pharmaceutics

Review
Topical Drug Delivery to the Posterior Segment of the Eye
Marina Löscher, Chiara Seiz, José Hurst and Sven Schnichels *

Centre for Ophthalmology Tübingen, University Eye Hospital Tübingen, Elfriede-Aulhorn-Str. 7,

72076 Tübingen, Germany; [email protected] (M.L.); [email protected] (C.S.);
[email protected] (J.H.)
* Correspondence: [email protected]; Tel.: +49-70712987888

Abstract: Topical drug delivery to the posterior segment of the eye is a very complex challenge.
However, topical delivery is highly desired, to achieve an easy-to-use treatment option for retinal
diseases. In this review, we focus on the drug characteristics that are relevant to succeed in this
challenge. An overview on the ocular barriers that need to be overcome and some relevant animal
models to study ocular pharmacokinetics are given. Furthermore, a summary of substances that were
able to reach the posterior segment after eye drop application is provided, as well as an outline of
investigated delivery systems to improve ocular drug delivery. Some promising results of substances
delivered to the retina suggest that topical treatment of retinal diseases might be possible in the
future, which warrants further research.

Keywords: ocular drug delivery; ocular pharmacokinetics; ocular barriers; permeability; drug
characteristics; retinal diseases





 1. Introduction
Citation: Löscher, M.; Seiz, C.; Hurst, Topical delivery is the safest and easiest method to apply ocular medication, as it can
J.; Schnichels, S. Topical Drug be applied non-invasively by the patients themselves. Nevertheless, the drug absorbance
Delivery to the Posterior Segment of and permeability are low and, as a consequence, the drug concentration in the eye drops is
the Eye. Pharmaceutics 2022, 14, 134. very high and can lead to side effects [1,2].
https://doi.org/10.3390/ Diseases of the anterior segment are generally treated with eye drops. Topical treat-
pharmaceutics14010134 ment of retinal diseases is also desired, as commonly used injections require frequent
Academic Editor: Monica application by a specialized ophthalmologist and are associated with various side effects.
M. Jablonski However, topical drug application faces various obstacles, especially if the drug needs to
reach the posterior segment. Besides eye drops, topical application via hydrogels, consist-
Received: 30 November 2021
ing of a network of natural or synthetic polymer chains, is also possible [3]. For glaucoma
Accepted: 28 December 2021
treatment, the application of hydrogel, in the form of contact lenses, has been investigated.
Published: 6 January 2022
Timolol containing nanoparticles were loaded onto contact lenses and showed timolol
Publisher’s Note: MDPI stays neutral release and IOP reduction over 5 days [4]. In situ gels are hydrogels applied as solutions,
with regard to jurisdictional claims in which can quickly transition from sol-to-gel due to chemical and/or physical crosslink-
published maps and institutional affil- ing [5]. The short duration of action, as well as the rapid excretion rate, is often the limiting
iations. factor with conventional eye drops. In situ gel systems may provide a potential solution to
these problems. However, it is still unclear what influence the in situ gels have on sustained
release behavior and tissue distribution in the eye. A recent study on ocular drug delivery
of thermosensitive in situ gels, loaded with betaxolol hydrochloride, showed prolonged
Copyright: © 2022 by the authors.
drug release [6]. Most of the ocular hydrogel research, however, is focusing on intravitreal
Licensee MDPI, Basel, Switzerland.
injections to obtain sustained drug release from a reservoir in the vitreous, and a recent
This article is an open access article
overview was given by Blessing et al. [3]. Another option for achieving prolonged posterior
distributed under the terms and
conditions of the Creative Commons
segment delivery are inserts and implants. An overview on new developments was given
Attribution (CC BY) license (https://
by Castro-Balado et al. [7]. As those applications still require an invasive procedure, they
creativecommons.org/licenses/by/ are not included in this review. Our focus is solely on eye drops as the most common,
4.0/). non-invasive application method.

Pharmaceutics 2022, 14, 134. https://doi.org/10.3390/pharmaceutics14010134 https://www.mdpi.com/journal/pharmaceutics

Pharmaceutics 2022, 14, 134 2 of 21

In this review, we will focus on eye drop medication against glaucoma and age-related
macular degeneration (AMD), since those diseases are among the leading causes for blind-
ness worldwide. With increasing age, the prevalence of both diseases rises tremendously [8].
In a large meta-analysis of the last two decades, the prevalence of primary open angle
glaucoma ranges from 0.4% at age < 40 to 9.2% at age > 80, with an overall global prevalence
of 2.4% [9]. Another large meta-analysis showed the overall prevalence of AMD in the age
group of 45–85 to be at 8.69%, ranging from 3.49% at age 45–49 to 24.96% at age 80–85 [10].
Due to the aging population, the number of patients suffering from AMD and glaucoma
will reach 288 million [10] and 111.8 million [8] in 2040, respectively.
Glaucoma comprises a group of neurodegenerative diseases, leading to optic nerve
damage and retinal ganglion cell death, which ultimately results in vision loss [11,12]. Most
forms of glaucoma are associated with an increased intraocular pressure (IOP) [13]. IOP
is currently the main line of therapy to slow down the neurodegenerative progression of
the disease [14]. Therefore, IOP unrelated therapies are increasingly investigated for the
treatment of glaucoma to stop neurodegeneration [15].
AMD is a multifactorial disease influenced by genetic disposition, life style factors,
and, especially, aging [16,17]. Although the early forms of AMD are typically without
symptoms, the two late forms, atrophic and exudative AMD, cause slow (atrophic) or
rapid (exudative) vision deterioration and result in severe visual impairment and even
legal blindness [18]. In spite of the introduction of the anti-vascular endothelial growth
factor (VEGF) therapy, which can slow the progression of exudative AMD, the therapeutic
options for AMD are far from satisfactory [19]. No therapeutic options exist to slow or
halt the progression from early to late AMD, and treatment possibilities for the early or
atrophic forms of AMD are lacking. Improving and prolonging treatment efficacy, as
well as reducing the number of injections needed, is the goal of several new therapeutic
developments, including targeting additional pathways, combination therapy, and new
drug delivery systems [20].
In this review, our focus is on non-invasive topical drug delivery. We will first provide
an overview of the ocular barriers that a topically applied drug has to overcome to reach the
back of the eye, as well as the different absorption pathways that a drug can take (Section 2).
Additionally, an insight into the animal models used for ocular drug delivery studies is
given and compared to the human morphology (Section 3). Next, several examples of anti-
glaucoma and -AMD drugs, which did show successful delivery to the posterior segment
after topical application, are presented (Section 4), as well as some promising drug delivery
systems that are applied to improve topical delivery to the back of the eye (Section 5).

2. Barriers of Topical Delivery to the Posterior Segment of the Eye and the Influence of
Drug Characteristics
To reach the posterior segment of the eye, a topically applied medication has to cross
various barriers. A schematic overview of drug absorption and elimination is given in
Figure 1. Drug penetration is hindered by static, as well as dynamic impediments. Static
obstacles include the various layers of the ocular tissues (cornea, conjunctiva, sclera, and
retina), as well as the vascular blood-retinal and blood-aqueous barriers [21]. Tear dilution,
lymphatic clearance, efflux pumps, and choroidal, as well as conjunctival blood flow, are
among the dynamic parameters responsible for impeded drug delivery [22].

2.1. Penetration of the Precorneal Layer

The first barrier, which already washes away up to 99% of the active ingredient, is the
precorneal layer, the tear film. The tear film consists of three components: the lipid layer (which
prevents evaporation), aqueous layer, and underlying mucosal layer (which is composed of a
variety of soluble and membrane-bound mucins). Whereby, according to the latest findings,
the last two are combined to form an aqueous mucous layer [23]. This tear film, as well as
the blinking of the eyelid, effectively prevent the penetration of substances and result in only
1–5% of the applied drug remaining on the ocular surface for a sufficiently long time to become
 Pharmaceutics 2022, 14, 134 3 of 21

effective there [1]. However, a large part of the flushed out drugs can be reabsorbed by the nasal
mucous membrane, which is the reason for systemic side effects [2]. Due to the
Pharmaceutics 2022, 14, x FOR PEER REVIEW 3 of amphiphilic
22
properties of the tear film, purely hydrophobic, as well as purely hydrophilic substances, have a
particularly difficult time penetrating this barrier.

FigureFigure 1. Schematic
1. Schematic overview
overview of drugof drug absorption
absorption and elimination
and elimination after
after topical topical application.
application.

2.2. Corneal
2.1. Penetration or Conjunctival
of the Precorneal Layer Absorption
Absorption
The first into the
barrier, which eye of washes
already topically administered
away up to 99%drugs of themayactiveoccur throughisthe cornea
ingredient,
and/or conjunctival epithelium. Following corneal absorption,
the precorneal layer, the tear film. The tear film consists of three components: the drugs may reach
lipidthe tissues
of the posterior segment of the eye by passing through
layer (which prevents evaporation), aqueous layer, and underlying mucosal layer (which the anterior segment. After drug
absorption
is composed of ainto the of
variety conjunctiva,
soluble andthe vitreous and retina
membrane-bound can beWhereby,
mucins). reached, according
either by diffusion
to thethrough the sclera
latest findings, theorlast
cornea or via
two are clearance
combined to into
formthe an systemic circulation
aqueous mucous layer[21].
[23].
At physiological
This tear film, as well as the pH the cornea
blinking behaves
of the eyelid, as a negatively-charged
effectively prevent the penetration membrane, of there-
fore positively-charged
substances and result in only 1–5% molecules
of the penetrate
applied drug more easily than
remaining on the negatively-charged
ocular surface com-
for a pounds [24].long
sufficiently Thetime
multilayered
to becomestructure
effective of the [1].
there cornea furthera complicates
However, large part ofthe theuptake of
active
flushed substances.
out drugs can be The epithelial
reabsorbed cellnasal
by the layermucous
has lipophilic
membrane, properties
which isand is generally the
the reason
for systemic side effects
rate-limiting barrier[2].
to Due to the amphiphilic
transcorneal transport. properties
The underlying of the stroma
tear film, purely
is hydrophilic and
hydrophobic, as well as purely hydrophilic substances, have a particularly
the endothelial layer below is lipophilic [25]. Lipophilic drugs take the transcellular route difficult time
penetrating
through this
thebarrier.
corneal epithelium, whereas hydrophilic compounds cross the epithelial barrier
via the paracellular pathway. Paracellular permeation is limited by the paracellular pore
2.2. Corneal
diameter.or Conjunctival
The cornealAbsorption
epithelium only allows hydrophilic compounds with a size < 500 Da
to permeateinto
Absorption duethetoeye
its of
paracellular pore diameter
topically administered drugsof 2.0
maynm ± 0.2.
occur Whereas
through the conjunc-
the cor-
nea and/or conjunctival
tiva allows permeationepithelium. Following
of molecules corneal
of size 5–10absorption,
kDa, due to drugs may reach the
its paracellular pore size of
tissues
3.0ofnmthe±posterior
1.6 [26] segment
(Table 1).of the eye by passing through the anterior segment. After
drug absorption into through
Absorption the conjunctiva,
the cornea the
andvitreous and retina
conjunctiva is easiercanforbe reached,drugs.
lipophilic eitherThe by lipophilic
diffusion through the sclera or cornea or via clearance into the systemic circulation
drug propranolol, for example, is absorbed 5–10-fold greater than sotalol, a hydrophilic drug [21].
At
withphysiological pH the cornea
the same molecular weightbehaves as a negatively-charged
[27]. Lipophilic compounds with membrane,
lipid/water there-partition co-
fore positively-charged
efficients (LogD values) of 2–3 are considered optimal for corneal permeation.com-
molecules penetrate more easily than negatively-charged Compounds
poundswith [24]. The
even multilayered
higher structure
values (logD of the lower
> 3) show corneapermeability,
further complicates the uptake
due to slower of
desorption from
activethe
substances. The epithelial cell layer has lipophilic properties and is generally
lipophilic epithelium to the hydrophilic stroma. Therefore, different lipophilicity is not the the
rate-limiting
determiningbarrier to transcorneal
factor for the cornealtransport. The underlying
permeability stroma is hydrophilic
of highly lipophilic and
pilocarpine prodrugs, but
the endothelial layer below is lipophilic [25]. Lipophilic drugs take the transcellular
rather the conversion rate of the prodrug to the more hydrophilic parent drug, which allows an route
through thetransfer
easier corneal from
epithelium, whereasto
the epithelium hydrophilic
the stromacompounds
[28]. cross the epithelial bar-
rier via theBesides
paracellular pathway. Paracellular permeation
passive diffusion, active transport also plays a role is limited byinthe paracellular
drug penetration of the
pore diameter. The corneal epithelium only allows hydrophilic compounds with a size <
cornea. As hydrophilic drugs only show low passive diffusion across the cornea, the impact of
500 Da to permeate due to its paracellular pore diameter of 2.0 nm ± 0.2. Whereas the
active transport may be more pronounced, compared to lipophilic drugs. For lipophilic drugs,
conjunctiva allows permeation of molecules of size 5–10 kDa, due to its paracellular pore
the transfer from the epithelium to the stroma is the limiting step; therefore, the efflux transport
size of 3.0 nm ± 1.6 [26] (Table 1).
in the basolateral side of the epithelium has a higher impact. Nevertheless, since drug absorption
Absorption through the cornea and conjunctiva is easier for lipophilic drugs. The
via transporters is saturable, passive diffusion is possibly the dominating mechanism [29].
lipophilic drug propranolol, for example, is absorbed 5–10-fold greater than sotalol, a hy-
drophilic drug with the same molecular weight [27]. Lipophilic compounds with li-
pid/water partition coefficients (LogD values) of 2–3 are considered optimal for corneal
Pharmaceutics 2022, 14, 134 4 of 21

Table 1. Influence of drug characteristics on transport of topically applied drugs across ocular tissues.

Drug Characteristics
Ocular Tissues Size and Radius of the Drug Charge of the Drug
Lipophilic Hydrophilic
Transcellular, 5–10× greater
Easier penetration of Paracellular
absorption than hydrophilic drugs,
Cornea <500 Da positively-charged <500 Da
transfer from epithelium to
molecules Low passive diffusion
stroma -> rate-limiting
Mainly through
Easier than for hydrophilic conjunctiva (9–17 times
Conjunctiva 5–10 kDa
compounds larger surface
area than cornea)
<70 kDa, better permeability Passage of Easier penetration than
RPE-choroid and sclera are equal
Sclera of globular proteins vs. negatively-charged solutes lipophilic compounds,
barriers
linear dextrans is facilitated RPE is rate-limiting
<500 nm Negatively-charged
Easier than for hydrophilic
Vitreous Easier diffusion of small particles diffuse better Longer half-life
compounds
molecules than cationic particles

Even though the conjunctiva is more permeable than the cornea, the presence of efflux
pumps impedes substance transport via this pathway. In addition, the existing vascularization
of the conjunctiva, as well as the episcleral, leads to a removal via the systemic circulation.
The bioavailability of drugs in the anterior chamber is, therefore, at best 5% [30]. Several
macromolecule transporters (for amino acids, nucleosides, d-glucose, monocarboxylate, and
dipeptides) are expressed in the conjunctiva that may be relevant for ocular drug delivery. The
amino acid transporter ATB0,+, for example, recognizes almost all amino acids, making it a
feasible target for amino acid derivatives (including prodrugs) [31].

2.3. Permeation through the Intraocular Tissues

After crossing the cornea and reaching the aqueous humor, the drug diffuses to
the surrounding intraocular tissues and vitreous humor [32]. Further barriers for drug
permeation are the removal via the aqueous humor and the intracellular degradation of
active substances by metabolizing enzymes, such as glutathione [33]. Diffusion across the
vitreous is easier for small molecules [34]. However, as the mesh size of the vitreous is
estimated at 500 nm, size is not the limiting factor for particle diffusion [35]. The influence
of charge on particle diffusion is much more pronounced [36]. Since negatively-charged
hyaluronic acid and glycosaminoglycan proteins exist in the vitreous body, negatively-
charged particles, such as polylactic co-glycolic acid (PLGA) or human serum albumin,
diffuse better than cationic particles [35,37,38]. To improve the migration of positively-
charged particles through the vitreous, attempts are being made to coat them with polymers,
such as polyethylene glycol (PEG) and hyaluronic acid [39–41]. Another dominating factor
influencing the passage through the vitreous body and, consequently, the intraocular half-life
is the lipophilic properties of a substance. Hydrophilic and large compounds remain in the
vitreous for a longer time and are typically removed via anterior elimination [42]. Whereas small
and lipophilic molecules mainly take the posterior route, as they can easily cross the retina [43].
Pitkänen et al. discovered that lipophilic β-blockers passed the outer blood-retinal-barrier much
more efficiently than more hydrophilic β-blockers of similar size [44].
Besides the transcorneal diffusion through the anterior chamber to the vitreous and
retina, topically applied drugs can also enter the systemic circulation and reach the retina
via retinal vasculatures [45]. This periocular drug absorption includes diffusion of the
drug through the conjunctiva to the Tenon’s capsule and, further, through the sclera and
choroid to the retina. Although most of the administered dose is removed into the systemic
circulation [46] (Figure 1).
Scleral permeability is strongly dependent on molecular weight and radius. Smaller
molecules are more permeable than bigger ones. The sclera can be crossed by molecules up
to a size of roughly 70 kDa [47]. Similarly, globular proteins have a better permeability than
Pharmaceutics 2022, 14, 134 5 of 21

linear dextrans of the same molecular weight. [48]. Some lipophilic drugs can enter the
posterior segment directly by lateral scleral diffusion and subsequently penetrate Bruch’s
membrane and the RPE [46]. Hydrophilic compounds can easily penetrate the sclera, as it
consists of porous spaces within a collagen aqueous network [48]. At physiological pH the
matrix structure of proteoglycans is negatively charged, which facilitates the permeation of
negatively-charged compounds through the sclera [49]. For hydrophilic compounds taking
the transscleral route, the RPE is most likely the rate-limiting factor. Whereas RPE-choroid
and sclera represent comparable barriers for lipophilic drugs [44] (Table 1).
Melanin binding can also effect drug distribution and lead to increased drug concen-
trations in RPE and choroid. This may, on one hand, reduce receptor binding of the drug;
however, on the other hand, melanin binding could prolong drug effects, due to sustained
drug release from the melanin depot [50,51].

3. Model Systems to Study Drug Delivery to the Retina

To study the absorption of new drugs or delivery systems, different animal models
are used. The goal of those preclinical investigations is to predict the clinical performance
of the drug candidates. To this end, the characteristics of the model systems have to be
carefully considered and compared to the human situation.
Choosing the suitable preclinical model is of utmost importance, as shown by repeated
failures of topically applied drugs in clinical investigations, in spite of promising preclinical
data in rodents [52]. Animal models used in ocular drug delivery studies include mice,
rats, rabbits, monkeys, and sometimes dogs and pigs [45]. There are various characteristics
that can be taken into account. The bioavailability of topically applied drugs at the back of
the eye can be influenced, among other factors, by differences in the thickness of cornea
and sclera, axial length, and vitreous volume. The human vitreous volume as an example
is one thousand times larger than in rodents, which may have a strong influence on the
intraocular concentration of small molecule drugs applied via eye drops [45].
In the following section, we will outline only a few important parameters and compare
the eyes of mice, rabbits, and pigs to the human eye (Table 2).

Table 2. Ocular characteristics of commonly used preclinical models vs. the human eye (modified
and supplemented according to Wang et al. [45]).

Parameter Mice Rabbit Pig Human

Blink intervals
300 [53] 360 [53] 10 [54] 5 [53]
[blinks per minute]
Central corneal thickness [µm] 123–134 [55] 349–384 [56] 543–797 [57] 548 ± 35 [58]
Anterior chamber depth/ocular axis [mm] 0.1 [45] 0.16 [45] 1.77 ± 0.27 [59] 3.05 [45]
Anterior chamber volume [µL] 2.39–3.08 [60] ~250 [45] ~260 [61] ~170 [62]
Aqueous humor production [µL/min] 0.18 ± 0.05 [63] 1.46 ± 1.71 [64] 3–4 * [65] 2.4 ± 0.6 [66]
Vitreous volume [µL] 4.4 ± 0.7 [67] ~1400 [68] 3300 [69] ~4400 [70]
Vascular area
Mean Retinal thickness [µm] 204 [71] 163–340, avascular 300 [73] 310 [73]
area 142–168 [72]
Average RGC density [cells/mm2 ] 4000 [74] 6000 [74] 5700 [74]
* aqueous flow rate.

Mice are the most commonly used species, presumably owing to cost effectiveness,
availability of genetically modified strains, and short reproduction cycles [75,76]. However,
some data indicate that the small eyes of rodents are not well suited to predict the clinical
efficacy of topical drugs [77,78].
Mice eyes have a central corneal thickness of 123–134µm, which is one-fifth to one-
fourth of the human cornea (548 µm). The anterior chamber is 0.1 µm deep (factor 30 shorter
Pharmaceutics 2022, 14, 134 6 of 21

than the human anterior chamber) and has a volume of around 2.5 µL (little more than
1 percent of the human anterior chamber volume). The difference in vitreous volume is
even more pronounced, with the vitreous volume in mice being factor 1000 smaller than
in humans (4.4 µL vs. 4400 µL). The cone-based performance in the mouse retina is also
not representative for the mammalian retina, with a rod-to-cone ratio of only 97:3 [79,80].
Furthermore, the mouse retina is devoid of a macula or similar retinal region, with a high
density of cones, retinal ganglion, and bipolar cells [81].
Ocular pharmacokinetic studies are mostly done in rabbits. Compared to the small rodents,
the rabbit eye is much closer to the human situation. Corneal thickness is 349 µm (compared
to 548 µm) and, although the anterior chamber depth is only 0.16 µm (compared to 3.05 µm),
the anterior chamber volume is more comparable (250 vs. 170 µL). The vitreous volume in
rabbit is one-third of the human vitreous (1400 vs. 4400 µL). The retinal thickness is in the
same range (at least in the vascular area). While some authors claim the rabbit is a poor model
of the human eye, due to the differences in vitreous volumes and vitreous diffusional path
length [82,83], others state that for intravitreal pharmacokinetics the rabbit model is clinically
predictable, as intravitreal distribution and clearance is quite comparable between rabbits and
humans [84]. Rabbits are known to have a visual streak (VS), in which the density of rod and
cone photoreceptors, retinal ganglion, and amacrine cells is highest [85]. The neural retina of
the rabbit is rather hypoxic and, as only a small area shows any retinal circulation, mainly
dependent on choroidal circulation [86]. In contrast to the human eye, optic nerve fibers in the
rabbit are already myelinated in the retina [87].
Besides studies in non-human primates, which come with a lot of ethical and financial
concerns, the porcine eye resembles the human situation quite well. Regarding morphology,
size, and vascularization, human and porcine eyes are comparable [88,89]. The central corneal
thickness is in the same range (543–797 µm in pigs vs. 548 ± 35 µm in humans). The anterior
chamber depth is one-half to two-thirds, compared to humans (1.77 ± 0.27 vs. 3.05), whereas
the anterior chamber volume is roughly 50% bigger, 260 vs. 170 µL. The vitreous volume is
3300 µL vs. 4400 µL in humans. The retinae of humans and pigs are quite comparable, 300 µm
thickness in pig vs. 310 µm thickness in humans [73]. Further, the porcine retina is a suitable
model for retinal research, as the photoreceptor mosaic of pigs and humans is quite similar. The
porcine retina is well provided with cones, an expansive vascular tree, and an area sufficiently
devoid of blood vessels, to suggest an area centralis near the posterior pole [54]. To study human
retinal diseases and drug delivery to the retina porcine (and similarly bovine) eyes and retinal
explants are increasingly applied [90,91]. Developing ex vivo models from these waste products
of the food industry offers the chance to overcome shortcomings of the currently used in vivo
models, while, at the same time, reducing the number of animal experiments [73,90]. Peynshaert
et al., for example, recently developed a bovine retinal explant model with an intact vitreoretinal
interface, where retinal penetration, following intravitreal injection, can be studied [91,92].

4. Retinal Delivery of Different Medical Compounds

As mentioned in the previous chapters, drug delivery to the retina after topical admin-
istration faces various barriers. Nevertheless, a certain variety of compounds have shown
successful topical delivery to the posterior segment in preclinical models, and some have
already been investigated in clinical trials (Table 3).
Pharmacokinetics and distribution can be influenced by different size of the drugs and
pH. Corneal penetration can be enhanced by increasing the lipophilicity of the compound.
Prodrugs like Latanoprost and Travoprost take advantage of this effect. They contain
ester groups, which increases their lipophilicity and, thereby, the uptake into the cornea,
where the prodrugs are then metabolized into the active drugs by esterase enzymes [93].
Compared to small molecules, biologics face greater hurdles in topical absorption due to
their size. However, they also require only a lower target concentration, as they exhibit
higher affinities for their target [52].
Pharmaceutics 2022, 14, 134 7 of 21

Table 3. Overview of topically applied drugs reaching the posterior segment in preclinical investigations.

Physiological
Compound Characteristics Size logP * Preclinical Investigations Cmax Retina Ref.
Charge
Drugs tested for glaucoma treatment
Japanese white rabbits: 1 drop of 1%
Dorzolamide inhibitor of carbonic
324.4 Da (360.9 Da) 1 Dorzolamide: −0.15 dorzolamide hydrochloride eyedrops -> Cmax 3.79 µg/g [94]
(hydrochloride) anhydrase
after 1 h
Monkeys: 14 days 0.5% brimonidine twice
daily (35 µL drop -> 8.4 µCi, 119µg Brim) ->
Alpha2-adrenergic Cmax of radioactivity in choroid/retina Monkeys: 30.600 µg-Eq/g
Brimonidine 292.13 Da 1 1.37 [95]
agonist Rabbits: twice daily 14 days 0.5% solution Rabbits: 20.8 µg-Eq/g
(35 µL drop -> 2 µCi, 113 µg Brim) -> Cmax of
radioactivity in choroid/retina
Humans: 0.25% betaxolol twice daily for
28 days or longer -> 1290 ± 1170 ng/g in the
Humans: 71.4 ± 41.8 ng/g
Betaxolol Selektiver ß-Blocker 307.4 Da 1 2.81 choroid [96]
Monkeys: 121 ng/g
Monkeys: 0.25% betaxolol twice daily
unilaterally for 30 days
Rabbits: single drop (35 µL of 14C-netarsudil
Netardusil ROCK-inhibitor 453 Da 1 4.73 80 (left) or 50 (right) ng ∗ eq/g [97]
0.02% -> Cmax of in Retina-choroid
Arterially perfused bovine eye model: 4 mL of
9.27 mM memantine hydrochloride solution
Antagonist Memantine: 3.5
Memantine (HCL) 179.2 Da (215.76 Da) 1 placed in reservoir on the eye (8.002 µg) -> 2046 ng/g [98]
to nmDA-Receptors (hmdb.ca)
Cmax retina 2046 ng/g vitreous 442 ng/g,
Choroid/RPE 3894 ng/g after 9 h of perfusion
Drugs tested for AMD treatment
recombinant
Pigmented rabbits: Bevacizumab eye drops
humanized monoclonal Negatively-charged Unknown (known to be 18.2 ± 4.2 ng/g in
Bevacizumab 149 kDa (1.25 mg/0.05 mL six times daily for the first [100]
antibody, inhibits at pH 7.4 [99] lipophilic) retina/choroid
7 days) -> 18.2 ± 4.2 ng/g in retina/choroid
VEGF-A
Rabbits: 10 mg/mL ESBA105, 50 µL eyedrop ->
1 day hourly drops up to 10 h (up to 5 mg/day)
-> Cmax: vitreous humor (295 ng/mL),
neuroretina (214 ng/mL) and RPE-choroid
anti-TNF-alpha Single drop: 214 ng/mL
(263 ng/mL)
ESBA105 single-chain antibody 26 kDa - - Multi-day treatment: [101]
multi-day treatment: 9.6 mg/mL, 5 drops per
fragment 917 ng/mL
day up to 6 days (up to 15 mg/6 days) ->
Cmax: RPE-choroid (1298 ng/mL) vitreous
humor (580 ng/mL) and neuroretina
(917 ng/mL)
Pharmaceutics 2022, 14, 134 8 of 21

Table 3. Cont.

Physiological
Compound Characteristics Size logP * Preclinical Investigations Cmax Retina Ref.
Charge
Innovative small molecules
Monkeys: single eye drop -> >100 nM in the
GAL-101 B-Amyloid aggregation
289 Da Computed logP-1.1 retina, via sclera and choroid >100 nM [102]
(MRZ-99030) modulator, dipeptide

Rabbits: Single eye drop of 50 µL 5% SF-0166

integrin αVβ3
SF-0166 475.5 Da Computed logP 2.7 (2.5 mg/eye) -> Cmax retina-choroid 5103 ng/g in retina-choroid [103]
antagonist
5103 ng/g
Laser-induced CNV rat model -> systemically
Inhibitor of VEGF,
administered squalamine lactate -> partially
PDGF, and bFGF
Squalamine lactate 718.04 Da 2 Squalamine: 3.24 reduced choroidal neovascular membrane [104]
through intracellular
development
mechanism
No PK
Inhibitors of receptor tyrosine kinases
Laser-induced CNV mouse model-> single
10 µL drop of 1% TG100801: Cmax (TG100801)
-> 242 nM (retina), 1680 nM (Sclera/choroid);
TG100801, inactive inhibits Src kinases and Cmax (TG100572) -> 97 nM (retina), 2460 nM
Mouse: TG100572 -> 97 nM
prodrug of selected receptor 580.1 Da (476) 1 7.64 (Sclera/Choroid); [105]
Rabbit: TG100572 -> 41 nM
TG100572 tyrosine kinases Dutch belted rabbits- > 1 40 µL drop of 0.6%
TG100801: nach 2 h TG100801 -> 46 nM
(retina), 34 nM (Choroid), TG100572 -> 41 nM
(retina), 169 nM (choroid)
Laser-induced CNV rat model -> twice daily
targets multiple topical eye drop treatment -> decreased
receptor tyrosine leakage from photocoagulated lesions by 89.5%
pazopanib 437.5 Da 0 3.55 [77]
kinases such as VEGF (p < 0.001); inhibited thickness of the
receptors developed CNV lesions by 71.7% (p < 0.001)
No PK
PK: brown Norway rats tid for 10 days (4 µL x
Acrizanib small-molecule
445.40 Da 1 2.93 0.3% suspension) and 1 drop on day 11 -> 1910 nM [106]
(LHA510) VEGFR-2 inhibitor
Cmax 1910 nM (retina)
Topical administration led to significant and
VEGFR2 tyrosine kinase sustained drug levels in retina and choroid, as
PAN-90806 532.4 Da 0 Computed logP 3.7 [107]
inhibitor well as suppression of neovascularization in
various models
* predicted physiological charge and logP obtained from https://go.drugbank.com (accessed on 21 December 2021); computed logP obtained from https://pubchem.ncbi.nlm.nih.gov
(accessed on 21 December 2021); in red: tested in clinical trials.
Pharmaceutics 2022, 14, 134 9 of 21

4.1. Anti-Glaucoma Drugs

Several glaucoma drugs have shown to reach the back of the eye. Dorzolamide
hydrochloride was topically applied in Japanese white rabbits. After only 15 min, the
drug concentration in the anterior segment of the eye and retina increased significantly
and peaked within an hour. This indicates efficient migration of the drug between ocular
tissues. It can, therefore, be assumed that carbonate anhydrase activity is immediately
suppressed by dorzolamide and that the drug is rapidly distributed in the ocular tract
after local administration. [94]. Topical delivery of brimonidine to rabbits and monkeys
yielded retinal drug levels sufficient to activate alpha2-adrenergic receptors [95]. Similarly,
Betaxolol could be delivered to the retina of patients with glaucoma and cynomolgus
monkeys [96].
Netarsudil, a Rho-associated protein kinase inhibitor, was developed as novel treat-
ment option for glaucoma. In preclinical studies, large intra ocular pressure (IOP) re-
ductions were obtained in rabbits and monkeys, and a favorable pharmacokinetic profile
was shown. In distribution studies in rabbits with a single topical dose (35 µL) of 0.02%
14C-netarsudil, a Cmax of radioactivity of 80 (left eye) and 50 (right eye) ng ∗ eq/g was
reached in the retina [97]. In December 2017, after completing various clinical trials, Ne-
tarsudil was approved by the FDA for the treatment of open-angle glaucoma or ocular
hypertension [108].
Memantine HCL, an antagonist to NMDA-receptors used for the treatment of Alzheimer’s
disease, has neuroprotective properties and might be beneficial in the treatment of glau-
coma. In an arterially perfused bovine eye model, memantine was observed to accumulate
in the posterior segment. Koeberle et al. hypothesized that melanin-binding may support
sustaining significant concentrations in the retina [98].

4.2. Anti-AMD Drugs

The greatest desire in ophthalmic drug-delivery development is to identify a topi-
cal treatment option for retinal diseases like age-related macular degeneration (AMD).
Currently, anti-VEGF antibodies are applied intravitreally to inhibit choroidal neovascular-
ization in patients with AMD [109]. Because of their molecular weight of roughly 150 kDa,
topical delivery is highly challenging. After topical application of the VEGF-A inhibitor be-
vacizumab in pigmented rabbits (1.25 mg/0.05 mL six times daily for the first 7 days), only
a small level of bevacizumab was detected in the iris/ciliary body and retina/choroid, not
sufficient for a therapeutical effect [100]. In contrast to that, the anti-TNF-alpha single-chain
antibody fragment ESBA105, with a molecular weight of only 26 kDa, was distributed to all
ocular tissues, following topical application, reaching a retinal concentration of 214 ng/mL
after single application and 917 ng/mL after multi-day treatment in rabbits. Systemic drug
exposure was reported to be very low [101].
Besides antibodies and antibody fragments, several innovative small molecules and in-
hibitors of receptor tyrosine kinases have been investigated for their posterior segment delivery.

4.2.1. Innovative Small Molecules

Several small molecule eye drops have shown to reach the posterior segment and
were able to develop their effect against choroidal neovascularization (CNV) in preclinical
models. In the following section, we outline the promising candidates that were further
investigated in clinical trials.
One promising approach is targeting misfolded Amyloid β aggregation, to prevent
their neurotoxic effect. Russ et al. have shown that a single topical delivery of GAL-101, a
small molecule inhibitor of Aβ, sustained concentrations >100 nm in the retina of monkeys
for >2 h. Daily eye drops in a glaucoma rat model achieved >90% neuroprotection [102].
Phase I trials for geographic atrophy have successfully been completed [110].
Recent investigations have demonstrated promising results for drugs targeting arginyl-
glycyl-apartic acid (RGD)-binding integrins in ocular tissues [111]. One of those drugs
is the integrin αVβ3 antagonist SF-0166 (OcuTerra Therapeutics, Boston, MA, USA; for-
Pharmaceutics 2022, 14, 134 10 of 21

merly SciFluor Life Sciences, Inc., Boston, MA, USA). Preclinical investigations in rabbits
showed a good pharmacokinetic profile, and the efficacy results of topical administration in
laser-induced and VEGF-induced CNV rabbit models were comparable to a bevacizumab
injection [103]. Furthermore, biological effects and good tolerability have been shown
in early clinical trials with diabetic macular edema [112] and neovascular AMD patients,
which underlines the potential of targeting RGD-binding integrins, to develop a next-
generation therapy for retinal diseases [111].
Most of the investigated therapeutical approaches, however, failed to confirm a posi-
tive preclinical outcome. Squalamine lactate, a very promising inhibitor of angiogenesis
by a novel intracellular mechanism, was able to reduce choroidal neovascularization in
a laser-injury model in the rat when applied intravenously [104]. An eye drop formula-
tion of 0.2% squalamine lactate (OHR-102) was later launched by Ohr Pharmaceutical Inc.
(New York, NY, USA). In a Phase II trial (NCT01678963), this formulation, with improved
trans-scleral permeability and increased choroidal retention, showed a trend towards better
visual acuity in patients with all types of naïve neovascular lesions. Subsequent Phase III
trials (NCT02727881) failed to meet the primary endpoint [110].

4.2.2. Inhibitors of Receptor Tyrosine Kinases

TG100801 is an inactive prodrug that generates TG100572 by de-esterification, which
inhibits Src kinases and selected receptor tyrosine kinases. Topical TG100801 significantly
suppressed laser-induced CNV in mice and reduced fluorescein leakage from the vascula-
ture and retinal thickening, measured by optical coherence tomography in a rat model of
retinal vein occlusion [105]. TG100801 could not demonstrate efficacy when investigated in
AMD patients (ClinicalTrials. gov identifier: NCT00509548).
Pazopanib is a tyrosine kinase inhibitor that inhibits angiogenesis. To test the inhibitory
effect of pazopanib on experimental choroidal neovascularization (CNV), CNV was induced
in rats by laser coagulation of Bruch’s membrane. Twice daily topical treatment with
pazopanib significantly (p < 0.001) reduced leakage from photocoagulated lesions by 89.5%
and significantly reduced the thickness of the resulting CNV lesions by 71.7% (p < 0.001).
In addition, VEGF immunoreactivity was decreased, compared with control eyes [77].
When tested in humans, the compound attained to improve best-corrected visual acuity
in a Phase II trial, including patients with sub-foveal CNV secondary to AMD [113]. In
a subsequent Phase IIb trial, pazopanib did not show additional benefit, compared to
ranibizumab injections and, thereby, failed to meet its primary endpoint [114].
Acrizanib, a VEGFR2 inhibitor, showed impressive in vivo efficacy in the mouse
CNV model, by leading to complete inhibition of neovascularization. Three times daily
administration of 4 µL × 1.0% suspension/eye in the rat CNV model also resulted in 90%
inhibition of neovascular area [106]. When investigated in a clinical trial Acrizanib failed to
demonstrate efficacy, compared to anti-VEGF injections [115].
Another VEGFR2 inhibitor, which reaches the retina and choroid via the trans-scleral
route, is PAN-90806, by the company PanOptica, Inc. (Mount Arlington, NJ, USA). Pre-
clinical studies demonstrated sustained drug levels in choroid and retina, as well as the
suppression of the formation of new abnormal blood vessels. In the Phase I/II trials, the
PAN-90806 eye drops were applied as monotherapy in patients with neovascular AMD
(once daily for 12 weeks), 51% of which did not need a rescue injection during trial or one
month post-treatment [107]. Further clinical investigation is needed to confirm this data.
There will be many reasons for the failure in clinical investigations, but the lack of
sufficient investigations in a larger species might be one of them. As has been shown for
regorafenib and pazopanib, the drug concentrations in the choroid and retina, after topical
application in rabbits and monkeys, were much lower than those in rats and, therefore, not
sufficient to inhibit angiogenesis [116].
In interpreting efficacy and distribution data, it has to be taken into account that
systemic distribution to the posterior segment, following topical drug application, may
vary due to blood volume of the investigated species. Considering the smaller blood
Pharmaceutics 2022, 14, 134 11 of 21

volume of rabbits vs. humans (~0.12 l vs. ~5 l), drug levels in the posterior tissues may
reach values that cannot be transferred to humans. Therefore, Rodrigues et al. advised to
take drug levels and/or efficacy data in the untreated contralateral eye into account when
evaluating drug distribution [52].
From these examples, showing promising preclinical data but often failing in clinical
studies, it is evident that further improvements are needed. Delivery systems, such as
nanoparticles, offer the chance to enhance uptake and permeation of drugs to get a sufficient
amount of drug to the retina to be effective there.

5. Delivery Systems and Formulation Approaches to Improve Topical Delivery to the Retina
Numerous drug-delivery systems have been investigated, in order to achieve effective
drug concentrations in the posterior segment of the eye by topical application (Table 4).
Nanoparticles are increasingly applied as drug-delivery systems, on one hand, to enhance
the bioavailability of drugs, by increasing their absorption or facilitating their passage
through the tissue and, on the other hand, to achieve controlled release of the drug [117].
Polymeric materials have great potential as NP precursors, since their properties can easily
be tailored through derivatization of biopolymers or preparation of synthetic polymers,
according to drug delivery needs [118]. Drug uptake can also be improved via formulation
development approaches, such as the addition of enhancers of viscosity and permeability,
as well as prodrug design [119]. In the following chapter, different drug delivery and
formulation approaches, based on amino acids/peptides, lipids, DNA, and carbohydrates,
will be presented. Figure 2 offers an overview of the delivery systems presented in Section 5,
including the drugs that are transported and ocular diseases that are addressed.

Table 4. Overview on drug delivery systems for topical delivery to the posterior segment.

Delivery System (Drug) Size Characteristics Pharmacokinetics Further Results Ref.

Amino acid/Peptid-based drug delivery
Rabbits: 2x daily for 5 days
Higher aqueous 50 µL THC-Val-HS in IOP-lowering
THC-Val-HS solubility, higher Tocrisolve emulsion (300 µg equivalent to
Valine-hemisuccinate ester
513.6 Da; THC polar surface area, THC) -> THC-Val-HS: pilocarpine in a [120]
prodrug: Val-HS (THC)
314.2 Da improved logD 15.5 ng/50 mg retina-choroid, rabbit glaucoma
(pH 7.4) THC: 5.2 ng/50 mg model
retina-choroid after 1 h
Rats: 40 µL drop (2 µg
Cell-penetrating peptide TAT is positively
TAT-aFGF-His) -> His+ cells Strong protection
(CPP) HIV transactivator of charged (11 amino
Tat-aFGF-His: peaked after 30 min, still against
transcription (TAT) (acidic acids: [122]
~17.3-kDa detectable after 8 h in the ischemia-reperfusion
fibroblast growth factor GRKKRRQRRRC)
retina (mainly retinal injury in rats
(aFGF)) [121]
ganglion cells)
TAT is positively
Rats: 7 days twice daily
CPP TAT (calpain inhibitory charged (11 amino Tat-µCl was diffusely
Tat-µCl: 2857.37 Da (20 µL of 1 mM Tat-µCl) ->
peptide) acids: distributed [123]
(23 amino acids) Cmax 15.3 pg/µg protein in
-> Tat-µCl GRKKRRQRRRC) throughout the retina
the retina 1 h after last drop
[121]
Mouse model of
Rat: single 20 µL eye drop of CNV: CPP and
bevacizumab (25 µg/µL) -> bevacizumab eye
(5[6]- Cmax 1.65 ± 0.26 in the retina drops (twice daily
CPP polyarginine-6
carboxyfluorescein- after 40 min; 5 µL for 10 days) [124]
(bevacizumab)
RRRRRR-COOH) Porcine eyes: 20 µL drop significantly reduced
(25 µg/µL) -> 0.10 ± 0.03 µg CNV lesions,
per retina comparable to
anti-VEGF injection
Pharmaceutics 2022, 14, 134 12 of 21

Table 4. Cont.

Delivery System (Drug) Size Characteristics Pharmacokinetics Further Results Ref.

Lipid-based drug delivery
On interaction with
PS containing
membranes, annexin Rats: Single 30 µL drop
V is reported to form (13 mg/mL Avastin) ->
Annexin V liposomes Mean diameter of higher order 127 ng/g in the posterior eye;
[126]
(bevacizumab) 163 nm structures that induce Rabbit: 30 µL (25 mg/mL
formation of Avastin) once daily for 5 days
actin-independent -> 18 ng/g in retina/choroid
endocytic vesicles
[125]
Surface charge of
Rabbits: 30 µL twice in 5 min
Annexin V liposomes Mean particle size liposomes became
(125 ng/mL TGF-ß1) -> Cmax [127]
(TGF-ß1) 157 nm more negative with
114.7 pg/mL in the vitreous
annexin V
Colloidal Rabbits: Two x 50 µL eye Improved
Solid lipid nanoparticles SLN
Particle size: IN-SLN nanoparticulate drops -> retinal-choroidal transcorneal
(Indomethacin);
226 ± 5 nm dispersions -> IN-concentrations of permeability and [128]
Nanostructured lipid carriers
IN-NLC 227 ± 11 nm biocompatible and 227 ng/g with IN-SLN und retention
NLC (indomethacin)
mucoadhesive 893 ng/g with IN-NLC characteristics of IN
DNA-based drug delivery
Amphiphil, lipophilic
core, and hydrophilic Higher IOP reduction
Lipid DNA-Nanoparticles corona; than Briminodine
NP alone: 10 nm [129]
(Brimonidine) Aptameric and alone in DBA/2J
hydrophobic drug mice
loading
Albino rats: single drop of
Amphiphil, lipophilic
Trav-NP or Trav (80 µM) ->
core, and hydrophilic
Lipid DNA-Nanoparticles travoprost after 1 h:
NP alone: 10 nm corona; [130]
(Travoprost) 434.9 pg/mg (Trav-NP)
Aptameric drug
compared to 230.3 pg/mg
loading
(Trav)
Carbohydrate-based drug delivery
Solution with
Sodium carprate and
penetration enhancer
hydroxypropyl
0.5% sodium caprate Rabbits: 50 µL eye drops at
methylcellulose solution
28 kDa and viscosity 20 min intervals over 12 h -> [131]
(28 kDa antibody fragment ->
enhancer 1.5% 50–150 ng/mL in the vitreous
specificity for the rat
hydroxypropyl
CD4 molecule)
methylcellulose
Shaped like Rabbits: 1.5% dex-
truncated cones, with amethasone/γCD eye drops
γ-cyclodextrin (CD) 100–300 nm
a hydrophilic outer (50 µL) 3 doses in left eye for
(dexamethasone) drug/CD complexes [132]
surface and a 15 days -> left eye:
nanoparticle CD: 1–2 kDa
somewhat lipophilic 201 ± 48 ng/g, right eye:
central cavity 64 ± 12 ng/g in the retina
CSO + Valylvaline
(VV) + stearic acid Rabbits: 3 × 50µL
Higher ocular
Chitosan oligosaccharide (SA); VV is targeting CSO-VV-SA -> at 0.5 and 1 h
retention time
(CSO) nanomicelles 100 nm PepT-1 -> faster Dex conc. reached [133]
compared with
(dexamethasone) crossing of therapeutic levels (>200 ng/g)
traditional eye drops
conjunctival and in sclera-choroid-retina
scleral barriers
Pharmaceutics 2022, 14, x134
FOR PEER REVIEW 1213ofof 22
21

Figure 2.
Figure Schematicpresentation
2. Schematic presentationofof drug
drug delivery
delivery (DD)
(DD) systems
systems andand formulation
formulation approaches
approaches to
to im-
improve topical delivery to the back of the eye. (A) Drug delivery systems presented
prove topical delivery to the back of the eye. (A) Drug delivery systems presented in this review. in this re-
view.
(B) (B) Drugs
Drugs thattransported
that were were transported bydelivery
by those those delivery
systems.systems. (C) Diseases
(C) Diseases that are addressed
that are addressed with the
DD system
with the and drug.
DD system and drug.TheThe molecular
molecular structures
structures werewereobtained
obtainedfromfrom https://pub-
https://pubchem.
chem.ncbi.nlm.nih.gov
ncbi.nlm.nih.gov (accessed (accessed
on 21on 21 December
December 2021).2021). Abbreviations:
Abbreviations: THC—tetrahydrocanna-
THC—tetrahydrocannabinol;
binol; aFGF—acidic
aFGF—acidic fibroblast
fibroblast growth
growth factor;
factor; µCL—calpain
µCL—calpain inhibitorypeptide;
inhibitory peptide; Bev—bevacizumab;
Bev—bevacizumab;
IR—retinal ischemia/reperfusion; PR deg—photoreceptor degeneration; AMD—age-related macu-
IR—retinal ischemia/reperfusion; PR deg—photoreceptor degeneration; AMD—age-related mac-
lar degeneration; IN—indomethacin; DR—diabetic retinopathy; TGF-β1—transforming growth fac-
ular degeneration; IN—indomethacin; DR—diabetic retinopathy; TGF-β1—transforming growth
tor beta 1; Brim—brimonidine; Trav—travoprost; scFv—single-chain variable-domain fragment;
factor beta 1; Brim—brimonidine;
Dex—dexamethasone. Created with Trav—travoprost; scFv—single-chain
BioRender.com (accessed variable-domain
on 21 December 2021). fragment;
Dex—dexamethasone. Created with BioRender.com (accessed on 21 December 2021).
Table 4. Overview on drug delivery systems for topical delivery to the posterior segment.
5.1. Amino Acid/Peptid-Based Drug Delivery
Delivery System To overcome the anterior segment of the eye, there are many formulation approaches.
Size Characteristics Pharmacokinetics Further Results Ref.
(Drug) Recently, cannabinoids, such as tetrahydrocannabinol (THC), have been applied as anti-
glaucomaAmino
drugs for their IOP lowering
acid/Peptid-based effect.
drug However, THC eye drops have poor ability to
delivery
cross the cornea, due to their high logPRabbits:
value (6.42) and low
2x daily for 5aqueous solubility (1–2 µg/mL).
To overcome this low bioavailability, a valine-hemisuccinate
days 50 µL THC-Val-HS (Val-HS) ester prodrug has
been developed. THC-Val-HS achieved significantly higher transcorneal permeability,
THC-Val-HS in Tocrisolve emulsion IOP-lowering equiv-
Valine-hemisuccinate Higher
mainly due aqueous
to its solubility,
larger polar surface area, relatively lower logD 7.4, and increased aqueous
513.6 Da; (300 µg THC) -> THC- alent to pilocarpine
ester prodrug: Val-HS higher
solubility. polar
Adelli surface
et al. area,significantly higher THC concentrations in the anterior
showed [120]
THC 314.2 Val-HS: 15.5 ng/50 mg in a rabbit glaucoma
(THC) segment of the eye by
improved THC-Val-HS-loaded
logD (pH 7.4) topical eye drops in anesthetized rabbits. Com-
Da
pared with marketed pilocarpine HCl retina-choroid,
and timolol THC: 5.2 eye drops,
maleate modelthe intraocular
ng/50 mg retina-choroid
pressure-lowering effect of THC-Val-HS was equivalent to that of pilocarpine [120]. In
further development efforts, the prodrug was after 1h
formulated in a nanoemulsion (NE), which
Rats: 40 µL
led to a prolonged IOP lowering effect. In normotensive drop (2 µg rabbits, the THC-Val-HS-NE
Cell-penetrating pep-
showed a better effect than commercial TAT-aFGF-His) -> His+ [134].
timolol or latanoprost Strong protection
tide (CPP) HIV transac- Tat-aFGF- TAT is positively charged
Another formulation approach forpeaked
cells the topical
afterdelivery
30 min, ofagainst
proteins and peptides are
ischemia-
tivator of transcription His: ~17.3- (11 amino acids: GRK- [122]
cell-penetrating peptides (CPPs). Herein, using theafter
still detectable CPP 8HIV
h intransactivator of transcription
reperfusion injury in
(TAT) (acidic fibroblast kDa KRRQRRRC) [121]
(TAT), Wang et al. delivered acidic fibroblast
the retinagrowth
(mainlyfactor (FGF) to ratrats
retinal retina after a single
growth factor (aFGF)) topical administration (2 µg in a 40 µL solution). TAT-aFGF-His proteins were detectable
ganglion cells)
in the retina for at least eight hours and mediated strong protection against ischemia-
Pharmaceutics 2022, 14, 134 14 of 21

reperfusion injury compared to the aFGF-His and PBS treated groups: the inner retinal
layer structure was better maintained, retinal ganglion cell apoptosis was reduced, and
retinal function improved [122].
Ozaki et al. topically delivered a calpain inhibitory peptide (which protects pho-
toreceptors in retinal dystrophic rats) conjugated with TAT to the posterior segment of
the rat eye. Application of 20 µL of 1 mM Tat-µCL twice daily for seven days yielded a
concentration of 15.3 pg/µg protein one hour after the final instillation [123].
Cogan et al. also succeeded in achieving therapeutic concentrations in the posterior
segment of the rat eye by combining bevacizumab with polyarginine-6, another CPP.
After a single 20 µL eye drop of bevacizumab (25 µg/µL), a maximum concentration of
1.65 ± 0.26 µg/mL was detected in the retina at 40 min after application. In ex vivo studies
on porcine eyes, a single 20 µL eye drop of bevacizumab (25 µg/µL), complexed with CPP,
yielded a concentration of 10.68 ± 3.57 µg/mL in the vitreous and 0.10 ± 0.03 µg per retina,
which is within the therapeutic range for humans (10–200 µg/mL) [124].

5.2. Lipid-Based Drug Delivery Systems

Davis et al. detected physiologically relevant concentrations of bevacizumab in the
posterior segment of the eye in rats and rabbits. Here, the antibody was delivered using
liposomes functionalized with the anionic protein Annexin A5. A single 0.03 mL dose
(containing 13 mg/mL Avastin) yielded a bevacizumab concentration of 127 ng/g in the
posterior eye of rats. Application of 0.03 mL eye drops (25 mg/mL Avastin) once per day
for five days resulted in 18 ng/g in the rabbit retina/choroid [126]. The comparison of those
results to bevacizumab concentrations during clinical treatment with intravitreal injections
revealed that the topical liposomal delivery resulted in 3–5 orders of magnitude lower
bevacizumab concentrations. Which underlines that major improvements are necessary to
achieve clinically relevant results [135].
An Annexin 5 complemented liposome formulation was also used by Platania et al. to
deliver growth factor beta 1 topically to the vitreous of rabbits. After topical application
(two times within five minutes) of 30 µL eye drops (TGF-ß1 concentration of 125 ng/mL),
a maximum concentration of 114.7 ± 12.40 pg/mL was delivered to the vitreous (tmax
60 min) [127].
For the delivery of lipophilic drugs, Balguri et al. have designed various solid lipid
nanoparticles (SLN) and nanostructured lipid carriers (NLC), where liquid lipids are incor-
porated in the solid lipid structure. The delivery of the non-steroidal, anti-inflammatory
drug indomethacin (IN) was investigated in albino rabbits. Two doses of 50 µL eye drops
to conscious rabbits yielded retinal-choroidal IN-concentrations of 227 ng/g with IN-SLN
und 893 ng/g with IN-NLC [128].

5.3. Lipid DNA-Based Nanoparticles

Lipid DNA nanoparticles (NPs) are made of alkyl-modified oligonucleotides that rep-
resent amphiphilic molecules. Due to microphase separation, these NPs self-assemble into
micelles in an aqueous environment. The hydrophobic part (the lipid modifications) forms
the core, while the hydrophilic DNA sticks out of the micelle [136,137]. The NPs exhib-
ited relatively low critical micelle concentrations, demonstrating their stability in aqueous
surroundings [138]. NPs composed of amphiphilic DNA strands, that were composed
from twelve nucleotides, whereof four were lipid-modified with an alkyl chain, adhered
best to the ocular surface among different lipid NPs [138]. Brimonidine loading to these
lipid-modified DNA-NPs via hydrophobic interactions or using specific aptamers caused
improved affinity to the cornea. Maintaining drug release from the NPs. Brimonidine-NPs
significantly reduced intraocular pressure in live animals, more than pure brimonidine [129].
With the same delivery system, aptameric loading of travoprost was also achieved. Travo-
prost delivery with this NP system resulted in longer adhesion to the corneal surface,
enhanced uptake, efficacy, and biocompatibility. For example, after four hours, the amount
Pharmaceutics 2022, 14, 134 15 of 21

delivered in the eyes of albino rats via these NPs was four times higher, compared to
pristine travoprost [130].

5.4. Carbohydrate-Based Drug Delivery Systems

A 28-kDa antibody fragment (in a solution supplemented with penetration and
viscosity enhancers) could be delivered to the vitreous of rabbits, at concentrations of
50–150 ng/mL 12 h, after topical administration of 50 µL eye drops (at 20 min intervals
over 12 h) containing 0.8–1.1 mg/mL protein. The antibody fragment was applied in a
solution with sodium carprate as penetration enhancer and hydroxypropyl methylcellulose
as viscosity enhancer [131].
γ-cyclodextrin-based (CD) nanoparticles have been developed, by the company Oculis
Switzerland (Lausanne, Switzerland), for the treatment of anterior and posterior segment
diseases. The cyclodextrins increase the solubility of lipophilic drugs and can enhance topi-
cal drug uptake, through constantly supplying dissolved drug molecules to the membrane
surface [139]. Multiday application of the anti-inflammatory corticosteroid dexamethasone-
loaded CD nanoparticles yielded high concentration in the retina of rabbits (201 ng/g in the
treated eye) [132]. In a clinical trial with DME patients, this formulation could significantly
improve visual acuity and result in decreased macular thickness, comparable to a posterior
subtenon injection of triamcinolone acetonide, a frequently-reported, off-label treatment
for DME [140]. Several other clinical studies using γCD eye drops have been conducted,
and the development is ongoing to find better treatment options for DME and postcataract
surgery inflammation [141].
Xu et al. reported the development of Chitosan oligosaccharide-valylvaline-stearic
acid (CSO-VV-SA) nanomicelles. CSO is responsible for increased retention at the ocular
surface, and VV is known to target the peptide transporter-1 (PepT-1), which can enhance
ocular uptake and penetration of conjunctival, as well as scleral tissue. In an in vivo study in
rabbits, therapeutic concentrations of dexamethasone were reached in retina-choroid-sclera
via CSO-VV-SA eye drop application [133].

6. Conclusions and Future Perspectives

Treating retinal diseases by applying simple eye drops is an attractive goal for oph-
thalmologists, patients, and the pharmaceutical industry likewise. The biggest challenge
in reaching this goal is to achieve sufficient drug bioavailability, while minimizing side
effects. Topical treatment of the posterior segment of the eye presents many anatomic and
physiologic hurdles. As a result, drug delivery via the eye is very complex. In many cases,
in vivo animal models are the most appropriate model to study the absorption of new
drugs or delivery systems and to best predict their clinical performance. However, novel
ex vivo and in silico models show compatible outcomes [73,142]. It is important that model
systems are carefully chosen, evaluated, and compared to the human situation.
Detailed physicochemical characterization of the compound and delivery system is
required to predict and evaluate ocular bioavailability and optimize the particle properties
accordingly. Additionally, production methods are becoming more and more elaborate,
with the goal to produce defined delivery systems, according to drug loading, size, shape,
and properties of the drug delivery systems. It is important to incorporate the requirements
for large scale production early in the development process to make sure the particles are
ready to be produced for later clinical investigations.
As topically delivered drugs take a long journey through the eye to reach the posterior
segment and different routes are possible, in the future, targeted delivery, for example, by
using aptamers, might be of importance to make sure the target cells/tissues are reached
and side effects are minimized. One way to go might be delivery systems comprising
of receptor targeting. Additionally, the development of innovative, new small molecule
therapeutics would be desirable, as the currently-applied antibody therapeutics are not
suitable for topical delivery, due to their size. Safety profiles of the particles are another
hurdle in bringing those new technologies from bench to bedside. The focus should lie
Pharmaceutics 2022, 14, 134 16 of 21

on the development of biocompatible and biodegradable drug delivery systems with safe
degradation products.
Some promising preclinical studies, in which effective drug concentrations were
achieved in the posterior segment of the eye by topical application, were highlighted in
this review, suggesting that topical treatment of retinal diseases may be possible. However,
clinical trials showed that innovative drug-tailored delivery systems are needed for an
efficient retinal drug delivery.
In conclusion, innovative delivery systems and more clinical data are needed to
sufficiently understand and tailor retinal drug delivery via topical application.

Author Contributions: Conceptualization, M.L. and S.S.; writing—original draft preparation, M.L.
and C.S.; writing—review and editing, J.H. and S.S.; visualization, M.L.; supervision, S.S.; funding
acquisition, S.S. All authors have read and agreed to the published version of the manuscript.
Funding: This research received no external funding.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Acknowledgments: S.S. acknowledges funding from the GO-Bio initial initiative of the Bundesmin-
isterium für Bildung und Forschung, Germany, grant number 16LW0025, project REVeyeVE; S.S.
further acknowledges funding of the Bundesministerium für Wirtschaft und Energie, Germany for
the EXIST Forschungstransfer: nano-I-drops; grant number 03EFDBW075.
Conflicts of Interest: The authors declare no conflict of interest.

References
1. Zhang, W.; Prausnitz, M.R.; Edwards, A. Model of transient drug diffusion across cornea. J. Control. Release 2004, 99, 241–258.
[CrossRef]
2. Fraunfelder, F.; Meyer, S.M. Systemic Side Effects from Ophthalmic Timolol and Their Prevention. J. Ocul. Pharmacol. Ther. 1987,
3, 177–184. [CrossRef] [PubMed]
3. Ilochonwu, B.C.; Urtti, A.; Hennink, W.E.; Vermonden, T. Intravitreal hydrogels for sustained release of therapeutic proteins. J.
Control. Release 2020, 326, 419–441. [CrossRef] [PubMed]
4. Jung, H.J.; Abou-Jaoude, M.; Carbia, B.E.; Plummer, C.; Chauhan, A. Glaucoma therapy by extended release of timolol from
nanoparticle loaded silicone-hydrogel contact lenses. J. Control. Release 2012, 165, 82–89. [CrossRef] [PubMed]
5. Choi, B.; Loh, X.J.; Tan, A.; Loh, C.K.; Ye, E.; Joo, M.K.; Jeong, B. Introduction to In Situ Forming Hydrogels for Biomedical
Applications. In In-Situ Gelling Polymers: For Biomedical Applications; Loh, X.J., Ed.; Springer: Singapore, 2015; pp. 5–35.
6. Wei, Y.; Li, C.; Zhu, Q.; Zhang, X.; Guan, J.; Mao, S. Comparison of thermosensitive in situ gels and drug-resin complex for ocular
drug delivery: In vitro drug release and in vivo tissue distribution. Int. J. Pharm. 2020, 578, 119184. [CrossRef]
7. Castro-Balado, A.; Mondelo-García, C.; Zarra-Ferro, I.; Fernández-Ferreiro, A. New ophthalmic drug delivery systems. Farm.
Hosp. 2020, 44, 149–157. [CrossRef]
8. Tham, Y.-C.; Li, X.; Wong, T.Y.; Quigley, H.A.; Aung, T.; Cheng, C.-Y. Global Prevalence of Glaucoma and Projections of Glaucoma
Burden through 2040: A systematic review and meta-analysis. Ophthalmology 2014, 121, 2081–2090. [CrossRef]
9. Zhang, N.; Wang, J.; Li, Y.; Jiang, B. Prevalence of primary open angle glaucoma in the last 20 years: A meta-analysis and
systematic review. Sci. Rep. 2021, 11, 13762. [CrossRef]
10. Wong, W.L.; Su, X.; Li, X.; Cheung, C.M.G.; Klein, R.; Cheng, C.Y.; Wong, T.Y. Global prevalence of age-related macular
degeneration and disease burden projection for 2020 and 2040: A systematic review and meta-analysis. Lancet Glob. Health 2014,
2, e106–e116. [CrossRef]
11. Song, W.; Huang, P.; Zhang, C. Neuroprotective therapies for glaucoma. Drug Des. Dev. Ther. 2015, 9, 1469–1479. [CrossRef]
12. Vrabec, J.P.; Levin, L.A. The neurobiology of cell death in glaucoma. Eye 2007, 21, S11–S14. [CrossRef] [PubMed]
13. Sharif, N.A. Therapeutic Drugs and Devices for Tackling Ocular Hypertension and Glaucoma, and Need for Neuroprotection and
Cytoprotective Therapies. Front. Pharmacol. 2021, 12, 729249. [CrossRef] [PubMed]
14. Gossman, C.A.; Christie, J.; Webster, M.K.; Linn, D.M.; Linn, C.L. Neuroprotective Strategies in Glaucoma. Curr. Pharm. Des. 2016,
22, 2178–2192. [CrossRef]
15. Shalaby, W.S.; Ahmed, O.M.; Waisbourd, M.; Katz, L.J. A Review of Potential Novel Glaucoma Therapeutic Options Independent
of Intraocular Pressure. Surv. Ophthalmol. 2021, 21. [CrossRef]
16. Heesterbeek, T.J.; Lorés-Motta, L.; Hoyng, C.B.; Lechanteur, Y.T.E.; Hollander, A.I.D. Risk factors for progression of age-related
macular degeneration. Ophthalmic Physiol. Opt. 2020, 40, 140–170. [CrossRef]
Pharmaceutics 2022, 14, 134 17 of 21

17. Lambert, N.G.; ElShelmani, H.; Singh, M.K.; Mansergh, F.C.; Wride, M.A.; Padilla, M.; Keegan, D.; Hogg, R.E.; Ambati, B.K. Risk
factors and biomarkers of age-related macular degeneration. Prog. Retin. Eye Res. 2016, 54, 64–102. [CrossRef] [PubMed]
18. Ma, H.H.; Liutkevičienė, R. Age-Related Macular Degeneration: What Do We Know So Far? Acta Med. Litu. 2021, 28, 36–47.
[CrossRef]
19. Fogli, S.; Del Re, M.; Rofi, E.; Posarelli, C.; Figus, M.; Danesi, R. Clinical pharmacology of intravitreal anti-VEGF drugs. Eye 2018,
32, 1010–1020. [CrossRef]
20. Patel, P.; Sheth, V. New and Innovative Treatments for Neovascular Age-Related Macular Degeneration (nAMD). J. Clin. Med.
2021, 10, 2436. [CrossRef]
21. Hughes, P.M.; Olejnik, O.; Chang-Lin, J.-E.; Wilson, C.G. Topical and systemic drug delivery to the posterior segments. Adv. Drug
Deliv. Rev. 2005, 57, 2010–2032. [CrossRef]
22. Urtti, A. Challenges and obstacles of ocular pharmacokinetics and drug delivery. Adv. Drug Deliv. Rev. 2006, 58, 1131–1135.
[CrossRef]
23. Willcox, M.D.; Argüeso, P.; Georgiev, G.; Holopainen, J.M.; Laurie, G.; Millar, T.J.; Papas, E.B.; Rolland, J.P.; Schmidt, T.A.; Stahl,
U.; et al. TFOS DEWS II Tear Film Report. Ocul. Surf. 2017, 15, 366–403. [CrossRef]
24. Malhotra, M.; Majumdar, D.K. Permeation through cornea. Indian J. Exp. Biol. 2001, 39, 11–24. [PubMed]
25. Gaudana, R.; Ananthula, H.K.; Parenky, A.; Mitra, A.K. Ocular Drug Delivery. AAPS J. 2010, 12, 348–360. [CrossRef]
26. Hämäläinen, K.M.; Kananen, K.; Auriola, S.; Kontturi, K.; Urtti, A. Characterization of paracellular and aqueous penetration
routes in cornea, conjunctiva, and sclera. Investig. Ophthalmol. Vis. Sci. 1997, 38, 627–634.
27. Wang, W.; Sasaki, H.; Chien, D.-S.; Lee, V.H.L. Lipophilicity influence on conjunctival drug penetration in the pigmented rabbit:
A comparison with corneal penetration. Curr. Eye Res. 1991, 10, 571–579. [CrossRef]
28. Suhonen, P.; Järvinen, T.; Rytkönen, P.; Peura, P.; Urtti, A. Improved Corneal Pilocarpine Permeability with O,O0 -(l,4-Xylylene)
Bispilocarpic Acid Ester Double Prodrugs. Pharm. Res. 1991, 8, 1539–1542. [CrossRef] [PubMed]
29. Mannermaa, E.; Vellonen, K.-S.; Urtti, A. Drug transport in corneal epithelium and blood–retina barrier: Emerging role of
transporters in ocular pharmacokinetics. Adv. Drug Deliv. Rev. 2006, 58, 1136–1163. [CrossRef]
30. Ramsay, E.; del Amo, E.M.; Toropainen, E.; Tengvall-Unadike, U.; Ranta, V.-P.; Urtti, A.; Ruponen, M. Corneal and conjunctival
drug permeability: Systematic comparison and pharmacokinetic impact in the eye. Eur. J. Pharm. Sci. 2018, 119, 83–89. [CrossRef]
31. Hosoya, K.-I.; Lee, V.H.; Kim, K.-J. Roles of the conjunctiva in ocular drug delivery: A review of conjunctival transport mechanisms
and their regulation. Eur. J. Pharm. Biopharm. 2005, 60, 227–240. [CrossRef] [PubMed]
32. Gadek, T.; Lee, D. Topical Drug Delivery to the Back of the Eye. In Drug Product Development for the Back of the Eye; Kompella, U.B.,
Edelhauser, H.F., Eds.; Springer: Boston, MA, US, 2011; pp. 111–124.
33. Ganea, E.; Harding, J.J. Glutathione-Related Enzymes and the Eye. Curr. Eye Res. 2006, 31, 1–11. [CrossRef]
34. Xu, J.; Heys, J.J.; Barocas, V.H.; Randolph, T.W. Permeability and diffusion in vitreous humor: Implications for drug delivery.
Pharm. Res. 2000, 17, 664–669. [CrossRef] [PubMed]
35. Xu, Q.; Boylan, N.J.; Suk, J.S.; Wang, Y.-Y.; Nance, E.A.; Yang, J.-C.; McDonnell, P.J.; Cone, R.A.; Duh, E.J.; Hanes, J. Nanoparticle
diffusion in, and microrheology of, the bovine vitreous ex vivo. J. Control. Release 2013, 167, 76–84. [CrossRef] [PubMed]
36. Del Amo, E.M.; Rimpelä, A.-K.; Heikkinen, E.; Kari, O.K.; Ramsay, E.; Lajunen, T.; Schmitt, M.; Pelkonen, L.; Bhattacharya,
M.; Richardson, D.; et al. Pharmacokinetic aspects of retinal drug delivery. Prog. Retin. Eye Res. 2017, 57, 134–185. [CrossRef]
[PubMed]
37. Kim, H.; Robinson, S.B.; Csaky, K.G. Investigating the Movement of Intravitreal Human Serum Albumin Nanoparticles in the
Vitreous and Retina. Pharm. Res. 2008, 26, 329–337. [CrossRef] [PubMed]
38. Bejjani, R.A.; Benezra, D.; Cohen, H.; Rieger, J.; Andrieu, C.; Jeanny, J.-C.; Golomb, G.; Behar-Cohen, F.F. Nanoparticles for gene
delivery to retinal pigment epithelial cells. Mol. Vis. 2005, 11, 124–132.
39. Peeters, L.; Sanders, N.N.; Braeckmans, K.; Boussery, K.; Van De Voorde, J.; De Smedt, S.C.; Demeester, J. Vitreous: A Barrier to
Nonviral Ocular Gene Therapy. Investig. Opthalmol. Vis. Sci. 2005, 46, 3553–3561. [CrossRef]
40. Martens, T.F.; Remaut, K.; Deschout, H.; Engbersen, J.F.; Hennink, W.E.; van Steenbergen, M.J.; Demeester, J.; De Smedt, S.C.;
Braeckmans, K. Coating nanocarriers with hyaluronic acid facilitates intravitreal drug delivery for retinal gene therapy. J. Control.
Release 2015, 202, 83–92. [CrossRef]
41. Ryals, R.C.; Patel, S.; Acosta, C.; McKinney, M.; Pennesi, M.E.; Sahay, G. The effects of PEGylation on LNP based mRNA delivery
to the eye. PLoS ONE 2020, 15, e0241006. [CrossRef]
42. Wilson, C.G.; Tan, L.E.; Mains, J. Principles of Retinal Drug Delivery from Within the Vitreous. Drug Prod. Dev. Back Eye 2011, 2,
125–158. [CrossRef]
43. Koevary, S.B. Pharmacokinetics of Topical Ocular Drug Delivery: Potential Uses for the Treatment of Diseases of the Posterior
Segment and Beyond. Curr. Drug Metab. 2003, 4, 213–222. [CrossRef]
44. Pitkänen, L.; Ranta, V.-P.; Moilanen, H.; Urtti, A. Permeability of Retinal Pigment Epithelium: Effects of Permeant Molecular
Weight and Lipophilicity. Investig. Opthalmol. Vis. Sci. 2005, 46, 641–646. [CrossRef] [PubMed]
45. Wang, L.; Ben Zhou, M.; Zhang, H. The Emerging Role of Topical Ocular Drugs to Target the Posterior Eye. Ophthalmol. Ther.
2021, 10, 465–494. [CrossRef] [PubMed]
Pharmaceutics 2022, 14, 134 18 of 21

46. Varela-Fernández, R.; Díaz-Tomé, V.; Luaces-Rodríguez, A.; Conde-Penedo, A.; García-Otero, X.; Luzardo-Álvarez, A.; Fernández-
Ferreiro, A.; Otero-Espinar, F.J. Drug Delivery to the Posterior Segment of the Eye: Biopharmaceutic and Pharmacokinetic
Considerations. Pharmaceutics 2020, 12, 269. [CrossRef] [PubMed]
47. Yadav, D.; Varma, L.T.; Yadav, K. Drug Delivery to Posterior Segment of the Eye: Conventional Delivery Strategies, Their Barriers,
and Restrictions. In Drug Delivery for the Retina and Posterior Segment Disease; Patel, J.K., Sutariya, V., Kanwar, J.R., Pathak, Y.V.,
Eds.; Springer: Cham, Switzerland, 2018; pp. 51–67.
48. Ambati, J.; Canakis, C.S.; Miller, J.W.; Gragoudas, E.S.; Edwards, A.; Weissgold, D.J.; Kim, I.; Delori, F.C.; Adamis, A.P. Diffusion
of high molecular weight compounds through sclera. Investig. Ophthalmol. Vis. Sci. 2000, 41, 1181–1185.
49. Cheruvu, N.P.S.; Kompella, U.B. Bovine and Porcine Transscleral Solute Transport: Influence of Lipophilicity and the Choroid–
Bruch’s Layer. Investig. Opthalmol. Vis. Sci. 2006, 47, 4513–4522. [CrossRef]
50. Rimpelä, A.-K.; Schmitt, M.; Latonen, S.; Hagström, M.; Antopolsky, M.; Manzanares, J.A.; Kidron, H.; Urtti, A. Drug Distri-
bution to Retinal Pigment Epithelium: Studies on Melanin Binding, Cellular Kinetics, and Single Photon Emission Computed
Tomography/Computed Tomography Imaging. Mol. Pharm. 2016, 13, 2977–2986. [CrossRef]
51. Robbie, S.J.; Von Leithner, P.L.; Ju, M.; Lange, C.A.; King, A.G.; Adamson, P.; Lee, D.; Sychterz, C.; Coffey, P.; Ng, Y.S.; et al. As-
sessing a Novel Depot Delivery Strategy for Noninvasive Administration of VEGF/PDGF RTK Inhibitors for Ocular Neovascular
Disease. Investig. Opthalmol. Vis. Sci. 2013, 54, 1490–1500. [CrossRef]
52. Rodrigues, G.A.; Lutz, D.; Shen, J.; Yuan, X.; Shen, H.; Cunningham, J.; Rivers, H.M. Topical Drug Delivery to the Posterior
Segment of the Eye: Addressing the Challenge of Preclinical to Clinical Translation. Pharm. Res. 2018, 35, 245. [CrossRef]
53. Weir, A.B.; Collins, M. Assessing Ocular Toxicology in Laboratory Animals; Springer: New York, NY, USA, 2012.
54. Gelatt, K.N. Essentials of Veterinary Ophthalmology, 3rd ed.; Wiley-Blackwell: Hoboken, NJ, USA, 2014; p. 720.
55. Henriksson, J.T.; McDermott, A.M.; Bergmanson, J.P.G. Dimensions and Morphology of the Cornea in Three Strains of Mice.
Investig. Opthalmol. Vis. Sci. 2009, 50, 3648–3654. [CrossRef]
56. Schulz, D.; Iliev, M.E.; Frueh, B.E.; Goldblum, D. In vivo pachymetry in normal eyes of rats, mice and rabbits with the optical low
coherence reflectometer. Vis. Res. 2003, 43, 723–728. [CrossRef]
57. Faber, C.; Scherfig, E.; Prause, J.; Sørensen, K. Corneal Thickness in Pigs Measured by Ultrasound Pachymetry In Vivo. Scand. J.
Lab. Anim. Sci. 2008, 35, 39–43.
58. Sadoughi, M.; Einollahi, B.; Einollahi, N.; Rezaei, J.; Roshandel, D.; Feizi, S. Measurement of central corneal thickness using
ultrasound pachymetry and Orbscan II in normal eyes. J. Ophthalmic Vis. Res. 2015, 10, 4–9. [CrossRef]
59. Menduni, F.; Davies, L.N.; Madrid-Costa, D.; Fratini, A.; Wolffsohn, J. Characterisation of the porcine eyeball as an in-vitro model
for dry eye. Contact Lens Anterior Eye 2018, 41, 13–17. [CrossRef] [PubMed]
60. Clough, J.D.; Parikh, C.H.; Edelhauser, H.F. Anterior Chamber, Lens and Globe Volumes in Balb/C and C57/BL6 Mice. Investig.
Opthalmol. Vis. Sci. 2003, 44, 648.
61. Johnson, M.; Caro, N.; Huang, J.-D. Adequacy of exchanging the content of the anterior chamber. Exp. Eye Res. 2010, 91, 876–880.
[CrossRef] [PubMed]
62. Thomasy, S.M.; Eaton, J.S.; Timberlake, M.J.; Miller, P.E.; Matsumoto, S.; Murphy, C.J. Species Differences in the Geometry of the
Anterior Segment Differentially Affect Anterior Chamber Cell Scoring Systems in Laboratory Animals. J. Ocul. Pharmacol. Ther.
2016, 32, 28–37. [CrossRef]
63. Aihara, M.; Lindsey, J.D.; Weinreb, R.N. Aqueous Humor Dynamics in Mice. Investig. Opthalmol. Vis. Sci. 2003, 44, 5168–5173.
[CrossRef]
64. Zhao, M.; Hejkal, J.J.; Camras, C.B.; Toris, C.B. Aqueous Humor Dynamics during the Day and Night in Juvenile and Adult
Rabbits. Investig. Opthalmol. Vis. Sci. 2010, 51, 3145–3151. [CrossRef] [PubMed]
65. Loewen, R.T.; Roy, P.; Park, D.B.; Jensen, A.; Scott, G.; Cohen-Karni, D.; Fautsch, M.P.; Schuman, J.; Loewen, N.A. A Porcine
Anterior Segment Perfusion and Transduction Model with Direct Visualization of the Trabecular Meshwork. Investig. Opthalmol.
Vis. Sci. 2016, 57, 1338–1344. [CrossRef]
66. Goel, M.; Pacciani, R.G.; Lee, R.K.; Battacharya, S.K. Aqueous Humor Dynamics: A Review. Open Ophthalmol. J. 2010, 4, 52–59.
[CrossRef] [PubMed]
67. Kaplan, H.J.; Chiang, C.-W.; Chen, J.; Song, S.-K. Vitreous Volume of the Mouse Measured by Quantitative High-Resolution MRI.
Investig. Ophthalmol. Vis. Sci. 2010, 51, 4414.
68. Kane, A.; Barza, M.; Baum, J. Intravitreal injection of gentamicin in rabbits. Effect of inflammation and pigmentation on half-life
and ocular distribution. Investig. Ophthalmol. Vis. Sci. 1981, 20, 593–597.
69. Jonckx, B.; Porcu, M.; Candi, A.; Etienne, I.; Barbeaux, P.; Feyen, J.H.M. Assessment of Ocriplasmin Effects on the Vitreoretinal
Compartment in Porcine and Human Model Systems. J. Ophthalmol. 2017, 2017, 2060765. [CrossRef] [PubMed]
70. Johnson, M.W. Posterior Vitreous Detachment: Evolution and Complications of Its Early Stages. Am. J. Ophthalmol. 2010, 149,
371–382. [CrossRef] [PubMed]
71. Ferguson, L.R.; Ii, J.M.D.; Balaiya, S.; Grover, S.; Chalam, K.V. Retinal Thickness Normative Data in Wild-Type Mice Using
Customized Miniature SD-OCT. PLoS ONE 2013, 8, e67265. [CrossRef]
72. Carpenter, C.L.; Kim, A.Y.; Kashani, A.H. Normative Retinal Thicknesses in Common Animal Models of Eye Disease Using
Spectral Domain Optical Coherence Tomography. Adv. Exp. Med. Biol. 2018, 1074, 157–166. [CrossRef]
Pharmaceutics 2022, 14, 134 19 of 21

73. Schnichels, S.; Paquet-Durand, F.; Löscher, M.; Tsai, T.; Hurst, J.; Joachim, S.C.; Klettner, A. Retina in a dish: Cell cultures, retinal
explants and animal models for common diseases of the retina. Prog. Retin. Eye Res. 2020, 81, 100880. [CrossRef]
74. Murali, A.; Ramlogan-Steel, C.A.; Andrzejewski, S.; Steel, J.C.; Layton, C.J. Retinal explant culture: A platform to investigate
human neuro-retina. Clin. Exp. Ophthalmol. 2018, 47, 274–285. [CrossRef]
75. Ericsson, A.C.; Crim, M.; Franklin, C.L. A Brief History of Animal Modeling. Mo. Med. 2013, 110, 201–205.
76. Verra, D.M.; Sajdak, B.; Merriman, D.; Hicks, D. Diurnal rodents as pertinent animal models of human retinal physiology and
pathology. Prog. Retin. Eye Res. 2019, 74, 100776. [CrossRef] [PubMed]
77. Yafai, Y.; Yang, X.M.; Niemeyer, M.; Nishiwaki, A.; Lange, J.; Wiedemann, P.; King, A.G.; Yasukawa, T.; Eichler, W. Anti-angiogenic
effects of the receptor tyrosine kinase inhibitor, pazopanib, on choroidal neovascularization in rats. Eur. J. Pharmacol. 2011, 666,
12–18. [CrossRef] [PubMed]
78. Singh, R.; Wurzelmann, J.I.; Ye, L.; Henderson, L.; Hossain, M.; Trivedi, T.; Kelly, D.S. Clinical evaluation of pazopanib eye drops
in healthy subjects and in subjects with neovascular age-related macular degeneration. Retina 2014, 34, 1787–1795. [CrossRef]
[PubMed]
79. Solovei, I.; Kreysing, M.; Lanctôt, C.; Kösem, S.; Peichl, L.; Cremer, T.; Guck, J.; Joffe, B. Nuclear Architecture of Rod Photoreceptor
Cells Adapts to Vision in Mammalian Evolution. Cell 2009, 137, 356–368. [CrossRef]
80. Peichl, L. Diversity of mammalian photoreceptor properties: Adaptations to habitat and lifestyle? Anat. Rec. Part A Discov. Mol.
Cell. Evol. Biol. 2005, 287, 1001–1012. [CrossRef]
81. Huber, G.; Heynen, S.; Imsand, C.; Hagen, F.V.; Muehlfriedel, R.; Tanimoto, N.; Feng, Y.; Hammes, H.-P.; Grimm, C.; Peichl, L.; et al.
Novel Rodent Models for Macular Research. PLoS ONE 2010, 5, e13403. [CrossRef]
82. Laude, A.; Tan, L.E.; Wilson, C.G.; Lascaratos, G.; Elashry, M.; Aslam, T.; Patton, N.; Dhillon, B. Intravitreal therapy for neovascular
age-related macular degeneration and inter-individual variations in vitreous pharmacokinetics. Prog. Retin. Eye Res. 2010, 29,
466–475. [CrossRef] [PubMed]
83. Rowe-Rendleman, C.L.; Durazo, S.A.; Kompella, U.B.; Rittenhouse, K.D.; Di Polo, A.; Weiner, A.L.; Grossniklaus, H.E.; Naash,
M.I.; Lewin, A.S.; Horsager, A.; et al. Drug and Gene Delivery to the Back of the Eye: From Bench to Bedside. Investig. Opthalmol.
Vis. Sci. 2014, 55, 2714–2730. [CrossRef] [PubMed]
84. Del Amo, E.M.; Urtti, A. Rabbit as an animal model for intravitreal pharmacokinetics: Clinical predictability and quality of the
published data. Exp. Eye Res. 2015, 137, 111–124. [CrossRef]
85. Juliusson, B.; Bergström, A.; Röhlich, P.; Ehinger, B.; van Veen, T.; Szél, A. Complementary cone fields of the rabbit retina. Investig.
Opthalmol. Vis. Sci. 1994, 35, 811–818.
86. Qiu, G.; Stewart, J.M.; Sadda, S.; Freda, R.; Lee, S.; Guven, D.; de Juan, E.; Varner, S.E. A new model of experimental subretinal
neovascularization in the rabbit. Exp. Eye Res. 2006, 83, 141–152. [CrossRef] [PubMed]
87. Balaratnasingam, C.; Kang, M.H.; Yu, P.; Chan, G.; Morgan, W.H.; Cringle, S.J.; Yu, D.-Y. Comparative quantitative study of
astrocytes and capillary distribution in optic nerve laminar regions. Exp. Eye Res. 2014, 121, 11–22. [CrossRef] [PubMed]
88. Lassota, N. Clinical and Histological Aspects of CNV Formation: Studies in an Animal Model. Acta Ophthalmol. 2008, 86, 1–28.
[CrossRef]
89. Sanchez, I.; Martin, R.; Ussa, F.; Fernandez-Bueno, I. The parameters of the porcine eyeball. Graefe’s Arch. Clin. Exp. Ophthalmol.
2011, 249, 475–482. [CrossRef]
90. Schnichels, S.; Kiebler, T.; Hurst, J.; Maliha, A.M.; Löscher, M.; Dick, H.B.; Bartz-Schmidt, K.-U.; Joachim, S.C. Retinal Organ
Cultures as Alternative Research Models. Altern. Lab. Anim. 2019, 47, 19–29. [CrossRef]
91. Peynshaert, K.; Devoldere, J.; De Smedt, S.C.; Remaut, K. In vitro and ex vivo models to study drug delivery barriers in the
posterior segment of the eye. Adv. Drug Deliv. Rev. 2018, 126, 44–57. [CrossRef]
92. Peynshaert, K.; Devoldere, J.; Forster, V.; Picaud, S.; Vanhove, C.; De Smedt, S.C.; Remaut, K. Toward smart design of retinal
drug carriers: A novel bovine retinal explant model to study the barrier role of the vitreoretinal interface. Drug Deliv. 2017, 24,
1384–1394. [CrossRef]
93. Janulevičienė, I.; Siaudvytyte, L.; Baršauskaitė, R. Ophthalmic Drug Delivery in Glaucoma—A Review. Pharmaceutics 2012, 4,
243–251. [CrossRef]
94. Inoue, J.; Oka, M.; Aoyama, Y.; Kobayashi, S.; Ueno, S.; Hada, N.; Takeda, T.; Takehana, M. Effects of Dorzolamide Hydrochloride
on Ocular Tissues. J. Ocul. Pharmacol. Ther. 2004, 20, 1–13. [CrossRef] [PubMed]
95. Acheampong, A.A.; Shackleton, M.; John, B.; Burke, J.; Wheeler, L.; Tang-Liu, D. Distribution of brimonidine into anterior and
posterior tissues of monkey, rabbit, and rat eyes. Drug Metab. Dispos. 2002, 30, 421–429. [CrossRef]
96. Holló, G.; Whitson, J.T.; Faulkner, R.; McCue, B.; Curtis, M.; Wieland, H.; Chastain, J.; Sanders, M.; DeSantis, L.; Przydryga, J.; et al.
Concentrations of Betaxolol in Ocular Tissues of Patients with Glaucoma and Normal Monkeys after 1 Month of Topical Ocular
Administration. Investig. Opthalmol. Vis. Sci. 2006, 47, 235–240. [CrossRef] [PubMed]
97. Lin, C.-W.; Sherman, B.; Moore, L.A.; Laethem, C.L.; Lu, D.-W.; Pattabiraman, P.P.; Rao, P.V.; Delong, M.A.; Kopczynski, C.C.
Discovery and Preclinical Development of Netarsudil, a Novel Ocular Hypotensive Agent for the Treatment of Glaucoma. J. Ocul.
Pharmacol. Ther. 2018, 34, 40–51. [CrossRef]
98. Koeberle, M.J.; Hughes, P.M.; Skellern, G.G.; Wilson, C.G. Pharmacokinetics and Disposition of Memantine in the Arterially
Perfused Bovine Eye. Pharm. Res. 2006, 23, 2781–2798. [CrossRef]
Pharmaceutics 2022, 14, 134 20 of 21

99. Li, S.K.; Liddell, M.R.; Wen, H. Effective electrophoretic mobilities and charges of anti-VEGF proteins determined by capillary
zone electrophoresis. J. Pharm. Biomed. Anal. 2011, 55, 603–607. [CrossRef] [PubMed]
100. Nomoto, H.; Shiraga, F.; Kuno, N.; Kimura, E.; Fujii, S.; Shinomiya, K.; Nugent, A.K.; Hirooka, K.; Baba, T. Pharmacokinetics of
Bevacizumab after Topical, Subconjunctival, and Intravitreal Administration in Rabbits. Investig. Opthalmol. Vis. Sci. 2009, 50,
4807–4813. [CrossRef] [PubMed]
101. Furrer, E.; Berdugo, M.; Stella, C.; Behar-Cohen, F.; Gurny, R.; Feige, U.; Lichtlen, P.; Urech, D.M. Pharmacokinetics and Posterior
Segment Biodistribution of ESBA105, an Anti–TNF-α Single-Chain Antibody, upon Topical Administration to the Rabbit Eye.
Investig. Opthalmol. Vis. Sci. 2009, 50, 771–778. [CrossRef]
102. Russ, H.; Parsons, C.; Barkana, Y.; Pearlman, A.L.; Heier, J.S.; Levin, L.A.; Weinreb, R.N.; Liebmann, J.M. Sustained neuroprotective
effect of novel Aβ aggregation modulator GAL-101 shown in dry AMD and glaucoma models with transient peak concentrations
using eye drops. Investig. Opthalmol. Vis. Sci. 2019, 60, 5393.
103. Askew, B.C.; Furuya, T.; Edwards, D.S. Ocular Distribution and Pharmacodynamics of SF0166, a Topically Administered αvβ3
Integrin Antagonist, for the Treatment of Retinal Diseases. J. Pharmacol. Exp. Ther. 2018, 366, 244–250. [CrossRef]
104. Ciulla, T.A.; Criswell, M.H.; Danis, R.P.; Williams, J.I.; McLane, M.P.; Holroyd, K.J. Squalamine Lactate Reduces Choroidal
Neovascularization in a Laser-Injury Model in the Rat. Retina 2003, 23, 808–814. [CrossRef]
105. Doukas, J.; Mahesh, S.; Umeda, N.; Kachi, S.; Akiyama, H.; Yokoi, K.; Cao, J.; Chen, Z.; Dellamary, L.; Tam, B.; et al. Topical
administration of a multi-targeted kinase inhibitor suppresses choroidal neovascularization and retinal edema. J. Cell. Physiol.
2008, 216, 29–37. [CrossRef]
106. Adams, C.M.; Anderson, K.; Artman, G.; Bizec, J.-C.; Cepeda, R.; Elliott, J.; Fassbender, E.; Ghosh, M.; Hanks, S.; Hardegger,
L.A.; et al. The Discovery of N-(1-Methyl-5-(trifluoromethyl)-1H-pyrazol-3-yl)-5-((6-((methylamino)methyl)pyrimidin-4-yl)oxy)-
1H-indole-1-carboxamide (Acrizanib), a VEGFR-2 Inhibitor Specifically Designed for Topical Ocular Delivery, as a Therapy for
Neovascular Age-Related Macular Degeneration. J. Med. Chem. 2018, 61, 1622–1635. [CrossRef] [PubMed]
107. PanOptica. Once-Daily Topical Anti-VEGF Eye Drop for Wet AMD and Other Neovascular Eye Disease. Available online:
www.panopticapharma.com (accessed on 2 September 2021).
108. Hoy, S.M. Netarsudil Ophthalmic Solution 0.02%: First Global Approval. Drugs 2018, 78, 389–396. [CrossRef]
109. Andreoli, C.M.; Miller, J. Anti-vascular endothelial growth factor therapy for ocular neovascular disease. Curr. Opin. Ophthalmol.
2007, 18, 502–508. [CrossRef]
110. Thomas, C.N.; Sim, D.A.; Lee, W.H.; Alfahad, N.; Dick, A.D.; Denniston, A.K.; Hill, L.J. Emerging therapies and their delivery for
treating age-related macular degeneration. Br. J. Pharmacol. 2021. [CrossRef] [PubMed]
111. Van Hove, I.; Hu, T.-T.; Beets, K.; Van Bergen, T.; Etienne, I.; Stitt, A.W.; Vermassen, E.; Feyen, J.H. Targeting RGD-binding
integrins as an integrative therapy for diabetic retinopathy and neovascular age-related macular degeneration. Prog. Retin. Eye
Res. 2021, 85, 100966. [CrossRef]
112. Edwards, D.; Boyer, D.S.; Kaiser, P.K.; Heier, J.S.; Askew, B. First-in human study of SF0166 Topical Ophthalmic Solution in
patients with diabetic macular edema. Investig. Opthalmol. Vis. Sci. 2018, 59, 1961.
113. Danis, R.; McLaughlin, M.M.; Tolentino, M.; Staurenghi, G.; Ye, L.; Xu, C.-F.; Kim, R.Y.; Johnson, M.W.; for the Pazopanib Eye
Drops Study Group. Pazopanib eye drops: A randomised trial in neovascular age-related macular degeneration. Br. J. Ophthalmol.
2013, 98, 172–178. [CrossRef] [PubMed]
114. Csaky, K.G.; Dugel, P.U.; Pierce, A.J.; Fries, M.A.; Kelly, D.S.; Danis, R.P.; Wurzelmann, J.I.; Xu, C.-F.; Hossain, M.; Trivedi, T.
Clinical Evaluation of Pazopanib Eye Drops versus Ranibizumab Intravitreal Injections in Subjects with Neovascular Age-Related
Macular Degeneration. Ophthalmology 2015, 122, 579–588. [CrossRef] [PubMed]
115. Poor, S.H.; Adams, C.M.; Ferriere, M.; Weichselberger, A.; Grosskreutz, C.L.; Weissgerber, G. Topical VEGF receptor inhibitor,
LHA510, did not demonstrate efficacy in a Proof-of-Concept study in patients with neovascular age-related macular degeneration
(nv AMD). Investig. Opthalmol. Vis. Sci. 2018, 59, 2394.
116. Horita, S.; Watanabe, M.; Katagiri, M.; Nakamura, H.; Haniuda, H.; Nakazato, T.; Kagawa, Y. Species differences in ocular
pharmacokinetics and pharmacological activities of regorafenib and pazopanib eye-drops among rats, rabbits and monkeys.
Pharmacol. Res. Perspect. 2019, 7, e00545. [CrossRef]
117. Löscher, M.; Hurst, J.; Strudel, L.; Spitzer, M.S.; Schnichels, S. Nanopartikel als Drug-Delivery-Systeme für die Ophthalmologie.
Der Ophthalmol. 2017, 115, 184–189. [CrossRef]
118. Begines, B.; Ortiz, T.; Pérez-Aranda, M.; Martínez, G.; Merinero, M.; Argüelles-Arias, F.; Alcudia, A. Polymeric Nanoparticles for
Drug Delivery: Recent Developments and Future Prospects. Nanomaterials 2020, 10, 1403. [CrossRef] [PubMed]
119. Lanier, O.L.; Manfre, M.G.; Bailey, C.; Liu, Z.; Sparks, Z.; Kulkarni, S.; Chauhan, A. Review of Approaches for Increasing
Ophthalmic Bioavailability for Eye Drop Formulations. AAPS PharmSciTech 2021, 22, 107. [CrossRef]
120. Adelli, G.R.; Bhagav, P.; Taskar, P.; Hingorani, T.; Pettaway, S.; Gul, W.; ElSohly, M.A.; Repka, M.A.; Majumdar, S. Development of
a ∆9-Tetrahydrocannabinol Amino Acid-Dicarboxylate Prodrug with Improved Ocular Bioavailability. Investig. Opthalmol. Vis.
Sci. 2017, 58, 2167–2179. [CrossRef]
121. Vivès, E.; Brodin, P.; Lebleu, B. A Truncated HIV-1 Tat Protein Basic Domain Rapidly Translocates through the Plasma Membrane
and Accumulates in the Cell Nucleus. J. Biol. Chem. 1997, 272, 16010–16017. [CrossRef] [PubMed]
Pharmaceutics 2022, 14, 134 21 of 21

122. Wang, Y.; Lin, H.; Lin, S.; Qu, J.; Xiao, J.; Huang, Y.; Xiao, Y.; Fu, X.; Yang, Y.; Li, X. Cell-penetrating peptide TAT-mediated
delivery of acidic FGF to retina and protection against ischemia–reperfusion injury in rats. J. Cell. Mol. Med. 2010, 14, 1998–2005.
[CrossRef] [PubMed]
123. Ozaki, T.; Nakazawa, M.; Yamashita, T.; Ishiguro, S.-I. Delivery of Topically Applied Calpain Inhibitory Peptide to the Posterior
Segment of the Rat Eye. PLoS ONE 2015, 10, e0130986. [CrossRef]
124. De Cogan, F.; Hill, L.; Lynch, A.; Morgan-Warren, P.J.; Lechner, J.; Berwick, M.R.; Peacock, A.F.A.; Chen, M.; Scott, R.A.H.;
Xu, H.; et al. Topical Delivery of Anti-VEGF Drugs to the Ocular Posterior Segment Using Cell-Penetrating Peptides. Investig.
Opthalmol. Vis. Sci. 2017, 58, 2578–2590. [CrossRef] [PubMed]
125. Kenis, H.; van Genderen, H.; Bennaghmouch, A.; Rinia, H.A.; Frederik, P.; Narula, J.; Hofstra, L.; Reutelingsperger, C.P.M. Cell
Surface-expressed Phosphatidylserine and Annexin A5 Open a Novel Portal of Cell Entry. J. Biol. Chem. 2004, 279, 52623–52629.
[CrossRef]
126. Davis, B.; Normando, E.M.; Guo, L.; Turner, L.A.; Nizari, S.; O’Shea, P.; Moss, S.E.; Somavarapu, S.; Cordeiro, M.F. Topical Delivery
of Avastin to the Posterior Segment of the Eye In Vivo Using Annexin A5-associated Liposomes. Small 2014, 10, 1575–1584.
[CrossRef]
127. Platania, C.B.M.; Fisichella, V.; Fidilio, A.; Geraci, F.; Lazzara, F.; Leggio, G.M.; Salomone, S.; Drago, F.; Pignatello, R.;
Caraci, F.; et al. Topical Ocular Delivery of TGF-β1 to the Back of the Eye: Implications in Age-Related Neurodegenerative
Diseases. Int. J. Mol. Sci. 2017, 18, 2076. [CrossRef]
128. Balguri, S.P.; Adelli, G.; Majumdar, S. Topical ophthalmic lipid nanoparticle formulations (SLN, NLC) of indomethacin for
delivery to the posterior segment ocular tissues. Eur. J. Pharm. Biopharm. 2016, 109, 224–235. [CrossRef] [PubMed]
129. Schnichels, S.; Hurst, J.; de Vries, J.W.; Ullah, S.; Frößl, K.; Gruszka, A.; Löscher, M.; Bartz-Schmidt, K.-U.; Spitzer, M.S.; Herrmann,
A. Improved Treatment Options for Glaucoma with Brimonidine-Loaded Lipid DNA Nanoparticles. ACS Appl. Mater. Interfaces
2021, 13, 9445–9456. [CrossRef] [PubMed]
130. Schnichels, S.; Hurst, J.; de Vries, J.W.; Ullah, S.; Gruszka, A.; Kwak, M.; Löscher, M.; Dammeier, S.; Bartz-Schmidt, K.-U.; Spitzer,
M.S.; et al. Self-assembled DNA nanoparticles loaded with travoprost for glaucoma-treatment. Nanomed. Nanotechnol. Biol. Med.
2020, 29, 102260. [CrossRef] [PubMed]
131. Williams, K.A.; Brereton, H.M.; Farrall, A.; Standfield, S.D.; Taylor, S.D.; Kirk, L.A.; Coster, D.J. Topically applied antibody
fragments penetrate into the back of the rabbit eye. Eye 2004, 19, 910–913. [CrossRef] [PubMed]
132. Johannsdottir, S.; Jansook, P.; Stefansson, E.; Kristinsdottir, I.M.; Fulop, Z.; Asgrimsdottir, G.M.; Thorsteindsottir, M.; Eiriksson,
F.F.; Loftsson, T. Topical drug delivery to the posterior segment of the eye: Dexamethasone concentrations in various eye tissues
after topical administration for up to 15 days to rabbits. J. Drug Deliv. Sci. Technol. 2018, 45, 449–454. [CrossRef]
133. Xu, X.; Sun, L.; Zhou, L.; Cheng, Y.; Cao, F. Functional chitosan oligosaccharide nanomicelles for topical ocular drug delivery of
dexamethasone. Carbohydr. Polym. 2019, 227, 115356. [CrossRef]
134. Sweeney, C.; Dudhipala, N.; Thakkar, R.; Mehraj, T.; Marathe, S.; Gul, W.; ElSohly, M.A.; Murphy, B.; Majumdar, S. Effect of
surfactant concentration and sterilization process on intraocular pressure–lowering activity of ∆9-tetrahydrocannabinol-valine-
hemisuccinate (NB1111) nanoemulsions. Drug Deliv. Transl. Res. 2020, 11, 2096–2107. [CrossRef] [PubMed]
135. Urtti, A. Comment on “Topical Delivery of Avastin to the Posterior Segment of the Eye In Vivo Using Annexin A5-Associated
Liposomes”: Topical Liposomal Bevacizumab Results in Negligible Retinal Concentrations. Small 2019, 15, e1805199. [CrossRef]
136. Rösler, A.; Vandermeulen, G.W.; Klok, H.-A. Advanced drug delivery devices via self-assembly of amphiphilic block copolymers.
Adv. Drug Deliv. Rev. 2012, 64, 270–279. [CrossRef]
137. Anaya, M.; Kwak, M.; Musser, A.J.; Müllen, K.; Herrmann, A. Tunable Hydrophobicity in DNA Micelles: Design, Synthesis, and
Characterization of a New Family of DNA Amphiphiles. Chemistry 2010, 16, 12852–12859. [CrossRef]
138. De Vries, J.W.; Schnichels, S.; Hurst, J.; Strudel, L.; Gruszka, A.; Kwak, M.; Bartz-Schmidt, K.-U.; Spitzer, M.S.; Herrmann, A. DNA
nanoparticles for ophthalmic drug delivery. Biomaterials 2018, 157, 98–106. [CrossRef] [PubMed]
139. Loftsson, T.; Hreinsdóttir, D.; Stefánsson, E. Cyclodextrin microparticles for drug delivery to the posterior segment of the eye:
Aqueous dexamethasone eye drops. J. Pharm. Pharmacol. 2007, 59, 629–635. [CrossRef] [PubMed]
140. Ohira, A.; Hara, K.; Jóhannesson, G.; Tanito, M.; Ásgrímsdóttir, G.M.; Lund, S.H.; Loftsson, T.; Stefánsson, E. Topical
dexamethasoneγ-cyclodextrin nanoparticle eye drops increase visual acuity and decrease macular thickness in diabetic macular
oedema. Acta Ophthalmol. 2015, 93, 610–615. [CrossRef]
141. Loftsson, T.; Stefánsson, E. Aqueous eye drops containing drug/cyclodextrin nanoparticles deliver therapeutic drug concentra-
tions to both anterior and posterior segment. Acta Ophthalmol. 2021. [CrossRef] [PubMed]
142. Hurst, J.; Fietz, A.; Tsai, T.; Joachim, S.C.; Schnichels, S. Organ Cultures for Retinal Diseases. Front. Neurosci. 2020, 14, 583392.
[CrossRef] [PubMed]