Glaucoma is the second leading cause of irreversible blindness that affects more
than 60 million people worldwide. Elevated intraocular pressure (IOP) is the primary
risk factor,[1,2] and reducing IOP is the only clinical approach to prevent further
glaucomatous vision loss.[3,4] All daily therapies, however, fail to achieve sufficient
IOP
reduction, likely because they do not target the conventional outflow pathway that
controls IOP and becomes diseased in glaucoma.[2]
The conventional outflow pathway is the primary route of aqueous humor outflow from
the
eye, and increased resistance deep in this pathway, where the trabecular meshwork
(TM)
and Schlemm's canal (SC) inner wall interact, causes IOP elevation in glaucoma. Nitric
oxide (NO), a key signaling molecule responsible for mediating a wide array of
physiological roles across multiple biological systems, decreases outflow resistance
and
lowers IOP in various animal models and human patients.[5–13] Due to the therapeutic
potential of NO, many efforts have focused on developing NO-donating compounds for
glaucoma. In particular, latanoprostene bunod (VESNEO; Bausch and Lomb), a modified
version of the existing glaucoma drug latanoprost, contains an NO-donating moiety
and is
modestly effective at reducing elevated IOP by ≈1.5 mmHg over latanoprost alone
in ocular hypertensive patients.[11–13] Despite the promise of NO as a glaucoma
therapy, significant challenges remain before the full IOP-reducing potential of NO
can
be realized.
NO plays a multifaceted role within the conventional outflow pathway such that
its therapeutic effect depends strongly upon the location and concentration of NO
delivered.[14] Some effects of NO may also be
counterproductive and tend to cause IOP elevation that opposes the desired therapeutic
outcome. For instance, NO delivery to the smooth muscle cells of the ciliary muscle
(CM)
causes CM relaxation.[15] As CM tension maintains
patency of the conventional outflow pathway, NO-induced relaxation of the CM increases
outflow resistance and elevates IOP.[15–17] Acting as a
vasodilator, NO may also increase IOP by increasing downstream episcleral venous
pressure[18] or by altering choroidal blood
volume.[19] Dosing is also important, as high
levels of NO can exacerbate off-target effects.[20] Furthermore, the reactive nature
of NO and its short half-life only
allows NO to diffuse over short distances,[21,22] which severely limits the
efficacy of topical drug preparations from reaching deep into the outflow pathway
where
resistance is generated. In order to fully exploit the IOP-reducing potential of NO,
it
is therefore necessary to achieve local and controlled delivery of NO deep in the
conventional outflow pathway. To our knowledge, NO-mediated IOP-lowering therapeutics
that primarily target the conventional outflow pathway do not currently exist.
Here, we develop and test an NO delivery platform that directly targets the
conventional outflow pathway and locally liberates a controlled dose of NO via enzyme
biocatalysis (Scheme 1a). In our approach, enzymes
are embedded at the desired sites and serve as biological machinery that can locally
convert externally administered NO donors into active therapeutics. To enmesh enzymes
deep within the TM, which is the principal resistance-generating region, we encapsulate
β-galactosidase in polymer carriers. We fabricate polymer
carrier capsules via layer-by-layer adsorption of interacting polymers onto sacrificial
particle templates,[23–28] a versatile technique that allows incorporation
of an extensive choice of materials within the multilayer structures and gives fine
control over the diffusion of molecules across the shell of the polymer capsules.
In
this study, we chose hydrogen-bonded polymer pairs based on thiol-functionalized
poly(methacrylic acid) (PMASH) and poly(N-vinylpyrrolidone)
(PVP) as the system to encapsulate enzyme β-galactosidase (Scheme 1b) due to their
high colloidal stability,
(bio)degradability, and biocompatibility.[29–31] These capsules have
previously been used to encapsulate a range of functional and active biomolecules,
including enzymes,[32–34] DNA,[35,36] anticancer drugs,[37,38]
antigenic peptides,[30,39] and intact proteins.[31,40] Successful delivery of
vaccines using PMA carriers have been demonstrated in vivo.[31] We encapsulate NO
donor, β-gal-NONOate,
in liposomes because these lipid nanocarriers allow for sustained release of
biomolecules and their properties can be modulated through the judicious choice of
the
lipid composition.[41–43] Liposomes have been used to encapsulate
latanoprost[44] and plasmid DNA,[45] and injected into eyes in vivo. In our study,
liposomes are delivered to the outflow pathway. Upon degradation of the lipid vesicles,
NO donors are slowly released to the TM, where the enzyme is enmeshed, and enzymatic
activity of β-galactosidase results in local delivery of active
therapeutic NO at the outflow resistance sites, thus achieving an on-site NO delivery
to
the conventional outflow pathway.
In the current study, we: i) assemble
β-galactosidase-loaded polymer capsules and
β-gal-NONOate-loaded liposomes, ii) demonstrate local
release of NO via biocatalysis of β-gal-NONOate by
β-galactosidase and investigate the reaction kinetics in
vitro, iii) demonstrate control over the dose and time of NO release via an
enzyme-prodrug mechanism, and iv) demonstrate the effect of localized delivery of
NO to
the conventional outflow pathway on outflow resistance in mouse models. Here, we present
direct evidence that on-site delivery of NO to the outflow resistance sites provides
efficacious therapeutic alteration of aqueous humor dynamics.
We first investigated NO release kinetics achieved via catalytic activity of
β-galactosidase on β-gal-NONOate in
mock aqueous humor solution (DBG solution: Dulbecco's phosphate buffered saline with
divalent cations and 5.5 × 10−3
m glucose). We used an NO-sensitive electrode immersed in
β-galactosidase solution (0.1 mg mL−1) and
measured changes in NO concentration over time in response to the addition of
β-gal-NONOate (50 × 10−6
m). β-Galactosidase catalyzes the release of NO by
hydrolysis of the glycosidic bonds. Upon addition of NO donors into the
β-galactosidase solution, an increasing production of NO was
detected, followed by a decline back to the original baseline signal (Figure 1a).
This reaction generated NO with an
average half-life (t
1/2) of ≈5 min at physiological
conditions (pH 7.4 and 37 °C), which refers to the time to reach 50% NO decay.
With the same concentration of enzyme (0.1 mg mL−1
β-galactosidase) and an increasing amount of added substrates,
NO can be released in a dose-dependent manner (Figure
1b). The concentration of NO generated by
β-galactosidase-mediated hydrolysis of
β-gal-NONOate was derived by correlation with a calibration
curve (Figure S1, Supporting
Information). The release of NO followed a 1:2 stoichiometry and exhibited a
linear relationship. These β-galactosidase (0.1 mg
mL−1)/β-gal-NONOate (50 ×
10−6
m) pairs released NO at physiologically relevant concentrations required to
activate soluble guanylate cyclase in the TM to increase outflow facility and decrease
elevated IOP in vivo.[8,21,46,47]
Using the same setup, we next investigated the kinetics of NO release when
enzymes and NO donors were spatially separated and encapsulated in polymer capsules
and
liposomes, respectively. Here, β-galactosidase was adsorbed onto
amine-functionalized silica particles, followed by the sequential deposition of four
bilayers of PMASH and PVP. The thiol groups within the polymer layers were
cross-linked with 2,2′-dithiodipyridine, and disulfidestabilized hollow PMA
capsules containing β-galactosidase were obtained upon
dissolution of the silica templates (Scheme 1b). To
confirm the encapsulation of β-galactosidase, we conjugated the
enzyme with Alexa Fluor 488 dye. Fluorescence microscopy imaging of capsules containing
Alexa Fluor 488-labeled β-galactosidase showed a homogeneous
green corona on the capsule membrane (Figure S2, Supporting Information). The hollow
capsules were intact,
non-agglomerated, and preserved the spherical shape of the particle templates. The
NO
donor, β-gal-NONOate, was encased within zwitterionic
1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)
liposomes.
Here, an NO-sensitive electrode was immersed and equilibrated in a suspension of
capsules containing β-galactosidase in DBG solution
(106 capsules μL−1), and we measured NO release
in response to the addition of β-gal-NONOate-loaded liposomes
(50 × 10−6
m
β-gal-NONOate, 106 liposomes
μL−1) within a glass vial. This reaction was performed at
37 °C on a hot plate with continuous mixing. Upon introduction of liposomes to
the capsule suspension, a maximal NO concentration of ≈900 ×
10−9
m was generated in 30 min, followed by a gradual decay over 6 h to the baseline
signal (Figure 2a). By spatial separation and
encapsulation of enzymes and NO donors into polymer capsules and liposomes,
respectively, the half-life of NO released increased 30-fold, extending the
t
1/2 from ≈5 min to ≈2.5 h, which is
highly beneficial for treatment that requires sustained delivery of NO. NO donors
were
gradually released from lipid vesicles over time, freely permeating through the shell
of
the PMA capsules and were subsequently hydrolyzed by
β-galactosidase. This led to a staggered generation of NO
greater than the depletion of NO in solution. Successful generation of NO indicated
that
core removal did not cause loss of enzyme activity in the capsules, as previously
reported.[33,34,36]
To demonstrate the ability to control the dose of released NO, we spiked varying
concentrations of β-gal-NONOate-loaded liposomes (25 ×
10−6 to 75 × 10−6
m
β-gal-NONOate, 106 liposomes
μL−1) into the same suspension of capsules containing
β-galactosidase in DBG solution (106 capsules
μL−1). We observed a dose-dependent generation of NO (250
× 10−9 to 1250 × 10−9
m, which corresponded to the maximal NO concentration generated in 30 min) as a
function of increasing concentration of β-gal-NONOate
encapsulated inside the liposomes (Figure 2b).
These data demonstrated that through enzyme biocatalysis, the desired amount of NO
delivery can be tuned independently via the choice of concentration of administered
NO
donors.
To demonstrate a sustained delivery of NO, we repeated the enzymatic catalysis
over three cycles by removing the hydrolyzed substrates and adding fresh
β-gal-NONOate-loaded liposomes (75 ×
10−6
m
β-gal-NONOate, 106 liposomes
μL−1) into the suspension of capsules containing
β-galactosidase in DBG solution (106 capsules
μL−1). Using methodology used in this work, over 42 h, the
enzyme retained at least 50% of its activity (Figure
2c), and this approach allows NO delivery that can be tuned at the desired
units of time through the rate of liposome administration. We are now investigating
means to extend the lifetime of the enzyme and to provide for greater duration of
treatment.
To simulate the effects of localized delivery of NO in vivo and to evaluate
their impact on a physiological parameter (conventional outflow), we measured outflow
facility (C
r) in enucleated eyes from C57BL/6 male wild-type
mice. Outflow facility is the mathematical inverse of outflow resistance. This study
consists of a two-step experimental protocol: i) delivery of
β-galactosidase-loaded capsules (1.5 × 106
capsules) to conventional outflow tissues via intracameral injections in living mice,
and ii) ex vivo delivery of β-gal-NONOate-loaded liposomes
(106 liposomes) during perfusion of enucleated eyes 48 h after
intracameral injections. This approach allowed for sufficient recovery time and for
the
capsules to be enmeshed into active filtration regions of the TM prior to administration
of β-gal-NONOate-loaded liposomes. Microparticles delivered into
the TM may induce IOP elevations in otherwise healthy mice.[48,49] However, this
response typically requires larger particles (e.g., 15 μm[48]) or the addition of
10 mg mL−1 hyaluronate
solution in addition to smaller particles (1–6 μm[49]) similar in size to those used
in the current study. To control
for potential IOP elevation, sham-treated eyes were injected with otherwise identical
capsules lacking enzyme. The use of enucleated eyes allowed for accurate measurements
of
outflow facility in mice with the iPerfusion system[50] by eliminating pressure-independent
outflow, aqueous humor inflow, and
episcleral venous pressure, which are only present in living mice. Previous studies
have
shown that ex vivo eyes remain pharmacologically responsive to NO for up to several
hours[6] and respond to receptor mediated
compounds for up to 24 h.[51]
All experiments used paired eyes, where the experimental eye received capsules
containing β-galactosidase, while the contralateral control eye
received empty capsules. Both eyes were enucleated for ex vivo mouse eye perfusions,
where the perfusate consisted of liposomes containing
β-gal-NONOate (50 × 10−6
m) in DBG solution, and were pressurized at 8 mmHg to allow the eyes to
acclimatize to the pressure and temperature environment. After the acclimatization
period, eyes were perfused over seven increasing pressure steps, from 6 to 15 mmHg.
Figure 3a shows a sample flow-pressure plot
comparing contralateral control and treated eyes, which indicates an apparent increasing
effect of NO delivery on the flow rate. To determine the effect of localized NO delivery
on C
r, pressure and flow data for each step were averaged,
and a power law regression was fit to the data according to Q =
C
r(P/P
r)
β
P,
where Q is the flow rate, P is the pressure,
P
r is the reference pressure,
C
r is the reference facility at
P
r, and β characterizes the
non-linearity of the data (see the Experimental
Section for a description of outflow facility analysis). The delivery of
β-gal-NONOate-loaded liposomes in the presence of capsules
containing β-galactosidase resulted in an average increase in
C
r by 84% [23, 177%] (mean [95% CI]) relative to
vehicle-treated contralateral eyes (p = 0.011, n = 7
pairs, weighted t-test, average facility values:
6.50
x
/1.57 nL min−1
mmHg−1 vs 3.55
x
/1.38 nL
min−1 mmHg−1, Figure 3b; see the Experimental Section
for a description of statistical reporting).
In the current study, the structural integrity of the
β-galactosidase-loaded PMA capsules allowed them to be localized
to, and enmeshed within, TM likely within the down-stream juxtacanalicular portion
of
the TM where the bulk of outflow resistance is thought to be generated.[52,53]
Provided there is a supply of NO donors, NO can be continuously and locally released
and
delivered to the outflow resistance sites within the lifetime of the enzymes. A portion
of NO donors might be cleared through aqueous outflow; however, segmental flow will
tend
to cluster β-gal-NONOate-loaded liposomes and
β-galactosidase-loaded capsules into the active filtration
regions of the TM, where their close proximity will minimize the diffusion distance
necessary for the substrate to reach the enzyme. By coupling an enzyme-prodrug therapy
mechanism[54–56] with smart biomaterial encapsulation, we localized the delivery
of NO to the target tissue, and the desired dose of therapeutics can be tuned by varying
the concentration of externally administered NO donors within the liposomes—two
highly desirable features for effective NO-mediated IOP-lowering therapy.
In conclusion, we have developed a platform for delivering NO to the targeted
outflow resistance sites within the conventional outflow pathway, utilizing the strategy
of on-site NO release via enzyme biocatalysis to increase outflow facility. Our platform
will allow future manipulation of NO delivery in a dose- and time-dependent manner
without concern of off-target effects, and potentially help compensate for the impaired
NO-regulatory machinery[57–59] within the conventional outflow pathway that
contributes toward the pathogenesis of glaucoma. The strategy developed is generic
and
may open new routes to the next generation of localized therapeutic delivery.
Experimental Section
In Vivo Intracameral Injections of
β-galactosidase-Loaded Capsules
All animal experiments were conducted in compliance with the Association
for Research in Vision and Ophthalmology Statement for the Use of Animals in
Ophthalmic and Vision Research under UK Home Office Project License approval for
research at Imperial College London (PPL 70/7306). Mice were first anesthetized
with ketamine (66.6 mg kg−1, Fort Dodge Animal Health) and
Domitor (medetomidine hydrochloride, 0.66 mg kg−1, Orion
Pharma) via intraperitoneal (IP) injection. Each mouse received dilation drops
(2.5% w/v phenylephrine hydrochloride and 1% w/v tropicamide, Bausch &
Lomb) to both eyes to minimize potential damage to the iris during injection.
This was followed by a subcutaneous injection of enrofloxacin antimicrobial (5
mg kg−1, Bayer Healthcare). Eyes were kept moist with
artificial tears (Vidisic, Bausch & Lomb) prior to intracameral
injections. For intracameral injections, mice were secured in place with the
assistance of a head holder (model 923-B, Kopf Instruments) and placed on a
warm-water place mat. Eyes were first cannulated with a pulled glass microneedle
(100 μm tip) positioned parallel to the iris and above the limbus, to
remove a portion of the aqueous humor in the anterior chamber by capillary
action. Intracameral injections were then carried out with a separate pulled
glass microneedle filled with 1.5 μL of
β-galactosidase-loaded PMA capsules (106
capsules μL−1, treated eyes) or empty PMA capsules
(106 capsules μL−1, control eyes) in
UltraPure DNase/RNAse-free dH2O (Invitrogen) connected to a 10
μL glass gastight syringe (Hamilton). The needle was inserted into the
same cannulation site with the assistance of a micromanipulator, and 1.5
× 106 PMA capsules were delivered to the anterior chamber of
the eye. Topical antibiotic ointment (1% w/w Fucithalmic, LEO Pharma) was then
applied to the eyes followed by slow removal of the needle from the eye to
reduce reflux. After the procedures, the mice were given Antisedan (atipamezole
hydrochloride, 1.5 mg kg−1, Orion Pharma) via IP injections
and placed in a heated chamber (maintained at 28 °C) to allow for faster
recovery from anesthesia. The mice were allowed to recover for 48 h
post-intracameral injections, before they were euthanized by overdose of
pentobarbital (100 μL, Euthatal, Merial Animal Health) and the eyes were
enucleated for ex vivo perfusions.
Ex Vivo Delivery of β-gal-NONOate-Loaded Liposomes
via Mouse Eye Perfusions
Eyes from mice (post-intracameral delivery of PMA capsules) were
enucleated within 10 min of death by overdose of pentobarbital and stored in
phosphate buffered saline (PBS) at room temperature until perfusion, typically
within 15 min. Experiments used paired contralateral eyes, which were perfused
simultaneously on two identical iPerfusion systems[50] under identical experimental
conditions. Our
cannulation method follows previously described techniques.[5,6,60] Briefly, the eye
was
affixed to a support using cyanoacrylate glue and submerged in PBS in a
thermoregulated bath at 35 °C. A 33-gauge beveled needle (Nanofil, World
Precision Instruments) was used to cannulate the eye, with the tip of the needle
positioned in the anterior chamber using a micromanipulator. The pressure and
flow rate inside the eye were measured using a wet–wet differential
pressure transducer (PX409, Omegadyne) and a thermal flow sensor (SLG64-0075,
Sensirion), respectively. The enucleated eyes were perfused with
β-gal-NONOate-loaded liposomes in DBG (50 ×
10−6
m
β-gal-NONOate, 106 liposomes). Both treated
and control eyes were pressurized from a reservoir at 8 mmHg for 45 min to allow
the eyes to acclimatize to the pressure and temperature environment. During this
acclimatization period, the anterior chamber was filled with liposomes which
flowed toward the filtration-active regions of the TM, where the
β-galactosidase-loaded PMA capsules were enmeshed.
After the acclimatization period, eyes were perfused over seven increasing
pressure steps, from 6 to 15 mmHg with a motorized reservoir.
Outflow Facility Analysis
Conventional outflow facility was defined as previously described by
Sherwood et al.[50] Briefly, for each
eye, the last 4 min of steady state pressure and flow data for each step were
averaged, and a power law regression was fit to the data according to
Q =
C
r(P/P
r)
β
P, where Q is the flow rate,
P is the pressure, P
r is the
reference pressure, C
r is the reference facility at
P
r, and β characterizes
the non-linearity of the data. This model is used rather than the more common
linear model, as it was shown that neither the assumption of
pressure-independent facility nor of a finite flow rate at zero pressure are
valid in enucleated mouse eyes.[50] We
therefore consider the facility at a “physiological” pressure drop
across the outflow pathway in vivo, IOP-EVP = P
r = 8
mmHg, and C
r acts as an indicator of the
physiological facility. It should be noted that although
C
r represents the total outflow facility, which
comprises trabecular facility and any pressure dependent components of
unconventional outflow, the latter are relatively diminutive and hence
C
r is a good representation of the conventional
outflow facility. Additionally, based upon population studies, the distribution
of outflow facility data is more accurately represented by a lognormal
distribution; therefore, outflow facility values are expressed as
Cx
/ME95, where ME95 is
the 95% margin of error, such that the 95% confidence intervals can be
calculated as [C/ME95, C×ME95]. In order to
determine whether the observed treatment effect was statistically significant, a
weighted paired t-test was used on the log-transformed
C
r values according to Sherwood et al.[50]
Detailed methods are available in the Supporting
Information.
Supporting Information
Supporting Information is available from the Wiley Online Library or from
the author.
Supplementary