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Nanoparticle Drug Delivery to Retinal Ganglion Cells in Glaucoma G.C. Shih1, M.G. Sternberg1, S.D. Crish1, E.M. Harth2, D.J. Calkins1
1Vanderbilt Eye Institute, 2Vanderbilt Department of Chemistry
Vanderbilt University Medical Center, Nashville, TN
.
Purpose
Methods
Patient Compliance (IOP data)
RGC Uptake
We constructed a 53nm nanosponge (Reti-Nano) with encapsulated DiO to measure retinal deposition following intravitreal injection in C57BL/6 mice. In addition, RGC DiO uptake was examined in phosphorylated heavy-chain neurofilament (SMI31)+ RGCs. To determine the efficacy of brimonidine- and travatan-loaded nanosponges in lowering IOP, C57BL/6 mice underwent microbead injection into the anterior chamber of both eyes to induce IOP elevation. Mice then received a single intravitreal injection of brimonidine- or travatan-loaded nanoparticles into one eye and PBS in the other. IOP was measured in both eyes up to 10 days post-injection.
Current marketed treatments for glaucoma include surgical alteration of eye structure and
medications administered topically, all of which aim to either improve aqueous drainage
or decrease aqueous production. However, current glaucoma treatments are problematic
for a variety of reasons, one of the largest problems being patient compliance. Also,
lowering IOP does not necessarily stop neurodegeneration, thus prompting exploration of
neuroprotective agents as treatments for glaucoma. The particular success of topical
neuroprotective agents has been variable, as drug delivery to the RGCs in the posterior
chamber of the eye is problematic.
Here we examined the feasibility of using nanoparticle-encapsulated agents administered
intravitreally in mice with elevated ocular pressure.
Ideally, the creation of an optimal retinal drug delivery system would provide more
consistent therapeutic outcomes and increase treatment success rates.
Conclusion
Reti-Nano nanoparticles reached the retina, remained there for a period of
several weeks, and showed no obvious toxic effects. The presence of
nanoparticles in the vitreal chamber, extracellular space, and intracellularly for
days to weeks enhances their long-term retinal drug delivery potential. DiO
localization to the nerve fiber layer and uptake by RGCs indicates that the
nanoparticles passed the inner limiting membrane, a major barrier to many
therapeutic compounds. Together with the reduction of IOP by nanoparticle-
encapsulated brimonidine or nanoparticle-encapsulated travatan, this data
suggests intravitreal injections of therapeutically-loaded nanoparticles may be
useful as a long-term drug delivery system in glaucoma.
Results
• The retina appeared free of pathology at all time points after NP injection.
• Retinal DiO deposition increased 3-fold after 14 days and 8-fold after 28 days
compared to baseline.
• About 40% of SMI31+ RGCs also contained DiO.
• Travatan NP treatment resulted in a reduction of elevated IOP to near baseline
levels over the course of 5 days.
• Brimonidine NP treatment also resulted in a reduction of elevated IOP near
baseline levels over the course of 10 days.
Travatan- and Brimonidine-Loaded Nanoparticle
Injection Treatments Reduce IOP to Near Baseline Levels
Sappington et al.: IOVS. 2010
3167
Evidence of DiO Uptake by Retinal Ganglion Cells
Retinal Deposition of DiO Increased Over Time
Left: Confocal
micrographs of
whole mount retina
showing
accumulations of
NPs and RGCs
stained for SMI31.
Individual RGC
uptake of DiO
molecules is
apparent (circles).
DiONP
0.1 mm
SMI31 + DiONP
0.1 mm
DiONP SMI31 DiONP + SMI31
25 µm
Top Left: Microbead injection into the anterior chamber of a rat eye
using a glass micropipette (approximately 100-μm diameter).
Top Center: Vertical section of flat-mounted anterior chamber from
an eye injected with 5 μL microbeads. Microbeads are apparent in
the iridocorneal angle and cluster near the point of aqueous outflow
(arrow).
Top Right: Photomicrograph of the anterior segment immediately
after injection with 5 μL microbeads.
Bottom Right: Photomicrographs of the anterior segment
immediately after injection with 5 μL saline.
. Multiple copies of
linear polyester
Controlled equivalents
of cross-linking unit
Intermolecular chain cross-linking
Above: Reti-Nano nanoparticle sponge structure
DESCRIPTION OF RETI-NANO
• A 53 nm NP that releases drug (or other
payload) over time as it degrades.
– Prepared from linear polyester
precursors coupled with cross-linking
units.
– By varying the amount of cross-linker
per polyester group, NPs with distinct
nanoscopic dimensions can be formed
(van der Ende et al. 2008 & 2009).
• Advantages of Reti-Nano
– NPs have amorphous properties at 37°C
that positively contribute to a linear
degradation and a controlled release of
therapeutics without a “burst effect”.
– Exhibit an increased drug load capacity
(3X greater than traditional polyester
particles).
(van der Ende et al.: J. Am. Chem. Soc. 2008)
Left: Hydrolytic degradation studies of (▲) 725.1 ± 94.3 nm AB1 nanoparticles; (◼) 115.6 ± 12.5
nm AB1 nanoparticles; and () 30.71 ± 2.21 nm AB1 nanoparticles.
Right: In vitro release profile of paclitaxel from particles loaded with 11.3% paclitaxel prepared
with the emulsification process. The cumulative release profile shows a desirable controlled and
sustained release of paclitaxel from the nanoparticles (van der Ende et al.: Polym. Chem. 2010).
0
10
20
30
40
50
60
0 5 10 15 20 25 30
Ret
inal
Su
rface
Cover
ed (
%)
Days Post-Treatment
Above: Confocal fluorescent
micrographs of whole-mount
retina immunostained for
phosphorylated heavy chain
neurofilament (SMI31) and
demonstrating DiO deposition.
Right: With an increase in
time following NP injection,
there is a linear increase in the
percentage of retinal surface
covered by DiO deposition.
Subjective Retinal Coverage vs. Time
Topical Travatan vs Intravitreal Travatan Nanoparticles
Days
0 2 4 6 8 10 12 14
Intr
ao
cu
lar
Pre
ssu
re (
mm
Hg
)
12
14
16
18
20
22
24
A B C
A: Bilateral microbead injectionB: 1st topical Travatan or intravitreal nanoparticle injectionC: 2nd topical Travatan
Topical Travatan
Intravitreal Travatan Nanoparticles
Intravitreal Lumigan Nanoparticles vs Control
Days
0 5 10 15 20 25 30
Intr
ao
cu
lar
Pre
ssu
re (
mm
Hg
)
10
12
14
16
18
20
22
24Intravitreal PBS
Intravitreal Lumigan nanoparticles
A B A: Bilateral microbead injectionB: Intravitreal injections
Intravitreal Lumigan Nanoparticles vs Control
Days
0 10 20 30 40 50
Intr
ao
cu
lar
Pre
ssu
re (
mm
Hg
)
10
12
14
16
18
20
22
24Intravitreal PBS
Intravitreal Lumigan nanoparticles
A B A: Bilateral microbead injectionB: Intravitreal injections
Cornea
Iris
Ciliary Body