H. Han, et al.
JournalofControlledRelease327(2020)676–687
(30 μM, calculated as Ce6). The samples were irradiated using 660 nm
light with a power of 500 mW/cm2 for 4 min. Finally, the biofilms were
stained with Live/Dead dye for 20 min and visualized with CLSM.
light irradiation (500 mW/cm2 power) with different light irradiation
times and further incubated for 24 h. Next, 20 μL of MTT (5 mg/mL)
was added to each well, and the cells were further cultured at 37 °C for
4 h. Finally, the medium was replaced with 150 μL DMSO to dissolve
the obtained crystals, and the OD values at 490 nm were measured
using a microplate reader.
2.14. P. aeruginosa-infected keratitis induction and topical ocular eye drop
administration
2.10. In vitro bactericidal activity of Ce6 and supramolecular nanoparticles
All animal procedures were performed in accordance with the
ARVO (Association for Research in Vision and Ophthalmology)
Statement for the Use of Animals in Ophthalmic and Vision Research
and the Guidelines for Principles of Laboratory Animal Care (National
Institutes of Health publication NO. 86–23, revised 1985). Twenty-five
healthy C57BL/6 mice (5–6 weeks old and weighing approximately
18 g) were provided by the animal center of Zhejiang Academy of
Medical Sciences. Bacterial inoculation was performed on the right
corneas of the mice. The mice were topically anesthetized with 0.5%
proparacaine hydrochloride (Alcaine; Alcon Laboratories, Inc.) fol-
lowed by initiation of three 1 mm abrasions on the cornea using a 25-
gauge needle. The mechanically damaged mice corneas were inoculated
with 10 μL of P. aeruginosa suspension (about 105 CFU/mL). After 12 h
of inoculation, mice with infected eyes were randomly divided into five
groups (CTRL, MMP-IS NPs + light, MMP-S NPs, MMP-S NPs + light,
and LVFX) with five mice each. Specifically, three groups were instilled
with 20 μL of MMP-IS NPs or MMP-S NPs (100 μM Ce6 equivalent)
every 5 min for 6 cycles and then washed with 10 mL of normal saline.
After 3 h of incubation, the corneas of the MMP-IS NPs + light and
MMP-S NPs + light groups were irradiated with 660 nm light and an
irradiation intensity of 250 mW/cm2 for 5 min. The CTRL group was
given normal saline only. The LVFX group was treated with commer-
cially available levofloxacin eye drop (Tarivid®, Santen
Pharmaceutical). All treatments were administered immediately from
12 h post-infection (H12). The MMP-IS NPs + light, MMP-S NPs, MMP-
S NPs + light groups were administrated once and the LVFX group was
continued for one consecutive week (four times daily).
The standard plate counting method was utilized for evaluating the
bactericidal activity of Ce6 and nanoparticles. First, 100 μL of P. aeru-
ginosa or S. aureus suspension (about 108 CFU/mL) was mixed with Ce6
or nanoparticles (10 μM Ce6 equivalent) in a 96-well plate. After
shaking and incubating in a 37 °C shaker for 4 h, the samples were
irradiated with a wavelength of 660 nm light and power of 500 mW/
cm2 for 0, 2, 4, and 8 min, respectively. Then, each sample was ap-
propriately diluted and incubated with LB solid medium. Bacterial co-
lonies were counted after 14 h of incubation at 37 °C. At the same time,
the bacterial colonies of P. aeruginosa treated with nanoparticles in the
dark were counted according to the aforementioned steps.
The morphologies of the bacteria treated with nanoparticles were
investigated under a SEM. The bacteria suspension was incubated with
MMP-S NPs solution (10 μM, calculated as Ce6). After shaking and
incubating in a 37 °C shaker for 4 h, the samples were irradiated with a
wavelength of 660 nm light and power of 500 mW/cm2 for 0, 2, 4, and
8 min, respectively. Then, the suspension was dropped on a clear glass
slide, fixed with glutaric dialdehyde for 2 h and dehydrated using
graded ethanol (10%, 20%, 40%, 60%, 80%, and 100% for 10 min
each). Gold was sputtered onto the samples before SEM tests.
2.11. The penetration and ROS generation behavior in biofilms
P. aeruginosa biofilms were cultured in glass dishes with LB broth at
37 °C. Two days later, the culture medium was removed, and the bio-
films were gently washed with PBS before testing. The MMP-S NPs and
MMP-IS NPs (30 μM, calculated as Ce6) were then added into the
dishes. After 4 h of incubation, the biofilms were washed with PBS three
times and further stained with DAPI. The fluorescence of the biofilms
and the nanoparticles were visualized with confocal laser scanning
microscopy (CLSM).
2.15. Therapeutic efficacy evaluation
In order to investigate therapeutic efficacy, ophthalmic evaluations
were performed by examining the infected eyes of mice following
various treatments using slit-lamp biomicroscopy at 12 h, 1 day, 3 days,
and 7 days after surgery. The clinical features of P. aeruginosa keratitis
were scored and graded according to the previously reported literatures
with a little modification [14,38]. A grade of 0 to 4 was assigned to
each feature based on these three criteria: area of corneal opacity,
density of opacity, and surface regularity. A normal untreated cornea
was given a score of 0 in each category and thus had a total score of 0.
The score for each eye could yield a possible total score ranging from 0
to 12. On behalf of determining the amount of viable bacteria in the
infected cornea tissues, the excised corneas were homogenized in
0.5 mL of sterile PBS. Aliquots of 0.1 mL of 10-fold diluted samples
were placed onto mannitol salt agar (MSA) plates. After incubation at
37 °C for 24 h, the colonies were counted and analyzed.
The ROS generation in P. aeruginosa biofilms was investigated using
ROS indicator, DCFH-DA. Briefly, the P. aeruginosa biofilms were first
cultured in 96-well plates. Then, PBS, MMP-S NPs (30 μM Ce6
equivalent), and MMP-IS NPs (30 μM Ce6 equivalent) was added into
the biofilm, respectively. After 4 h of incubation, the biofilms were
washed with PBS three times and further stained with DCFH-DA. The
biofilms were irradiated by a 660 nm light (500 mW/cm2 power) for
4 min and the fluorescence of DCF was visualized with fluorescent
microscopy.
2.12. Antibacterial effects against biofilms
P. aeruginosa biofilms were cultured in 96-well plates for 2 days.
Then, the culture medium was removed, and the biofilms were gently
washed with PBS. The MMP-S NPs and MMP-IS NPs (30 μM, calculated
as Ce6) were then added into each well. Four hours later, the samples
were irradiated with a wavelength of 660 nm light and power of
500 mW/cm2 for 0, 2, 4, and 8 min, respectively. PBS incubated bio-
films were used as controls (CTRL). The biofilms were then dispersed by
sonication for 10 min. Each sample was appropriately diluted and in-
cubated with LB solid medium for plate counting.
2.16. Histological analysis
At the end of the experiment, mice with different treatments were
sacrificed, and their tissues were harvested. The tissues were then fixed
in 4% paraformaldehyde in PBS, dehydrated in a graded series of
ethanol solutions, embedded in paraffin, and cut into 5 μm-thick sec-
tions.
In order to investigate the histological changes and inflammatory
response induced by the topical application of supramolecular nano-
particles or antibiotics, the mice corneal tissues were stained with he-
matoxylin and eosin (H&E), interleukin 1 beta (IL-1β), and tumor ne-
crosis factor alpha (TNF-α) and examined visually under an optical
microscope.
2.13. Live/dead staining of biofilms
P. aeruginosa biofilms were cultured in glass dishes for 2 days. After
washed with PBS, the biofilms were incubated with the nanoparticles
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Han, Haijie
