Ocular Delivery of Therapeutic Proteins: A Review: Comparison
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Please note this is a comparison between Version 1 by Divyesh H Shastri and Version 2 by Rita Xu.

Therapeutic proteins, including monoclonal antibodies, single chain variable fragment (ScFv), crystallizable fragment (Fc), and fragment antigen binding (Fab), have accounted for one-third of all drugs on the world market. In particular, these medicines have been widely used in ocular therapies in the treatment of various diseases, such as age-related macular degeneration, corneal neovascularization, diabetic retinopathy, and retinal vein occlusion. The formulation of these biomacromolecules is challenging due to their high molecular weight, complex structure, instability, short half-life, enzymatic degradation, and immunogenicity, which leads to the failure of therapies. Various efforts have been made to overcome the ocular barriers, providing effective delivery of therapeutic proteins, such as altering the protein structure or including it in new delivery systems. These strategies are not only cost-effective and beneficial to patients but have also been shown to allow for fewer drug side effects. Researchers discuss several factors that affect the design of formulations and the delivery of therapeutic proteins to ocular tissues, such as the use of injectable micro/nanocarriers, hydrogels, implants, iontophoresis, cell-based therapy, and combination techniques. In addition, other approaches are briefly discussed, related to the structural modification of these proteins, improving their bioavailability in the posterior segments of the eye without affecting their stability. Future research should be conducted toward the development of more effective, stable, noninvasive, and cost-effective formulations for the ocular delivery of therapeutic proteins. In addition, more insights into preclinical to clinical translation are needed.

Therapeutic proteins, including monoclonal antibodies, single chain variable fragment (ScFv), crystallizable fragment (Fc), and fragment antigen binding (Fab), have accounted for one-third of all drugs on the world market. In particular, these medicines have been widely used in ocular therapies in the treatment of various diseases, such as age-related macular degeneration, corneal neovascularization, diabetic retinopathy, and retinal vein occlusion. However, the formulation of these biomacromolecules is challenging due to their high molecular weight, complex structure, instability, short half-life, enzymatic degradation, and immunogenicity, which leads to the failure of therapies. Various efforts have been made to overcome the ocular barriers, providing effective delivery of therapeutic proteins, such as altering the protein structure or including it in new delivery systems. These strategies are not only cost-effective and beneficial to patients but have also been shown to allow for fewer drug side effects. In this review, we discuss several factors that affect the design of formulations and the delivery of therapeutic proteins to ocular tissues, such as the use of injectable micro/nanocarriers, hydrogels, implants, iontophoresis, cell-based therapy, and combination techniques. In addition, other approaches are briefly discussed, related to the structural modification of these proteins, improving their bioavailability in the posterior segments of the eye without affecting their stability. Future research should be conducted toward the development of more effective, stable, noninvasive, and cost-effective formulations for the ocular delivery of therapeutic proteins. In addition, more insights into preclinical to clinical translation are needed.

  • ocular diseases
  • sustained ocular delivery
  • therapeutic proteins

1. Introduction

Millions of people worldwide are affected by the diabetic retinopathy (DR), a neurodegenerative disorder of retina, which is one of the most common causes of blindness involving other complications, such as retinal vein occlusion (RVO) and corneal neovascularization (CNV) [1].
DR is caused by the damage of blood vessels at the back of the eye and does not show initial symptoms that lead to early DR or nonproliferative DR, where no new blood vessel growth occurs and patients have dilation of pre-existing capillaries, oedema, capillary occlusion, microaneurysms, and intraretinal neo-angiogenesis, leading to tortuous blood vessels formation [2]. Such proliferative vascular changes subsequently turned to severe-type damage to blood vessels and showed growth of fragile leaky blood vessels (neo-angiogenesis) in the retina called proliferative DR that leak a jelly-like substance, filling the center of the vitreous, leading to detachment of the retina from the back of the eye. Patients might observe black spot or floating strings in the vision, blurring or fluctuating vision, dark or empty areas in the vision, hemorrhage in the vitreous, and glaucoma, leading to gradual weakening of the vision [1][2][3].
A common cause of vision loss in older people is age-related macular degeneration (AMD), in which patients show degeneration of the retinal pigment epithelial cells and choroidal neovascularization [3]. In dry AMD, the macula thins (atrophic) with age in some patients. In wet AMD, known as neo-vascular AMD, the new vessel growth is the major cause that occurs with abrupt onset of central RVO, leading to capillary occlusion and inducing tissue hypoxia, increasing vascular endothelial growth factor (VEGF) expression and resulting in retinal proliferation of new vessels [3][4][5]. Thus, researchers are investigating new therapies that involve the use of monoclonal antibodies, vascular growth factors, oligonucleotides, genes, and anti-VEGF agents (e.g., ranibizumab, bevacizumab, aflibercept), for the prevention of neo-angiogenesis and stabilization of vascular leakage and, thereby, reducing the oedema.
Several therapeutic proteins have recently been approved on the market for the treatment of ocular diseases. Although many of these proteins have low molecular weight (<50 kDa) and short half-life, the physiological and anatomical barriers of the ocular tissues limit their efficacy when administered to the posterior segments of the eye. In addition, the ocular environment makes them unstable and inactive, leading to the failure of the treatment. Among the factors that contribute to this is the presence of proteolytic enzymes, such as trypsin, in the vitreous, which can increase with aging, resulting in degradation of injectable proteins Moreover, various static, dynamic, and metabolic barriers are responsible for short half-lives of therapeutic proteins [6][7].
Anti-VEGF delivery to the posterior segment of the eye by the intravitreal route is very painful, involving the use of a needle to penetrate the globe and release the drug into the vitreous. Moreover, repeated injections are required during the treatment, leading to increased further complications such as cataracts, retinal tears, endophthalmitis, and retinal detachment [7].
Thus, the research focus should be directed at reducing the dosing frequency (e.g., novel prolonged release formulations) and development of novel noninvasive methods or devices for drug administration (e.g., through nonparenteral routes). So far, researchers worldwide have investigated several strategies for the treatment of retinal diseases to minimize the limitations or gap within the current therapies involving therapeutic proteins, reducing patient administration pain while improving compliance. The use of depot formulations of injectable carriers containing drug-loaded micro- or nanoparticles, injectable in situ hydrogels, implants, and cell-based systems are among the most useful approaches to provide safe and sustained ocular delivery of therapeutic proteins [8]. These formulations can improve the ocular drug bioavailability and help reduce the frequency of drug administration, providing an increased drug residence time within the intraocular tissues and improving the treatment efficacy with good patient compliance. In addition, cell-based systems and cell-penetrating peptides (CPPs) are also offering good ocular bioavailability indicated from the phase III clinical trials on an anti-inflammatory peptide conjugated CPP delivery [9].
Ideal therapeutic protein ocular delivery systems should provide stable delivery of encapsulated proteins, sustained release, maintenance of effective concentrations at the target tissues, and minimal invasiveness with low systemic exposure. A usual practice is to combine technologies, such as injectable hydrogels containing nano- or microparticles, liposomes, or nanoparticles containing therapeutic proteins coated with bioadhesive polymers [8][9]. Advantages of sustained delivery of therapeutic protein formulations include improved patient compliance, adherence to chronic therapy, and local delivery with fewer side effects and a reduction in dosage and dosing frequency [10].
Currently, great attention is being focused on the development of a more effective noninvasive, sustained drug delivery in the treatment of ocular disorders for the anterior and posterior segments of the eye.

2. Routes of Ocular Drug Administration

Achieving an efficient ocular bioavailability of different therapeutic proteins remains a challenge due to presence of multiple ocular barriers (Figure 1). Moreover, diseases such as age-related macular degeneration, diabetic retinopathy, and cytomegalovirus (CMV) retinitis require therapeutic proteins to be delivered to the back of the eye. Herein, static barriers (different layers of cornea, sclera, and retina including blood aqueous and blood–retinal barriers), dynamic barriers (choroidal and conjunctival blood flow, lymphatic clearance, and tear dilution), and efflux pumps, in combination, constitute a significant challenge for drug delivery to the posterior segment of the eye [11].
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Figure 1. Schematic representation of various formulation approaches and routes of administration to the ocular tissues. 1. Transcorneal permeation into the anterior chamber, 2. Noncorneal drug permeation across conjunctiva to sclera into anterior uvea, 3. Drug distribution into anterior chamber from blood stream through blood aqueous barrier, 4. Drug elimination from anterior chamber by aqueous humor to trabecular meshwork and Schlemm’s canal, 5. Elimination of drug from aqueous humor into systemic circulation across blood aqueous barrier, 6. Distribution of drug from blood into posterior segment across the blood retinal barrier, 7. Intravitreal route, 8. Drug elimination from vitreous via posterior route across blood retinal barrier, 9. Elimination of drug from vitreous via anterior route to posterior chamber, 10. Intracameral route, 11. Intrastromal route, 12. Subconjunctival route, 13. Subtenon route, 14. Suprachoroidal route, 15. Subretinal route.
The elimination of therapeutic proteins from the body is similar to the endogenous peptide molecules, i.e., enzymatic cleavage from liver, kidney, blood, and small intestine, although those that show enzymatic resistance can be eliminated via liver or kidney based on their lipophilicity. Only less than 1% of therapeutic proteins with molecular weight >4000 Da show undesirable immune response after administration, which led to the failure of some clinical trials [12]. Among the main barriers present in the eye that hinder the ocular delivery of therapeutics are static barriers and dynamic barriers. Static barriers include different layers of cornea, sclera and retina, and blood aqueous barriers (BAB) (Figure 2), while dynamic barriers comprise tear film, choroidal and conjunctival blood flow, and lymphatic clearance, which hinder the movement of drug molecules from the anterior part of the globe to the posterior tissues [12][13]. High selectivity of blood retinal barriers (BRB) limits the movement of topically instilled drugs to the posterior segment. Moreover, systemically administered drugs have to cross the blood ocular barriers, i.e., BAB and BRB, to reach the retina (Figure 2). The use of the systemic route for the delivery for ocular therapeutics has several limitations related to the need of high doses due to systemic metabolism and poor permeability across the BRB. Moreover, exposure to nontargeted organs may cause systemic toxicity and severe adverse effects [13].
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Figure 2. Different routes for ocular drug clearance/elimination.
The topical route is preferred for the delivery of drugs to the anterior chamber of the eye for the treatment of cataract, dry eye, and corneal and conjunctival inflammatory and infectious diseases [13]. The topical ocular delivery route is not commonly used for the delivery of therapeutic proteins for retinal tissues due to the presence of ocular barriers; only <5% of the instilled dose enters through anterior segment to the posterior segment via the tear film and cornea (epithelium, endothelium, and stroma) to the anterior chamber of the eye [8][9][10][14][15]. The extent of absorption of drug molecules from the corneal surface is severely limited by different physiological barriers, such as: (1) Corneal epithelium that selectively inhibits the diffusion of hydrophilic and high molecular weight molecules such as proteins and peptides through the paracellular route, and it selectively prevents ion transport. Permeability of macromolecules is severely limited by the presence of tight junctions of the cornea and the lipophilic nature of the corneal epithelium. (2) The endothelium, which is responsible for corneal hydration. (3) Inner stroma, which presents a hydrophilic nature and inhibits the permeation of more lipophilic molecules [15][16]. These barriers protect the eyes from the entry of toxic entities and pathogenic substances and maintain homeostasis. Moreover, due to the high shear rate, tear turnover, and tear dilution, most (>95%) of the instilled dose is eliminated via the nasolacrimal duct to the gastrointestinal tract, leading to other systemic side effects (Figure 1). The presence of enzymes in the ciliary body digest the drug from the aqueous humor, and the corneal permeability is also limited depending on molecular size, surface charge, and hydrophilicity of drugs [17]. Large and hydrophilic drugs showed poor permeability compared to small and lipophilic peptides from the corneal epithelial tight junction (about 2 nm) [17]. Positively charged molecules can pass easily due to binding with the negatively charged corneal membrane [15]. Lipophilic drugs are distributed to corneal tissues via the transcorneal pathway (i.e., cornea > aqueous humor = iris = ciliary body > anterior sclera > lens), while hydrophilic drugs tend to move toward the posterior chamber via the conjunctival–sclera pathway. Large molecular drugs that show poor corneal permeability bypass the corneal epithelium penetration route and undergo noncorneal absorption [18]. Lipophilic peptides with molecular size >700 Da exhibit good membrane permeability [19][20].

2.1. Intraocular

Intraocular delivery involves delivery through injection or implants of sterile solutions or devices in the ocular tissues via (1) intravitreal, (2) subretinal, or (3) suprachoroidal routes.
(a)
Intravitreal
The intravitreal route targets drugs to the retina, providing higher drug bioavailability directly into the posterior segment, avoiding several ocular barriers, and eliminating problems associated with systemic toxicities. The vitreous has a mesh size of 500 nm that provides a loose barrier and allows diffusion and convection of large and small molecules as well as nanoparticles [21][22][23]. Molecular mobility in the vitreous also depends on the charge of the protein molecules, i.e., neutral and anionic molecules can diffuse more easily compared to cationic ones due to electrostatic interactions with the anionic hyaluronic acid polymer network in the vitreous [24]. Metabolic activity in the RPE determines the bioavailability of protein molecules injected intravitreally due to degradation by the presence of enzymes, i.e., cytochrome P450 and esterases [25]. PEGylation attachment of a high molecular weight hydrophilic moiety to the drug molecules, i.e., polyethylene glycol, either by covalent or noncovalent linkage or encapsulation in the nanoparticles, can dramatically reduce the enzymatic degradation [22]. Clearance observed between the posterior segment and anterior segment after intravitreal injection depends on the size, property, and concentration gradient. Molecules from the posterior segment diffuse to the inner limiting membrane (ILM) and finally reach to retina (Figure 2). The clearance efficiency also depends on penetration efficiency through the tight junction of RPE as the small and lipophilic ones can be transported easily compared to large and hydrophilic proteins [26]. From the retinal layer, the molecules pass through the choroidal blood vessels to the systemic circulation. Molecules that diffuse toward anterior side can be drained away into blood or lymphatic vessels via trabecular meshwork or Schlemm’s canal [26].
(b)
Subretinal
From subretinal injection, direct administration of molecules to the retina can be possible, so it is the most preferable and efficient route for the delivery of therapeutic proteins with low membrane permeability and of retinal gene therapy [27]. Drugs administered via this route to the inner layer of the retina are cleared via the anterior segment and not through choroidal vessels as RPE tight junctions limit the movement of drugs toward the outer layer of the retina and lead to damage to the RPE and retina (Figure 2) [28].
(c)
Suprachoroidal
With the help of microneedles or cannulas, drugs can be administered via this route beneath the sclera into the suprachoroidal space, allowing the drug to be available at the choroidal site. Drug distribution is uneven due to restricted movement from ciliary arteries of the choroid. Moreover, due to high blood flow in the choroidal blood vessels, most of the administered drug is lost to the systemic circulation, which leads to short half-lives. Macromolecules such as dextran of molecular weight 40 kDa have an experimental half-life of 3.6 h compared to 5.6 h obtained with a molecular weight of 250 kDa [28]. Bevacizumab (149 kDa) showed even greater half-life (7.9 h) indicating that, apart from molecular weight the charge, flexibility and lipophilicity can also affect the clearance [28]. Through rapid clearance, the particles containing therapeutic proteins form injectable implants with a long retention time that can last up to months. Thus, the suprachoroidal injections of implants containing therapeutics exhibit great scope for effective retinal delivery.

2.2. Periocular

It is a less invasive method where drugs are administered directly into the eye via injection into the subconjunctival, subtenon, peribulbar, retrobulbar, and posterior juxtascleral spaces, without any risk of cataract and endophthalmitis. Compared to the topical route, this route provides excellent drug bioavailability by avoiding corneal barriers. Injected drugs reach the posterior segment through the conjunctival sclera, but the bioavailability is much lower (0.1%) than that of the topical route (Figure 2) [29]. Drugs rapidly clearing (80–95%) into systemic circulation through choroidal vessels and multiple barriers between the retina and subconjunctival space leads to poor bioavailability. This route is less invasive and eliminates the drug permeation through sclera. Moreover, in the case of retinal diseases, for drug administration in large volumes, this route is preferred due to the high volume of the injection (100–500 µL) compared with the suprachoroidal route (50–200 µL) [30].

3. Ocular Barriers and Approaches to Ocular Administration

3.1. Ocular Barriers

Ocular distribution of protein therapeutics to the eye depends on several factors such as membrane permeability, ocular elimination, nontarget binding, and degradation by proteolytic enzymes. Membrane permeability and ocular elimination closely depend on their size, surface charge, and hydrophilicity and lipophilicity [31]. However, complexity of the ocular tissues in deciding parameters for ocular pharmacokinetics is a major obstacle in the designing of an effective delivery system for therapeutic proteins due to the presence of various ocular barriers.

3.1.1. Tissue Conditions

Collagen fibers from the hydrophilic stroma also limit the penetration of therapeutic proteins, which usually takes place via pinocytosis or endocytosis (active transport mechanism) [31][32]. The tight junctions present in the cornea, sclera, and retina significantly prevent the diffusion of hydrophilic large macromolecules [32][33]. The tight junctions in the conjunctival epithelium are usually wider than those in the corneal epithelium but are still unable to provide penetration of large molecules [34][35]. The vitreous humor is a highly viscous fluid-like gel composed of 98 to 99% w/v water content, salts, sugars, a network of collagen-type II fibrils with hyaluronan, glycosaminoglycan, and a wide array of proteins located in the posterior segment of the ocular globe [36]. Drugs administered intravitreally will have direct access to the vitreous cavity and retina and may take several hours to diffuse across the entire vitreous humor. The clearance of macromolecules from the vitreous cavity is very slow due to hindrance by RPE, whereas diffusion from the vitreous to the retina is restricted by ILM [37]. Because several other factors are involved such as initial dose, volume of distribution, and the rate of elimination [38][39], it also depends on size, surface charge, and characteristics of the macromolecules injected [40][41][42][43]. The vitreous can allow the diffusion of small, anionic macromolecules, restricting the bigger size or cationic macromolecules that exhibit nondiffusion kinetics and distribution profile. Molecules can be eliminated through anterior and/or posterior routes [], which is influenced primarily by volume of distribution and elimination half-life [39]. A large number of diseases uveitis, cytomegalovirus retinitis, and retinitis and proliferative vitreoretinopathy affect the ocular pharmacokinetics of various topically instilled molecules and their formulations. The diseased conditions produce certain physiological changes in the corneal stroma composed of collagen and water, leading to poor bioavailability of hydrophobic molecules [44]. Fungal keratitis involving chronic inflammation of corneal tissues leads to poor permeation [45]. To solve this problem, drugs are administered with a vehicle/emulsion to avoid evaporation of the limited natural tears in dry-eye patients, as well as the use of the iontophoretic technique to permeate the ionized molecules into ocular tissues. BRB breakdown as well as choroidal and retinal neovascularization were observed in glaucoma, leading to blindness in a large population. Pharmacokinetic parameters need to be determined in such conditions using animal models to prove efficacy. In one study [46] of measuring the pharmacokinetic parameters, using healthy and diseased animal models, it was observed that the AUC and Cmax were significantly lower in diseased models compared to normal animal models due to BRB breakdown and exposure of drugs to ocular tissues. Therefore, dose calculation needs to be performed to avoid dose-related toxicity.

3.1.2. Physicochemical Characteristics of Drug Molecules

Various physicochemical parameters of macromolecules such as solubility, hydrophilicity/lipophilicity, molecular weight, size and shape, surface charge, and degree of ionization affect the selection of the route and rate of drug permeation through the cul-de-sac [47]. Small and lipophilic molecules can diffuse and distribute rapidly and largely through RPE, inner limiting membrane (ILM), and outer limiting membranes (OLM), exhibiting efficient distribution to (and even faster elimination from) ocular tissues. Large and lipophilic molecules have poor membrane permeability, showing relatively longer retention time at the site of injection with poor ocular tissue distributions [48][49]. For example, the particles with a size of 200 nm were found to be retained in the retinal tissues for two months after injection [48][49]. The vitreal clearance rate is rapid for smaller particles and can also be observed from their half-lives, i.e., particles of size 50 nm, 200 nm and 2 µm showed half-lives of 5.4 ± 0.8, 8.6 ± 0.7, and 10.1 ± 1.8 days, respectively [50]. Most of the therapeutic proteins have complex structure, large size with molecular weight > 1000 Da, and large hydrogen bonding donor/acceptor groups and show poor membrane permeability across the ocular tissues and barriers [51]. Human retinal tissues prevent the permeation of macromolecules of size > 76 kDa due to inner and outer plexiform layers. Macromolecules greater than 150 kDa cannot reach the inner retinal tissues, while molecules such as brolucizumab (smaller size) can penetrate the retina and choroid tissues more effectively than other anti-VEGF [33][52]. Brolucizumab showed 2.2-fold higher concentrations in the retinal tissues and 1.7-fold higher concentrations in RPE/choroid tissues than ranibizumab in rabbits [53]. These macromolecular proteins, when traversing through the choroid, may wash out through the choriocapillaris, leading to a reduction in therapeutic concentrations, and, due to the large complex molecular structure, may increase the risk of their degradation at the physiological environment of pH and temperature resulting into shorter half-lives. Macromolecules showed half-life in the range of days to a week (Table 1) in the vitreous humor, i.e., bevacizumab had a half-life of 4.32 days with a minimum concentration of 162 μg/mL in the vitreous [54]. Frequent intravitreal injections of ranibizumab 0.3–2.0 mg/eye biweekly or monthly is required to maintain the therapeutic levels as it showed vitreous elimination of 9 days and intrinsic systemic elimination half-life of 2 h, making it noncompliant and often associated with other complications such as cataract, retinal hemorrhage, and detachment and endophthalmitis [55][56]. One comparative study showed brolucizumab clearance from the ocular tissues with a mean terminal half-life of 56.8 ± 7.6 h; ranibizumab took 62 h, and aflibercept was cleared with a half-life of 53 h in the same model [57][58][59]. The rapid clearance is presumed to be due to smaller molecular size and absence of the Fc domain in the case of brolucizumab. Unlike aflibercept, which has full-length antibodies, leading to the conservation mechanism, molecules without the Fc region are more prone to degradation and do not show a cumulative effect even after multiple injections [60]. The surface charge being a complex and heterogenous property of amino acid sequence of the therapeutic proteins along with pH of the surroundings are important criteria to be considered. Deamination, isomerization, or post-translational modification of the therapeutic proteins in a particular environment lead to formation of charge variant species in a mixture of therapeutic proteins [61]. Most therapeutic proteins are found to be positively charged at an isoelectric point (pI) of 7–9, leading to charge interactions with other molecules and ocular membranes and showing good penetration compared to negatively charged proteins [61]. Although the undesired entrapment of the polymeric network of the vitreous (negatively charged) should not be ignored, positively charged molecules tend to remain clumped in the vitreous without diffusion, while anionic particles diffuse to the retina [62][63]. The effect of surface charge on the particles was studied on human serum albumin (HSA) and showed that anionic particles of size 114 nm with an overall zeta potential of −33.3 mV can easily diffuse through the vitreal collagen fibrils to the retina within 5 h after injection, while cationic particles of size 175.5 nm with mean zeta potential of +11.7 mV showed aggregation in the vitreous [63]. An inflammatory condition of the vitreous showed accelerated diffusion and clearance of HSA [64].

3.1.3. Viscosity and pH of the Formulation

Most of the protein formulations are available with high and variable viscosity as sustained release of therapeutic proteins for longer duration needs very high quantities to be injected in single-dose administration, which is often associated with high viscosity and difficulty of the syringe to handle the formulation and is not allowed by FDA. A high concentration of therapeutic proteins is very difficult to pass through an 18 mm, 27–30 G needle [65]. Use of viscosity builders required in the formulation of small molecules helps the proteins reach the anterior chamber of the eye in contrast to macromolecules, which helps provide sufficient viscosity to the formulation up to 20 cps, prolong the corneal residence time, enhance the transcorneal absorption into the anterior chamber, and thereby increase bioavailability [66]. pH and osmolarity play a vital role in the ocular therapeutics. For drug delivery to the anterior segment, maximum therapeutic benefits can be achieved when the pH of the formulation matches the lacrimal fluid. The pH of the formulation is a critical parameter that needs to be observed as proteins become denatured and unstable due to irreversible conformational changes at both high and low pH values. Apart from pH, the type and concentration of buffer used can also influence the protein degradation pathways, i.e., deamination, disulfide bond formation/exchange, isomerization, and fragmentation [67][68]. A weak acidic buffer is optimal for the storage of antibodies, i.e., adalimumab (pH 5.2), ranibizumab (pH 5.5), and bevacizumab (pH 6.2), below their isoelectric points (~8.3–8.8) for ocular treatments [69][70]. Though buffers play a crucial role in providing stability and preservation of macromolecules, their use must be carefully considered to avoid associated complications such as immunogenicity and local toxicity [71]. Buffers used also must be within the osmolarity range (280–300 mOsm/kg) to be compatible with ocular tissues as they also impair tonicity. Moreover, hypotonic solutions originate clouding and cause edema of the corneal tissues, while hypertonic solutions desiccate the corneal tissues in the anterior chamber [72]. Therefore, to facilitate protein delivery, proper understanding of the formulation pH and viscosity, selection of buffer system, and use of chemical chaperones are of the utmost importance. This helps to control the behavior and characteristics of the therapeutic proteins and also avoid protein misfolding [73][74].

3.1.4. Protein Binding

Protein binding shows less effect on ocular distribution of therapeutic proteins as the level of protein in the eye (0.5–1.5 mg/mL) is significantly less compared to that of plasma (60–80 mg/mL) [75][76]. Vasotide® administered in genetically modified mouse model showed significant reduction in retinal angiogenesis in AMD [77]. Intravitreally administered molecules required to cross the ILM to reach the retina after diffusion through the vitreous body, which contains a high-density extracellular matrix made up of collagen, laminin, and heparin sulphate proteoglycan (composition changes with age), affect the drug permeability [78]. Higher drug penetration was observed with high binding affinity to the extracellular matrix, which led to effective penetration to the ILM, making the drug available to the retina. For example, adeno-associated virus serotype-2 showed excellent transduction to the retina after intravitreal injection due to high heparin sulphate proteoglycan binding affinity, while other serotypes and modified serotypes failed to transfect (low affinity with proteoglycan) [79].

3.1.5. Enzymatic Degradation

Different metabolic pathways also cause the loss of therapeutic activity or inactivation of the macromolecules by protein denaturation, aggregation, precipitations, adsorption, and proteolytic degradation, denaturation by temperature, pH, salt or ionic concentrations, and complexations with enzymes/coenzymes. Enzymatic degradation by proteolytic enzymes depends on concentrations of the enzymes in the vitreous (levels may rise with age and tissue conditions) and on the hydrolytic enzymes and esterases in retina [80][81]. Structural changes in the active form of complex primary, tertiary, or quaternary structures of protein molecules or chemical modification lead to irreversible aggregation and finally inactivation. The main routes of drug administration and fate from ocular tissues are shown schematically in the Figure 1 and Figure 2, respectively. Peptides are highly susceptible to enzymatic degradation (proteolytic cleavage) [82]. The proteolytic cleavage and breakdown to small peptides leads to lower half-lives. The drug pharmacokinetic properties and thereby therapeutic efficacy can be achieved by improving bioavailability to the ocular tissues, and that can be achieved by chemical or physical modification of the molecules using various formulation strategies, i.e., coadministration, conjugation of functional moieties, particle formulation, encapsulation into implant or hydrogel, and chemical modification/substitutions. Proteolytic stabilization of macromolecules and membrane permeability can be achieved by a prodrug approach or using biological analogues [31][83][84]. Similarly, lipophilicity or hydrophobicity can also be increased by covalent conjugation with hydrophobic moiety or by noncovalent interactions with any hydrophobic compound. Solubility improvements can also be achieved using a conjugation with cyclodextrin and PEG, eliminating enzymatic degradation [15][16][32][51]. Thus, the pharmacokinetic properties of therapeutic proteins can be optimized, keeping in mind these changes must not affect their biological efficacy. Therapeutic proteins need protection against enzymatic attack from the various proteolytic enzymes present in the vitreous such as matrix metalloproteinase and serine/cysteine protease. The level of enzyme concentration in the vitreous changes with the age and disease conditions, so the formulation targeted to the retinal diseases needs to be optimized against such enzymatic attack [85][86]. Use of D-form peptides or peptoid type has been shown to have good enzymatic resistance [87] in addition to chemical modifications at the N and C terminus; for example, C-terminal amidation or N-terminal acetylation will make the peptides more difficult to be recognized and targeted by the enzymatic attack [88][89]. Apart from proteolytic enzymes, certain metabolic enzymes such as cytochrome P450 reductases and lysosomal enzymes are also found in large amounts in the ocular tissues that maintain homeostasis and protect the ocular tissues [90][91][92]. Encapsulation of retinal drugs in a nanoparticulate system or implant matrix can improve the protection against the enzymatic degradation [93], as discussed later in formulation approaches.

3.2. Use of Penetration Enhancers

Different therapeutic approaches have been investigated for the improvement of drug bioavailability and providing sustained drug release to the corneal tissues. Bioavailability improvement to the anterior segment of the eye can be achieved by maximizing corneal absorption and reduction in precorneal drug loss, which can be achieved by using viscosity enhancers, penetration enhancers, and prodrug approaches [56][57][58]. The presence of tight junctions in the stratified epithelium allows only ions to be transported across the tissues, offering high resistance to therapeutic proteins; thus, the addition of absorption promotors or penetration enhancers can be more helpful to improve the permeability across the corneal tissues or membrane [29][57][94]. Permeation enhancers alter the integrity of the corneal epithelium, leading to the promotion of the corneal uptake and thus a rate-limiting step in the transport of macromolecules from the corneal tissues to the receptor site [58]. Inclusion of cetylpyridinium chloride [95], lasalocid [96], benzalkonium chloride [52], parabens [94], tween® 20, saponins [40], Brij® 35, Brij® 78, Brij® 98 ethylenediaminetetraacetic acid, bile salts [59], bile acids (such as sodium cholate, sodium taurocholate, sodium glycodeoxycholate, sodium taurodeoxycholate, taurocholic acid, chenodeoxycholic acid, and ursodeoxycholic acid), capric acid, azone, fusidic acid, hexamethylene lauramide, saponins [60], hexamethylene octanamide, and decyl methyl sulfoxide [97] in different formulations has shown a significant enhancement of corneal drug absorption. Moreover, the ability to catalyze the degradation of hyaluronic acid by hyaluronidase is also utilized since it has taken decades to improve the permeability across the ocular tissue barriers [98]. In the vitreous, hyaluronic acid provides a key role in maintaining structural integrity, volume expansion, and viscosity of the vitreous body [99]. Keeping in mind the associated toxicity and irritation, penetration enhancers should be used precisely and carefully.

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