Mild temperature photothermal assisted anti-bacterial and anti-inflammatory nanosystem for synergistic treatment of post-cataract surgery endophthalmitis

Rationale: Endophthalmitis, which is one of the severest complications of cataract surgeries, can seriously threaten vision and even lead to irreversible blindness owing to its complicated microenvironment, including both local bacterial infection and severe inflammation. It is urgent to develop a comprehensive treatment for both anti-bacterial and anti-inflammatory effects. Methods: Herein, we developed AuAgCu2O-bromfenac sodium nanoparticles (AuAgCu2O-BS NPs), which was designed to combine anti-bacterial and anti-inflammatory effects for integrated therapy of endophthalmitis after cataract surgery. The AuAgCu2O-BS NPs could eradicate methicillin-resistant Staphylococcus aureus (MRSA) bacterial strain relied on their photodynamic effects and the release of metal ions (Ag+ and Cu+) by the hollow AuAgCu2O nanostructures mediated mild photothermal effects. The anti-inflammatory drug, bromfenac sodium, released from the nanoparticles were able to significantly reduce the local inflammation of the endophthalmitis and promote tissue rehabilitation. In vivo bacterial elimination and anti-inflammation were confirmed by a postcataract endophthalmitis rabbit model. Results: Excellent antibacterial ability of AuAgCu2O-BS NPs was verified both in vitro and in vivo. Ophthalmological clinical observation and pathologic histology analysis showed prominent treatment of inflammatory reaction. Importantly, the mild temperature photothermal effect not only promoted the release of metal ions and bromfenac sodium but also avoided the thermal damage of the surrounding tissues, which was more suitable for the practical application of ophthalmology due to the complex structure of the eyeball. Moreover, superior biocompatibility was approved by the preliminary toxicity investigations, including low cytotoxicity, negligible damage to major organs, and stable intraocular pressure. Conclusions: Our studies of nanosystem provide a promising synergic therapeutic strategy for postcataract endophthalmitis treatment with favorable prognosis and promise in clinical translations.


Materials.
All chemicals except bromfenac sodium were purchased from Sigma-Aldrich Corporation and used without further purification. Bromfenac sodium was obtained from Senju Pharmaceutical Co.,
The morphologies of AuAgCu 2 O NPs were measured using Tecnai 20 transmission electron microscopy (TEM, FEI Tecnai F20, USA). The absorption of AuAgCu 2 O-BS NPs were recorded using a UV−vis spectrometer (SHIMADZU UV-2600, Japan). Hydrodynamic size measurements were performed at room temperature by a dynamic light scattering system (Malvern Panalytical Zetasizer Nano ZS90, UK). XRD patterns were characterized by an x-ray diffractometer (Panalytical X'PERT PRO, Netherl1.0ands). The mass ratios of the metal components (Au, Ag, and Cu) in the nanoparticles evaluated by ICP-MS were 18.3:12.3:69.4.

Photothermal Conversion Effect.
The photothermal effect of NPs induced by the 808-laser system (Changchun Optoelectronics MDL-N-808-10W, China) in vitro or in vivo was examined. The experimental conditions were performed at various power densities (0.25, 0.50, and 0.75 W/cm 2 ) and AuAgCu 2 O-BS NPs concentrations (0, 10, 20, 40, and 80 μg/mL) with a duration of 10 min. The infrared thermal imaging camera (FLIR A350, USA) recorded corresponding temperature changes.

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In Vitro Antibacterial Activity Analysis.
Then, the upper suspension was added into a 96-well plate, and the 600 nm wavelength optical density value (OD600) was measured by a microplate reader (SpectraMax M5). After being diluted 10 6 -fold, the 100 μL suspension was cultured on LB agar plates with a spreader for 24 h at 37 °C. The amount of the colony-forming unit (CFU) was calculated based on CFUs emergence. The laser (0.75 W/cm 2 , 10 min), AuAgCu 2 O NPs group, AuAgCu 2 O NPs plus laser treatment group, AuAgCu 2 O-BS NPs group, and the blank control group were also examined in the above manner.

Morphological Characterization of Bacteria.
The bacterial suspensions of MRSA (10 9 ) that were treated with AuAgCu 2 O-BS NPs (21.6 μg/mL, 0.75 W/cm 2 for 10 min laser irradiation) were collected, centrifuged (3000 rpm, 5 min), and washed with a phosphate buffer (0.1 M, pH = 7.0). The samples were fixed by a 2.5% glutaraldehyde solution in a phosphate buffer (0.1 M, pH 7.0) overnight and washed three times with a phosphate buffer (0.1 M, pH = 7.0). Then, the samples were fixed again with 1% OsO 4 for 1 h and washed with a phosphate buffer (0.1 M, pH = 7.0) three times, followed by dehydrating with a graded series of ethanol (30%, 50%, 70%, 90%, 95%, and 100%) for 15 min. The sample was then dehydrated again in a Hitachi Model HCP-2 critical point dryer. The samples were collected and coated with goldpalladium for the SEM morphological analysis. For the TEM analysis, the process of dehydration part until a graded series of ethanol dehydrated the samples was the same as the procedure for SEM and were transferred to absolute acetone for 20 min. After being placed in a 1:1 mixture of absolute acetone and the final Spurr resin mixture for 1 h at room temperature, the samples were transferred to a 1:3 mixture of absolute acetone and the final resin mixture for 3 h and then to a final Spurr resin mixture overnight. The samples were stained by uranyl acetate and alkaline lead citrate for 10 min, respectively, and observed for the TEM morphological analysis.

Bacterial Fluorescent Assay.
To further evaluate the bacterial viability and membrane integrity, the bacterial suspension was centrifuged and washed by a 1 mL PBS solution after being treated with AuAgCu 2 O-BS NPs (21.6 μg/mL, 0.75 W/cm 2 for 10 min laser irradiation). Then, 2 μL of SYTO 9 and propidium iodide (PI) was added and incubated for 20 min at 37 °C in the dark. Fluorescent images were recorded using a fluorescence inversion microscope (Olympus IX71, Japan).

ROS Effect Assay.
The intracellular ROS level was evaluated by the oxidative conversion of cell-permeable 20,70dichlorodihydrofluorescein diacetate (DCFH-DA). The bacterial suspension was centrifuged and washed by a 1 mL PBS solution after being treated with AuAgCu 2 O-BS NPs (21.6 μg/mL, 0.75 W/cm 2 for 10 min laser irradiation). Then, the suspension was incubated in a 96-well plate with 1 mL DCFH-DA (10 μM) at 37 °C in 5% CO 2 for 30 min and washed three times with PBS. The fluorescence images were then taken using a fluorescence inversion microscope (Olympus IX71, Japan).

In Vitro Cell Migration.
HCEC and HConEpic were seeded in a 24-well plate (5.0 × 10 4 cells/well) and cultured with an FBS-free medium for 24 h. Each well was scratched with a straight line by a 200 μL pipette tip and washed with PBS to remove debris. Cells were divided into five groups as follows: untreated, Laser (0.75 W/cm 2 , 10 min), bromfenac sodium (1.0 mg/mL), AuAgCu 2 O NPs (21.6 μg/mL), AuAgCu 2 O-BS NPs (21.6 μg/mL), and AuAgCu 2 O-BS NPs (21.6 μg/mL) irradiated with an 808 nm laser (0.75 5 W/cm 2 , 10 min) and incubated at 37 °C with the medium containing 1% FBS for 24 h. Afterward, each well of the cells was photographed, and the migration rate was calculated.
Cell viability was measured as a percentage of the absorbance at 570 nm to that of the blank control group without treatment.

In vitro anti-inflammatory Test.
To evaluate the anti-inflammatory capacity of bromfenac sodium in vitro, inflammation of HCEC, HConEpic, and ARPE-19 cells were activated by LPS. Cells were seeded into 96-well plates and were allowed to adhere overnight, followed by treated with LPS + PBS, LPS + bromfenac sodium, LPS + AuAgCu 2 O NPs, LPS + AuAgCu 2 O-BS NPs and LPS + AuAgCu 2 O-BS NPs +Laser (0.75 W/cm 2 , 10 min) for 24 h. Cells without any treatment were used as control. The concentrations of IL-1β and IL-6 in the culturing medium were detected by Elisa kits (USCN Business Co., Ltd., China) according to the manufacturer's instructions and the experiments were carried out in parallel and in triplicate.

Immunohistochemical Analysis.
The slides were obtained as mentioned and incubated in 10% goat serum for 30 min to block nonspecific binding sites. After incubation with IL-1β (Abcam, ab8320, 1:100) and IL-6 (Abcam, ab9324,1:250) at 4 °C overnight, the signal-labeled antibody was added to the slides. The slides were stained by diaminobenzidine (DAB) and counterstained with hematoxylin.

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In vivo toxicity assays were conducted with New Zealand White rabbits. The blood was drawn for a routine blood examination and liver and kidney functions. Five types of organs, including the heart, lungs, liver, spleen, and kidneys, were excised for pathological analysis. Tissue samples were fixed in 4% paraformaldehyde, dehydrated, embedded in paraffin, and sectioned into 5 μm slices.
These slides were stained with hematoxylin and eosin (H&E) for the toxicology analysis.

Hemolytic Test
5 mL fresh blood was collected in anticoagulant tubes and centrifuged at 1500 rpm for 15 min to obtain red blood cells (RBCs). After washed several times with PBS, RBCs were diluted into 50 mL PBS as stock dispersion. 0.2 mL diluted stock suspension was mixed with AuAgCu 2 O NPs (7.2, 14.4, 21.6, 28.8 μg/mL) dispersions (0.8 mL in PBS) while DI water and PBS were served as the positive (+) and negative controls (-) group, respectively. After incubated at room temperature for 4 h, the solution was centrifuged at 12,000 rpm for 15 min, and then the absorbance of the supernatant at 540 nm was measured using an M5microplate reader (Molecular Devices, USA). Here, The hemolysis percent was calculated by the following way: Percentage of hemolysis (%) = (As-An)/(Ap-An )×100%, where As, An and Ap is the absorbance of nanoparticles groups, the water, and PBS control group, respectively.

ICP-MS Analysis.
The rabbits were weighed and euthanized after 6 days, 12 days and 30 days. Blood samples, urine and feces were then collected at these different times. The eye, heart, liver, spleen, lung and kidneys were excised and washed thoroughly with phosphate-buffered saline (PBS) to remove residual blood.
After the residual water on the organ surface had been removed with filter paper, each sample was weighed and placed in separate beakers and were predigested with aqua regia for Au or HNO 3 for Ag and Cu overnight, followed by digestion with HNO 3 on a hot plate for several h and then cooled and diluted. After that the diluted solution were prepared for quantitative analysis by ICP-MS (PerkinElmer NexION 300X, USA). The samples were placed in separate beakers and were predigested with aqua regia for Au or HNO 3 for Ag and Cu overnight, followed by digestion with            Single asterisks (*) indicate p < 0.001 compared to the control group.  . The average content of total metal elements eye, major organs, blood, urine and feces at different time points. All data were deducted by the background value from the blank control.