. The injection of pDNA carrying the vascular endothelial growth factor (VEGF) gene effectively induced corneal and anterior chamber neovascularization.

To achieve higher transfection efficiency, physical methods like electroporation, magnetofection, sonoporation, etc., are incorporated into the therapy. Electroporation is a preferred method when it comes to ocular transfection *in vivo* [156]. During the electroporation, short electrical impulses increase cell plasma membrane permeability, allowing pDNA to easily penetrate target cells. This non-viral method has been utilized to mediate the successful delivery of pDNA into the retinal cells [157]. One study combined the suprachoroidal injection of plasmid DNA with the application of an electrical field. Suprachoroidal electroporation of a plasmid encoding a soluble VEGF receptor-1 (sFlt-1) significantly inhibited choroidal neovascularization (CNV) in a rat model, highlighting its potential therapeutic applications. In the context of corneal endothelium electroporation, naked DNA has been used in *in vitro* settings; however, *in vivo* tests are needed to study the efficacy of this method to be used for the transformation of this cell layer.

### CRISPR-Cas9 technology

Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) technology represents a major advancement in gene editing, with the potential to transform treatments for genetic eye disorders [158]. The most widely known CRISPR-Cas9 system consists of the Cas9 enzyme, which acts as "molecular scissors" to split DNA at a specific nucleotide sequence. This location is provided by guide RNA (gRNA) via binding to the Cas9 enzyme. The gRNA consists of 20 nucleotides that are complementary to the target DNA allowing Cas9 to recognize and bind to the desired genomic location. Once bound, the Cas9 enzyme creates a double-strand break in the DNA at the target site. This allows the removal, addition, or alteration of DNA segments [159], making it a highly versatile and efficient tool for gene editing. As with any other system, CRISPR-Cas9 has its limitations. A key limitation of CRISPR is the requirement for a protospacer adjacent motif (PAM) sequence next to the target site, which restricts the range of targetable sequences. Another limitation of this tool is the potential off-target effect, when the Cas9 enzyme can edit unintended sections of the genome, causing unwanted alterations.

Several different studies have been conducted towards the application of CRISPR-Cas9 technology for the treatment of LCA [160,161], HSV keratitis [162] and FECD [163]. Regarding the application of gene editing tools against corneal endothelial disorders, the CRISPR-Cas9 system has been tested *in vivo* against Fuchs' endothelial corneal dystrophy FECD. In the FECD mice model with mutated Col8a2<sup>Q455K</sup>/Col8a2<sup>Q455K</sup> gene, researchers used CRISPR-Cas9 to disrupt the start codon of the mutant Col8a2 gene of early-onset FECD. A single intraocular injection of an adenovirus carrying Cas9 and a guide RNA targeting Col8a2 efficiently knocked down the mutant gene in corneal endothelial cells. This prevented endothelial cell loss, rescued CECs pumping function, and prevented vision loss in the mice without adverse effects (Fig. 6). These results hold great promise for a non-surgical strategy to treat early-onset FECD by restoring function in post-mitotic corneal endothelial cells. This could potentially lead to a significant reduction of FECD cases that need corneal transplants. Further research is needed to optimize the approach and test it in human cells and larger animal models before clinical trials. Summarising, these studies demonstrated the promise of using CRISPR-Cas9 gene editing to treat ocular diseases, including corneal endothelial disorders like FECD by disrupting disease-causing mutations and restoring function.
<figure><img src="image_10.png" /><figcaption>**Fig. 6.** CRISPR-Cas9 knock out of mutated *Col8a2* gene after intracameral injection. (a) Images of corneal endothelium from the control group, non-injected Col8a2<sup>Q455K/Q455K</sup> mice and Col8a2<sup>Q455K/Q455K</sup> mice injected intracameraly with Ad-Cas9-Col8a2gRNA stained with Alizarin red. Scale bar = 200 µm. (b) Average corneal endothelium densities. Error bars show standard deviation. (c) Periodic Acid-Schiff (PAS)-stained corneas from non-injected and Ad-Cas9-Col8a2gRNA-injected Col8a2<sup>Q455K/Q455K</sup> mice. (d) De-swelling of central corneal thickness was measured from 0 to 50 min. Non-injected Col8a2<sup>Q455K/Q455K</sup> corneas displayed significantly delayed de-swelling compared to C57BL/6 J corneas. In contrast, Ad-Cas9-Col8a2gRNA injection significantly improved the corneal de-swelling rate similar to that of C57BL/6 J controls (n = 6). \*p < 0.05 by Student's t-test (reprint with permission from Uehara et al., Elife. 2021 Jun 8;10:e55637).</figcaption></figure>

### Nanoparticle-mediated delivery

Nanoparticle-mediated drug administration has been a successful approach with several formulations currently in clinical trials [164, 165]. NPs for clinical use have several advantages, such as multi-vacancy, with the ability to integrate multiple therapeutic moieties on one particle and allow specific cell targeting due to the attachment of peptides and proteins on the surface of the NPs. They can be scaled up efficiently and relatively cheaply and can prolong the half-life and stability of therapeutics, as exemplified before for RNA delivery. Nanoparticle-mediated cell delivery thus becomes also a promising approach for enhancing corneal endothelial regeneration. The aim of these strategies is to improve cell survival and integration, as well as therapeutic efficacy while minimizing off-target effects and immune responses.

The interaction between NPs and the corneal endothelial cells is essential in advancing corneal regeneration and drug delivery strategies. One open question relates to NPs' uptake mechanism by the eye in contrast to that of free therapeutics (Fig. 1), which have shown until now

limited diffusion capability to the anterior part of the eye. Fostering the interaction of soft nanoparticles such as polymer and hydrogel-based ones can promote the growth and functionality of stem cells crucial for corneal regeneration, something not achievable with drugs only. Assessing poly(lactic-co-glycolic acid) (PLGA) NPs to encapsulate VEGFA-specific siRNA for targeting corneal vascularization by reduction of VEGFA expression proved to be promising. The inhibitory effect of siRNA on corneal neovascularization was comparable to that of mononuclear antibodies against VEGF-A [166]. It has also been suggested for use against herpetic stromal keratitis targeting the VEGF pathway. Visudyne, a verteporfin-loaded liposomal formulation, remains one example of the treatment of macular degeneration. Mucus-penetrating particles for enhanced penetration of loteprednol etabonate have been more recently FDA-approved [36] and employed for dry eye symptoms. More recently, extracellular vesicles (EVs) emerged as a possible key player for eye diseases. Burnouf's team demonstrated that corneal endothelial cells treated with blood-derived EVs show improved viability, and accelerated wound-healing [167,168]. These data open the perspectives of using EVs as eye drop formulations for the repair of
the corneal endothelium. Studies demonstrated furthermore that silica nanoparticles do not induce cytotoxicity in corneal endothelial cells, underscoring their potential for intraocular drug delivery [169]. These examples underscore the promise of harnessing nanotechnology for enhanced corneal treatment possibilities.

Charge and size are essential parameters to be tuned as the cornea carries a negative charge with chitosan, gelatine, or poly-L-arginine-based NPs most adapted for ocular and transepithelial drug absorption. The TJs disruption properties of chitosan result in an increase in the cornea epithelium permeability [34]. The increased presence of cyclosporin A (CyA) in cornea/conjunctiva/aqueous humour after topical administration of CyA-loaded chitosan NPs rather than bulk chitosan is underlying enhanced permeability [170]. BMP7 gene transfer via polyethylenimine-conjugated gold nanoparticles (PEI2-Au NPS) modulated corneal fibrosis in stromal tissue [171]. Recently, PEI2-Au NPs have been beneficial for the delivery of genes to rabbit corneas in vivo using a topical application [172] (Fig. 7A) with high levels of gold detected in the PEI2-Au-NPs treated rabbit corneas. These findings suggest that PEI-Au-NPs could serve as a safe and efficient platform for delivering therapeutic genes to specific corneal cells. Additionally, these nanoparticles can be loaded with large therapeutic genes, making them a promising candidate for the development of corneal nanomedicine.

Carbon quantum dots (CQDs) have been more widely considered for eye-related applications [173-177]. Their nanometric size, ease of synthesis from different carbon precursors using carbonization approaches and the subsequent variety of properties motivated their application in the context of ocular nanomedicine. Positively charged

CQDs showed the possibility of destroying vitreous opacities [174], and permeating the corneal epithelium. CQDs [176,178], prepared from spermidine showed corneal penetration until the endothelium mostly via the paracellular transport route (Fig. 7B). They are believed to act as permeation enhancers as they showed the delivery of dextran of 150 kDa. Integration of VEGF aptamer onto CQDs inhibited VEGF-stimulated angiogenesis in choroidal blood vessels in a comparable manner to two commercially available anti-VEGF drugs, bevacizumab and aflibercept, making the nanoparticulate approach versatile to treat age-related macular degeneration and diabetic retinopathy [177].

Regulatory considerations are paramount in utilizing nanostructures for biomedical means. The regulatory safety evaluation is particularly critical to ensure safe utilization [35]. The literature remains rather contradictory on the toxicity issue. Gold nanoparticles of 20 and 80 nm were shown to cause oxidative stress and apoptotic damage to retinal cells with potential visual impairment [179]. AuNPs of 160 nm, upon intravitreal injection, showed, on the other hand, no toxic effects on retinal integrity and function in a mouse model [180]. Titanium dioxide (TiO₂) nanoparticles of < 45 nm caused ocular surface damage and reduced conjunctival goblet cell area in rabbits in vivo; however, when using TiO₂ NPs of 12 nm in zebrafish no retinal neurotoxicity was observed [181]. On the other hand, both graphene oxide and magnetic NPs showed no toxicity and great potential for future developments [182,183]. Clearly, a more comprehensive approach is essential to give safety guidelines to ensure that innovation is safeguarding the wellbeing of the individual.

<figure><img src="image_11.png" /><figcaption>**Fig. 7.** Nanoparticles for the treatment of eye disorder: (A) PEI2-Au NPs transfection of corneal cells. (b) Fluorescence images of human corneal fibroblast cells treated with PEI2-Au NPs compared to control. Nuclei are stained blue with DAPI. Scale bar 100 µm. (c) Slit-lamp microscopic images of rabbit corneas treated with PEI2-Au NPs compared to untreated eye at 12 h and 7 days post application (reprint with permission from Sharma, et al., Nanomedicine. 2011 Aug; 7(4): 505-513). (d) TEM image of CQDs along with fluorescence confocal microscopy images of *ex vivo* bovine cornea's epithelium and endothelium before and after topical application of CQDs for 1 h (reprint with permission for deHoon et al. ACS App. Mater. Inter. 2023, 15, 3).</figcaption></figure>
## Conclusion

This review aimed to summarise recent progress in various treatment strategies for corneal disorders, specifically emphasizing the non-invasive treatments of corneal endothelial diseases. While the ocular diseases linked to the anterior corneal layers have a vast selection of effective topical treatments, the diseases of the corneal endothelium are predominantly treated using extremely invasive techniques, like full or partial corneal transplantation. The multilayer anatomy of the cornea is one of the main reasons for the complicated delivery of effective treatment dose concentrations when applied topically (Fig. 1). However, with an extreme shortage of available corneal donors, more research was devoted to the development of effective topical treatments. Chemical drugs are suggested as an alternative non-invasive treatment that can be applied for various corneal endothelial diseases. While some of these therapeutics are derived from formulations that have been used against other eye diseases, like glaucoma, others are developed to regulate signalling pathways of cell proliferation, migration and apoptosis (Table 3). Therapeutic formulations like Y-27632, ripasudil, and netarsudil, have been approved for use in some cases of FECD. Other chemical formulations exhibited promising results in *in vitro* and *in vivo* studies, showing potential for the expansion of the pharmaceutical market with new non-invasive treatments of specific corneal endothelial disorders. The challenge in using these treatments topically, and not as intraocular injections, is related to preserving their working concentration after the corneal penetration, as their effectiveness is directly proportional to the concentration of the drug at the corneal endothelium site. Therefore, firstly, it is important to discover and test small chemical molecules that can effectively penetrate corneal layers in a short period. Secondly, development of biocompatible carriers that can mediate fast and effective drug passage through the outer corneal tissue area holds great importance in enabling the progress of topical eye treatments.

The exploration of next-generation therapeutic approaches for ocular diseases, particularly corneal disorders, highlights the promising potential of gene therapy and innovative delivery systems. As traditional treatments often fall short in efficacy or involve invasive procedures, emerging strategies, such as CRISPR-Cas9 gene editing, antisense oligonucleotides (ASOs), and nanoparticle-mediated delivery, offer non-invasive alternatives that could significantly improve patient outcomes. Novel therapeutic strategies that involve manipulation of protein expression levels and correction of genetic mutations are getting approved for the treatment of severe diseases, including ones related to ophthalmology. Section 4 described distinct types of gene therapy approaches in the frame of their application in studies related to corneal diseases. A number of them targeted specifically corneal endothelial diseases, like the combination of adenovirus delivery system and CRISPR-Cas9 machinery to knock down the expression of the mutant COL8A2 protein in corneal endothelial cells of a mouse model of early-onset FECD by Uehara et al. There is a long way for gene therapy medicine to be approved in general medical practice; however, the increasing number of successful medical cases treated using gene therapy proves the need for more research and clinical trials directed towards developing such therapies, particularly towards corneal endothelial disorders. The utilization of gene therapy has the potential to prevent the development of many genetic disorders at the early stages, reducing drastically the need for corneal transplantation.

Nanoparticles have started to revolutionize ocular drug delivery in a different manner for a variety of eye diseases, owing to the versatile character of these structures. Several clinical challenges have still to be overcome in nanoparticle-mediated drug delivery for ocular applications, which is paramount for advancing this field. Next to safety concerns surrounding nanoparticle-based drug delivery systems, accessibility, upscaling and quality control remain technical challenges to be addressed. As NP ocular therapy moves to clinical implementation, it remains imperative to include a larger variability of stakeholders and include the vision of healthcare professionals and patients alike.

Comprehensive preclinical and clinical trials to evaluate the long-term efficacy and safety of these therapies is becoming essential for the success of the translation of research to clinical practice.

The development of non-invasive ocular treatment approaches to reach deep eye tissues, including the corneal endothelium, is interlinked with the efforts to use different strategies for penetration of these treatments through the exterior eye tissues. While some strategies include the use of contact lenses, ointments, and gels to prevent the fast washing off the treatment, the application of nanoparticles for enhancing the treatment penetration speed holds great potential for increasing the treatment effect. Distinct types of these nanostructures are suitable for packaging and delivery of treatment depending on their chemical or biological nature and the target tissues. The ability of nanoparticles to incorporate multiple therapeutic agents, target specific cells, and enhance the stability and half-life of drugs makes them an attractive option for clinical applications. Recent research has demonstrated the potential of various nanoparticles to enhance drug penetration and therapeutic efficacy. Additionally, CQDs have shown promise in permeating the corneal epithelium and enhancing drug delivery. The development of hybrid nanoparticles, such as PEI2-Au-NPs, for gene delivery further illustrates the versatility and potential of these systems in treating eye disorders. However, the translation of these technologies into clinical practice requires a deeper understanding of the uptake mechanisms, optimization of nanoparticle properties, and addressing patient-specific factors. As research progresses, the integration of nanoparticles with existing therapeutic strategies could lead to more effective and personalized treatments for ocular diseases, ultimately improving patient outcomes. The continued exploration of nanoparticle-mediated delivery systems holds the potential to revolutionize the field of ophthalmology and beyond, offering new solutions for previously intractable medical challenges.

In summary, while challenges remain in optimizing these therapies for broader clinical application, the ongoing research and development in gene therapy and innovative drug delivery systems represent a transformative step in the management of ocular diseases. Continued investigation and refinement of these technologies will be crucial in realizing their full potential, ultimately leading to improved treatment options and enhanced quality of life for patients suffering from corneal and other eye disorders.

## CRediT authorship contribution statement

**Szunerits Sabine:** Writing – review & editing, Writing – original draft, Supervision. **Boukherroub Rabah:** Writing – review & editing. **Ngyliukh Nataliaia:** Writing – review & editing, Writing – original draft.

## Declaration of Generative AI and AI-assisted technologies in the writing process

During the preparation of this work, the author(s) used Perplexity.ai to improve the readability and language of the manuscript. After using this tool/service, the author(s) reviewed and edited the content as needed and take(s) full responsibility for the content of the published article.

## Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

## Acknowledgements

Financial support from the Danube Private University (DPU), the Centre National de la Recherche Scientifique (CNRS), and the University of Lille are acknowledged. Support of the government of Lower Austria
and Tyrol under the COMET Project «PI-SENS» (Personalized Medicine enabled by intelligent sensing systems) FFG grant no. FO 999915477 is acknowledged.

## Appendix A. Supporting information

Supplementary data associated with this article can be found in the online version at doi:10.1016/j.nantod.2025.102740.

## Data availability

No data was used for the research described in the article.

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**Dr. Nataliia Gnyliukh** obtained her Master's diploma in Biochemistry from the Ivan Franko National University of Lviv, Ukraine. She continued her studies as a PhD candidate at the Institute of Science and Technology Austria (ISTA), where she was co-affiliated with two groups of prof. Jiri Friml and prof. Martin Loose for in-depth studying of Clathrin-Mediated Endocytosis. After she defended her PhD in 2023, Nataliia joined the LiST group in 2024 as a researcher to develop various applications of personalised nanomedicine, including endogenous biomarker detection and non-invasive therapeutics for corneal diseases.

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**Dr. Rabah Boukherroub** received a PhD in chemistry from the University Paul Sabatier in Toulouse, France. He is currently a research director at the Institute of Electronics, Microelectronics and Nanotechnology (IEMN), CNRS & University of Lille, France. His research interests are in the area of functional materials, surface chemistry, and photophysics of semiconductor/metal nanostructures with emphasis on biosensors, nanomedicine, and electrocatalysis. He published many research publications, wrote 43 book chapters and is co-editor of 10 books in subjects related to nanotechnology, materials chemistry, and biosensors. He has 13 patents or patents pending.

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**Dr. Sabine Szunerits** is since 2009 Full professor in Chemistry at the University of Lille, and attached scientifically to the Institute of Electronics, Microelectronics and nanotechnology (IEMN). Since July 2024, she is on Sabbatical leave at the Danube Private University (DPU). Her research focus is on the use of material science and chemistry to tackle unmet medical-related problems such as the development of therapies against viral and bacterial infections using nanoparticles, sensitive bacterial and viral diagnostics, or transdermal/buccal/ocular delivery of therapeutic drugs. The work is published in > 500 peer-reviewed papers, and 24 book chapters. She has edited 4 books and has 10 patents pending (two licensed).