this download - University of Utah

2010 ARVO
Nanoparticles Delivering Anti-VEGF-
A Plasmid Regress Murine Corneal
Neovascularization
B.C. Stagg 1 , Y. Qazi 2 , S. Singh 3 , N. Singh 2 ,
E. Pearson 1 , U. Kompella 3 , B.K. Ambati 2 .
1 University of Utah School of Medicine,
Salt Lake City, UT; 2 Moran Eye Center,
Salt Lake City, UT; 3 Department of
Pharmaceutical Sciences, University of
Colorado - Denver, Denver, CO.
Purpose. To determine the efficacy of
pSEC.siRNA.VEGFA loaded Poly Lactic
Co-Glycolic Acid (PLGA) nanoparticles
(NPs) in the regression of murine corneal
neovascularization (KNV).
Methods. Plasmid-loaded nile red PLGA
nanoparticles were prepared using the double
emulsion solvent evaporation method.
KNV was induced in BALB/C mice by mechanical-alkali
injury using 2 ul of 0.15M
NaOH for 10 seconds followed by scraping
of the corneal epithelium with a Tooke
corneal knife. Vessels were allowed to
mature over 4 weeks after which the mice
were randomly divided into 4 groups, each
of which received one of the following
interventions: pSEC.siRNA.VEGFA NR
PLGA NPs (2ug plasmid), naked pSEC.
siRNA.VEGFA plasmid (2ug plasmid),
blank NR PLGA NPs, and DMSO. The
plasmid-loaded NPs were prepared in sterile
DMSO to a concentration of 1 ug/ul and
2 ul were injected intracorneally using a 33
gauge needle. 4 weeks after intervention,
the mice were sacrificed and the corneas
were harvested for flatmounts, RT-PCR,
and VEGF-A ELISA. Flatmounted corneas
were immunostained for CD31 (endothelial
cell marker) and the neovascular area
was quantitated using Scion Image. VEGF-
A gene expression was evaluated using RT-
PCR. Protein levels were determined using
VEGF-A ELISA.
Results. siRNA.VEGFA loaded PLGA
NPs showed significant regression of KNV
compared to naked plasmid and controls.
siRNA.VEGFA loaded PLGA NPs regressed
KNV to 12.5%. Naked plasmid
treatment resulted in a KNV area of 28%.
The two control groups had highly vascular
corneas with 53% KNV for DMSO and
55% KNV for blank NPs. VEGF-A protein
and RNA expression were reduced significantly
in siRNA.VEGFA loaded PLGA
NP-treated corneas.
4
Conclusion. pSEC.siRNA.VEGFA -loaded
PLGA NPs are an effective, non-viral,
non-toxic and sustainable form of gene
therapy for the regression of murine KNV.
Program#/Poster#: 440/D1144
Noncnzo10/ltj Mouse, a Model of Type
2 Diabetes, May Not Be Suitable for
Diabetic Retinopathy
H. Uehara, J. Cahoon, S. Oblad, J. Simonis,
L. Luo, B.K. Ambati.
Ophthalmology, University of Utah, Salt
Lake City, UT.
Purpose. Diabetic retinopathy is one complication
of diabetes mellitus. Many rodent
models of diabetes have been used to understand
the mechanism of, and improve,
diabetic retinopathy. In this study, we
characterized retina of NONcNZO10/Ltj
males, which was recently generated as a
mouse model of Type 2 diabetes mellitus.
Methods. NONcNZO10/Ltj males were
obtained from Jackson laboratory and fed
with a high fat diet. To observe retina vascularity
in vivo, fluorescein angiography
was used. Retina flatmounts were stained
with isolectin GS-IB4 for vessel endothelial
cells and α-SMA for pericytes. They
were then observed with confocal microscopy.
To determine retina thickness and
examine retina layer composition, we used
optical coherence tomography (OCT) and
cryosection. In addition, we examined expression
of PDE6α, PDE6β, and PDE6γ in
the retina by RT-PCR and checked rd1 mutation
by genotyping.
Results. Fluorescein angiography and retina
flat mount analysis showed that NONcNZO10/Ltj
retina has less vasculature
compared with control mouse, but pericytes
still exist with retina blood vessels. In
addition, high backgrounds were observed
in NONcNZO10/Ltj mouse by fluorescein
angiography. Retina thickness of NONcNZO10/Ltj
was dramatically thinner than
control mouse. OCT showed the thickness
of NONcNZO10/Ltj and control retina as
137±13μm and 281±25 μm respectively.
Cryosection of NONcNZO10/Ltj eye also
showed thin retina (NONcNZO10/Ltj retina:
93±12μm; Control retina: 203±18μm).
Nuclear observation indicated that the
outer nuclear layer of NONcNZO10/Ltj
was atrophic. From RT-PCR, PDE6α and
PDE6β did not express in NONcNZO10/
Ltj retina, but PDE6γ was still expressed.
Genotyping for rd1 indicated NONcN-
ZO10/Ltj is a rd1/rd1 mouse.
Conclusions. From these results, we confirmed
NONcNZO10/Ltj mouse retina
degeneration comes from rd1 mutation.
Although fluorescein angiography indicated
NONcNZO10/Ltj retina has leakage,
which is a manifestation of diabetic
retinopathy, NONcNZO10/Ltj mouse
may not be suitable for diabetic retinopathy
studies because the outer nuclear layer
progressively atrophies.
Program#/Poster#: 118/A246
Ocular Bioimaging of a Murine Model
of Macular Degeneration
C.A. Mamalis 1 , L. Luo 2 , S.A. Molokhia 1 ,
K. Jackman 1 , M. Romanowski 3 , B.K.
Ambati 4 . 1 Ophthalmology, Moran Eye
Center, Salt Lake City, UT; 2 Ophthalmology,
University of Utah, Salt Lake City,
UT; 3 Biomedical Engineering, University
of Arizona, Tucson, AZ; 4 Ophthalmology,
John Moran Eye Center, Salt Lake
City, UT.
Purpose. To determine if anti-Vascular
Endothelial Growth Factor antibody
fragments (fabs) conjugated with Indocyanine
Green (ICG) and gold nanorods
reveal the presence of subretinal injection
-induced choroidal neovascularization
(CNV) in a murine model.
Methods. Using the Thermo Scientific
Fab Preparation kit (#44885), anti- mouse
VEGF IgG underwent papain digestion
into Fab fragments which were isolated
from the remaining IgG and Fc particles.
Purified Fab fragments were lyophilized
and conjugated to ICG and aminated gold
nanorods in separate aliquots. The prepared
bioconjugates were combined and
injected systemically into the tail vein of
Balb/C mice which had previously been
injected with AAV.siRNA.sFlt to induce
CNV. The posterior segment was evaluated
using Ocular Coherence Tomography
(OCT) and ICG imaging capabilities
of the Heidelberg Spectralis. Images were
obtained immediately, 2 hours, 4 hours,
24 hours, and 2 weeks post injection, for
evidence of contrast indicating focally elevated
concentrations of VEGF.
Results. The bioconjugation of ICG and