Download Supplemental Data and Figures

Nanoparticles for diabetic retinopathy
SUPPLEMENTAL RESEARCH DESIGN AND METHODS
Measurement of leukostasis: Leukocytes adhesion to retinal vasculature was detected as
described previously, with slightly modifications (41). Briefly, the blood in the anesthesia rats
were first perfused out with phosphate-buffered saline (PBS) via a cannula introduced into the
aorta. Blood vessels were then fixed, following blocking and staining with fluorescent
isothiocyanate (FITC)–labeled Con A lectin (25 µg/mL in PBS, pH 7.4; Vector Laboratories,
Burlingame, CA). Unbound FITC-Con A was removed by thorough washes. The retinas were
then dissected, flat-mounted and visualized under fluorescence microscope. The total number of
adherent leukocytes per retina was determined, averaged and compared with Student’s t test.
Measurement of the retinal thickness: Retinal thickness was measured on 5 µm hematoxylin
and eosin (H&E)–stained sections following a documented protocol (42). Briefly, retinal sections
from non-diabetic or diabetic rats 4 weeks after the injection of Control-NP and K5-NP were
stained with H&E. Digitized images of the entire retina were captured using a CKK41 digital
imaging system (Olympus, U.S.A.) at 20× magnification with 1300 × 1300 pixels. To cover the
entire retina, six images were captured from each section. The retinal thickness was measured at
25% (S1), 50% (S2), and 75% (S3) of the distance between the superior pole and the optic nerve
and 25% (I1), 50% (I2), and 75% (I3) of the distance between the inferior pole and the optic
nerve (40), using SPOP advanced image software (Sterling Heights, MI). The mean retina
thickness was also calculated.
TUNEL assay: TUNEL was performed to examine apoptotic cells in the retina using APO-BrdU
TUNEL Assay Kit with Alexa Fluor 488 anti-BrdU from Molecular Probes (Carlsbad, CA,
Invitrogen). Briefly, 5-µm frozen ocular sections were blocked in equilibration buffer, and
labeled for 2 h at 37˚C with 50 µl of TUNEL reaction mixture containing the fluorescent
nucleotide deoxy-UTP and terminal deoxynucleotidyl transferase. The sections were
counterstained with DAPI, and visualized under fluorescent microscope.
Nanoparticles for diabetic retinopathy
SUPPLEMENTAL FIGURE LEGENDS
Supplemental Figure 1. Effect of K5-NP on leukostasis in the retinal vasculature. Diabetic
rats at 2 weeks after the onset of diabetes received an intravitreal injection of 5 μg/eye of K5-NP
in the treatment group and the same dose of Control-NP in the control group. Four weeks after
the injection, the animals were perfused to remove circulating leukocytes. The retinal vasculature
and adherent leukocytes were stained by FITC-conjugated concanavalin A. The retinas were then
dissected and flat-mounted. (A-D) Fluorescence photographs of retinal vasculature and adherent
leukocytes in non-diabetic rats (A), STZ-induced diabetic rats (STZ) (B), STZ-induced diabetic
rats injected with Control-NP (C), and K5-NP (D). (E) Leukostasis was quantified by counting
the adherent leukocytes in the retina vasculature (mean±SD, n=6). The result showed
significantly increased leukostasis in diabetic rats compared to non-diabetic control, and K5-NP
decreased leukostasis in diabetic rat retina compared to that injected with Control-NP. Scale bar:
20 µm.
2
Nanoparticles for diabetic retinopathy
Supplemental Figure 2. No significant change in retinal thickness after the K5-NP
injection. Diabetic rats at 2 weeks after the onset of diabetes received an intravitreal injection of
5 μg/eye of K5-NP in the treatment group and the same dose of Control-NP in the control group.
Four weeks after the injection, the animals were euthanized, and the eye sections were stained
with H&E. The retinal thickness was measured by image analysis at six locations, at 25% (S1),
50% (S2), and 75% (S3) of the distance between the superior pole and the optic nerve and 25%
(I1), 50% (I2), and 75% (I3) of the distance between the inferior pole and the optic nerve
following a documented procedure (40). (B) Retinal thickness at the six locations was averaged
in each group (mean±SD, n=6). There is no significant difference in retinal thickness (P>0.05).
3
Nanoparticles for diabetic retinopathy
Supplemental Figure 3. TUNEL analysis in the retina. (A,B) For TUNEL positive control,
retinal sections from OIR rats at age of P18 were stained for apoptotic cells using the TUNEL
method, and the nuclei counterstained with DAPI (blue). A number of TUNEL positive nuclei
(green) were observed in a representative OIR retinal section, primarily in the inner retina as
indicated by arrows. (C-F) Normal adult rats received an intravitreal injection of PBS or 5 μg of
K5-NP. Retinal sections were stained with the TUNEL assay at the same conditions as for the
OIR rats. No TUNEL positive cells were detected in the retina injected with PBS or K5-NP. (A,
C, E), TUNEL staining; (B, D, F) merged image of DAPI and TUNEL staining. Scale bar: 20
µm.
4
Nanoparticles for diabetic retinopathy
Supplemental Figure 4. Double immunostaining of K5-His positive cells and retinal Müller
cells in the rat retina. To reveal if the recombinant K5 is expressed in the Müller cells after K5NP injection, sagittal sections of the retina were immunostained with the anti-His-tag (red) and
anti-GFAP (green) antibodies, 7 days after the injection of K5-NP. The nuclei were
counterstained with DAPI (blue). (A) K5-His signal; (B) GFAP staining; (C) merged image of
the K5-His, GFAP and DAPI staining. Arrows indicate co-localization of K5-His with GFAP in
retinal Müller cell endfeet. Scale bar: 10 µm.
5