An Antibiotic Nanobomb Constructed from pH‐Responsive Chemical Bonds in Metal‐Phenolic Network Nanoparticles for Biofilm Eradication and Corneal Ulcer Healing

Abstract In the treatment of refractory corneal ulcers caused by Pseudomonas aeruginosa, antibacterial drugs delivery faces the drawbacks of low permeability and short ocular surface retention time. Hence, novel positively‐charged modular nanoparticles (NPs) are developed to load tobramycin (TOB) through a one‐step self‐assembly method based on metal‐phenolic network and Schiff base reaction using 3,4,5‐trihydroxybenzaldehyde (THBA), ε‐poly‐ʟ‐lysine (EPL), and Cu2+ as matrix components. In vitro antibacterial test demonstrates that THBA‐Cu‐TOB NPs exhibit efficient instantaneous sterilization owing to the rapid pH responsiveness to bacterial infections. Notably, only 2.6 µg mL−1 TOP is needed to eradicate P. aeruginosa biofilm in the nano‐formed THBA‐Cu‐TOB owing to the greatly enhanced penetration, which is only 1.6% the concentration of free TOB (160 µg mL−1). In animal experiments, THBA‐Cu‐TOB NPs show significant advantages in ocular surface retention, corneal permeability, rapid sterilization, and inflammation elimination. Based on molecular biology analysis, the toll‐like receptor 4 and nuclear factor kappa B signaling pathways are greatly downregulated as well as the reduction of inflammatory cytokines secretions. Such a simple and modular strategy in constructing nano‐drug delivery platform offers a new idea for toxicity reduction, physiological barrier penetration, and intelligent drug delivery.

scanning transmission electron microscope (HAADF-STEM, FEI Talos™ F200S 200 kV, USA).The chemical composition and structure of NPs were analyzed by X-ray photoelectron spectroscopy (XPS, Kratos AXIS Ultra DLD, UK) and Fourier transform-infrared spectroscopy (FT-IR, Bruker Tensor II, German).The release of Cu 2+ from NPs in the solution was measured by inductively coupled plasma mass spectrometry (ICP-MS, Agilent 7850, USA).The optical density (OD) value was measured by a microplate reader (Thermo Fisher Varioskan LUX, USA).The retention time of NPs on the ocular surface was estimated by a multimode optical in vivo imaging system (PerkinElmer IVIS Lumina XRMS Series III, USA).The fluorescence spectra and pathological section results were assessed by an upright/inverted fluorescence microscope (DM4 B/DMi8, Leica, Germany).The results of biofilm penetration and eradication of NPs were obtained by confocal laser scanning microscopy (CLSM, Zeiss LSM 880 Ariyscan, Germany).The bacteria and cells were identified automatically by flow cytometry (Beckman Coulter CytoFLEX, USA).The images of bacterial colonies were captured with a Nikon camera and colored with Photoshop.

Dissipative Particle Dynamics (DPD) Simulation:
The self-assembly process of EPL, THBA, TOB, and Cu 2+ in solution was simulated by the DPD method.In DPD, a box size of 15×15×15 nm 3 with a three-dimensional periodic boundary was constructed.
We synthesized several atoms or groups of atoms into a bead, reducing the degree of freedom in each molecule by interacting with the particles through an effective force field.The details of bond length and bond angle in the compound structure were ignored in the simulation method.The temperature was set at 298 K, the time step was 10 fs, the bead force field was martini, [S1] and the total simulation time of the self-assembly process was 20 ns.

Tissue Sections and Staining:
Immunofluorescent Staining: [S2] After the sections of corneal tissue were dewaxed, goat serum blocking solution was dropped at 37 °C for 1 h.Then, the primary antibody (anti-TNF-α, IL-6, and IL-1β antibody: 1:100) was added, and the sections were incubated in the wet box overnight at 4 °C.Subsequently, the sections were incubated for 60 min in a wet box at room temperature after dropping fluorescence-labeled secondary antibody.After being cleaned, DAPI staining was employed to seal the sections, and the staining results were observed and photographed under a fluorescence microscope (DM4 B, Leica).

RNA Isolation, cDNA Synthesis, and Real-Time Quantitative Polymerase Chain
Reaction (qPCR) Analysis: [S3] RNA from macrophages or corneal tissues was extracted by Trizol (Invitrogen, Carlsbad, CA, USA) method and its concentration was measured by NanoDrop 2000 (Thermo Scientific). 1 µg RNA was taken from each sample and cDNA was synthesized by reverse transcription via enzymatic reaction using PrimeScript™ RT reagent Kit (RR037A, TaKaRa).qPCR analysis was performed by TB Green® Premix Ex Taq™ II reagents (RR802A, TaKaRa, Japan) and conducted using a 7500 Fast Real-Time PCR System (Applied Biosystems): Holding Stage: 95 °C for 30 s. Cycling Stage: 40 cycles at 95 °C for 3 seconds, 60 °C for 30 s. Quantitative analysis was performed using the comparative (ΔΔCT) method and each assay was normalized by amplifying the housekeeping cDNA R-GAPDH.Table S1 listed the cytokines and sequences of forward and reverse primers.
Western Blotting Analysis: [S3] RAW264.7 cells or corneal tissues were cleaned with PBS.In an ice bath, the protein was extracted with RIPA lysis buffer (P0013B, Beyotime) containing 1 mM phenylmethanesulfonyl fluoride (PMSF).After being treated by low temperature and high-speed centrifugation (4 °C, 25 min, 15000 rpm), the supernatant, namely the total protein extract, was resulted and measured by Pierce™ BCA Protein Assay Kit (23225, Thermo Scientific).30 µg of protein sample was added to 10% SDS-Page gel and transferred to PVDF membrane (IPVH00010, Immobilon).
After being sealed with 5% BSA for 1 h, an appropriate diluted primary antibody was added to incubate overnight at 4 °C.Then, the secondary antibody was added and incubated at room temperature in the dark for 1 h.The specific protein bands were displayed using an ultra-sensitive ECL chemiluminescence substrate (A38555, Thermo Scientific) and imaged using Bio-Rad ChemiDoc MP, a commercial imaging system.
Image intensity was calculated using ImageJ software.β-Actin and GAPDH were used as internal reference proteins.All antibodies are listed in Table S2.

In Vitro Cytotoxicity Evaluation: 1) CCK-8 Assay:
The cytotoxicity of THBA-Cu and THBA-Cu-TOB NPs was detected by the CCK-8 method.Briefly, mouse fibroblast cells (L929 cells, 10 4 cells/well) and human corneal epithelial cells (HCECs, 10 4 cells/well) were seeded onto 96-well plates, respectively.Then, various concentrations of NPs (10, 20, 40, 80, and 160 µg/mL) at pH 7.4 and 5.5 were added and co-cultured with cells for 24 h, respectively.After that, CCK-8 solution (10%, 10 μL) was added into each well and incubated for 1 h.The optical density (OD) value of each well was measured at 450 nm using a microplate Bio-Rad reader.Cell viability (%) = [(ODNPs -ODcontrol)/ODcontrol] × 100%, data were presented as mean ± standard deviation (SD) (n = 5).2) Hemolysis Assay: A hemolysis test was used to detect the rupture of red blood cells.For positive control of the hemolysis test, we added an equal volume of distilled water into red blood cell suspension to trigger low permeability hemolysis.
Alternatively, we chose a solution that destroys the red cell membrane, such as 1% Triton-X 100, which was able to remove proteins from the red cell membrane and destroy red blood cells in a relatively short time.In the negative control experiment, erythrocytes were placed in a PBS buffer solution (pH 7.4) to maintain the isotonic environment required by erythrocytes.The cationic NPs were added into red blood cell suspension and co-incubated at 37 °C for 2 h, and then the mixture was centrifuged at 1000 rpm for 15 min to get supernatant with an absorbance value of 545 nm.Hemolysis (%) = [(ODNPs -ODPBS)/(ODTriton-X -ODPBS)] × 100%, data were presented as mean ± standard deviation (SD) (n = 5).
In Vivo Safety Evaluation: Six rats were randomly divided into two groups and treated with one drop of sterilized PBS (control) and THBA-Cu-TOB NPs eye drops in the morning, afternoon, and evening (right eye), respectively, for 7 days.Rats' blood was collected after anesthesia on day 8 to perform routine blood examination that mainly included blood cell examination, covering white blood cell (WBC), red blood cell (RBC), hemoglobin (HGB), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), and platelets (PLT), to determine occurrence of infection, anemia, hematological diseases or even platelet diseases.The biochemical examination was mainly used for checking liver and kidney blood biochemical indicators, including aspartate aminotransferase (AST), alanine aminotransferase (ALT), urea (UREA), and creatinine (CREA), for the detection of the viscera diseases and abnormalities.Finally, all rats were anesthetized and sacrificed for toxicological examination of the main organs, including the cornea, heart, liver, spleen, lung, and kidney.The biosafety of the THBA-Cu-TOB NPs in vivo was comprehensively evaluated by the above indicators and detection analysis.The clinical scoring standard refers to Table S4.

Figure S1 .
Figure S1.A) TEM and B) corresponding element (C, N, O, and Cu) mapping images for the THBA-Cu NPs.Inside A: schematic representation of the different morphology of NPs (upper-right corner) and photographs of NPs solution with the Tyndall effect (bottom-right-corner).Inside B, line-scan STEM elemental distribution (yellow arrow) of the THBA-Cu NPs.

Figure S2 .
Figure S2.A) TEM and B) SEM images of the THBA-Cu-TOB NPs.Inset: the size of the THBA-Cu-TOB NPs calculated by Image J software.

Figure S5 .
Figure S5.Determination of TOB in NPs by fading spectrophotometric method with aniline blue aqueous solution.A) The UV-vis spectra of TOB at various concentrations.B) The standard curve of UV-vis absorbance at 608 nm to TOB concentration.C) The UV-vis absorbance curve of THBA-Cu and THBA-Cu-TOB.

Figure S9 .
Figure S9.Checkerboard dilution results performed on P. aeruginosa to evaluate the synergy between the TOB and A) THBA, B) Cu 2+ , and C) EPL, respectively.

Figure S10 .
Figure S10.Changes of pH as P. aeruginosa growth in TSB solution.

Figure S15 .
Figure S15.CLSM images of the P. aeruginosa biofilm incubated with free TOB (Green: live bacteria; Red: dead bacteria).

Table S3 . MIC and MBC results of the different materials.
MIC: Minimum Inhibitory Concentration; MBC: Minimum Bactericidal Concentration.