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Potentiating hypoxic microenvironment for antibiotic activation by photodynamic therapy to combat bacterial biofilm infections



Metronidazole (Pharmaceutical Secondary Standard), vancomycin (Van, >90%), 1-(3-dimethylaminopropyl)−3-ethylcarbodiimide hydrochloride (EDC, >99%), N-hydroxysulfosuccinimide sodium salt (NHS, >98%), hyaluronidase (Hyal, 400 units/mg), fibrinogen, thrombin (~120 units/mg), dimethyl sulfoxide (DMSO, >99.9%), dimethylformamide (DMF, >99.9%), 9, 10-anthracenediyl-bis(methylene)-dimalonic acid (ABDA, >90%), and ethylenediamine (>99.5%) were obtained from Sigma-Aldrich; sodium hyaluronate (200 kDa) was purchased from SunlidaBio; and Ce6 (>99%) was bought from JenKem Technology.

Preparation of HA-Ce6-MNZ nanoparticles (HCM NPs)

Firstly, the sodium hyaluronate was dissolved in PBS (10 mM, pH 7.4) to form homogeneous HA solution (50 mg/mL). EDC (16.7 mg) and NHS (12.1 mg) were dissolved in PBS (10 mM, pH 7.4, 6 mL), which was mixed with 4 mL HA solution and stirred for 30 min. Then, ethylenediamine (0.2 g, dispersed in PBS) was mixed with the above solution for 12 h at room temperature (RT), dialyzed against PBS for 2 d, and further freeze-dried to obtain A-HA. EDC (1.6 mg), NHS (1.3 mg), and Ce6 (20 mg) were dissolved in DMSO solution (10 mL) and stirred for 30 min, followed by adding 10 mL A-HA solution (DMF: H2O = 1:1, 4 mg/mL) and reacting for 12 h at RT. The reaction mixture was diluted with 40 mL PBS, ultrasonicated for 15 min, and dialyzed against PBS for 2 d to form HC NPs. Subsequently, 10 mg freeze-dried HC NPs were dissolved in 10 mL DMSO-H2O solution (1:1) and mixed with MNZ (100 mg) for 12 h at RT, then dialyzed against PBS for 2 d. In this process, the DMSO was gradually removed from the solution and the solubility of Ce6 in HC NPs and MNZ decreased in the meantime, which induced their aggregation and the formation of HCM NPs. The hydrodynamic size and zeta potential of HCM NPs were measured on a ZetaPALS Potential Analyzer (Brookhaven Instruments, USA) in PBS at RT. The amount of Ce6 and MNZ were determined by UV-vis-NIR spectroscopy on a UV-3600 spectrophotometer (Shimadzu, Japan). The grafting ratio of Ce6 in HA-Ce6 is about 6.93%. The encapsulation efficiency of Ce6 and MNZ is about 24.64% and 0.35%, respectively.

Preparation of HCM gel

To form the HCM gel, PBS buffers containing HCM NPs (Ce6: 200 μg/mL, MNZ: 100 μg/mL) and fibrinogen (10 mg/mL) were mixed as solution A. Thrombin solution (10 NIH U/mL) in CaCl2 (0.1 M) was used as solution B. After spraying solution A and solution B at an equal volume by using a dual-cartridge sprayer, the HCM gel was formed in several minutes.

Hyal-responsive drug release of HCM NPs

HCM NPs (Ce6: 40 μg/mL; MNZ: 20 μg/mL) were dispersed in PBS with different pH values (7.4 and 5.5) with or without Hyal (200 unit/mL) and gently shaken at 37 °C. The Ce6 and MNZ released from HCM NPs were separated from the mixtures by ultrafiltration. The released Ce6 and MNZ were quantified by using UV-vis-NIR absorption spectroscopy.

Detection of singlet oxygen

The ABDA was used to detect 1O2. Briefly, different agents and ABDA (10 μg/mL) were dissolved in PBS and exposed with 635 nm laser (20 mW/cm2) for different times. The 1O2 can react with ABDA, which can be monitored via the decrease in absorbance at 380 nm.

Cell culture

Human normal liver (L-O2) cells and mouse smooth muscle cells (SMCs) were purchased from KeyGen BioTech. These cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS, Gibco) at 37 °C under 5% CO2.

Bacterial culture

Methicillin-resistant Staphylococcus aureus (MRSA, ATCC43300) were grown in chemically defined medium57, and then incubated in Luria-Bertani (LB) medium (containing 1% glucose, 107 CFU/mL) at 37 °C for 2 d to form MRSA biofilms.

Cytotoxicity of HCM NPs

L-O2 cells were grown in 96-well plates with 104 cells per well for 1 d. Then, L-O2 cells were incubated with HCM NPs dispersions with different concentrations for 1 d. Cell viability was determined by using LDH Cytotoxicity Colorimetric Assay Kit (BioVision).

Evaluation of the nitroreductase activity of MRSA

The activity of nitroreductase from MRSA in biofilms was analyzed according to the previous work36. Briefly, MRSA biofilms were incubated under hypoxic (5% O2) or normoxic conditions for 48 h. The MRSA were collected, washed three times with Tris-HCl buffer (20 mM, pH 7.6) at 4 °C, incubated with lysostaphin (50 μg/mL) and deoxyribonuclease I (DNase I, 50 μg/mL) for 30 min at 37 °C, respectively, and centrifuged (13000 × g) for 0.5 h at 4 °C. The nitroreductase activity in cell extracts was determined by using nitrofurantoin (50 μM) and nitrofurazone (50 μM) as substrates, and nicotinamide adenine dinucleotide phosphate (NADPH, 250 μM) or nicotinamide adenine dinucleotide (NADH, 250 μM) as electron donors. The absorbance at 400 nm for nitrofurazone and absorbance at 420 nm for nitrofurantoin were measured by using a microplate reader (PowerWave XS2, BioTek) to evaluate the nitroreductase activity.

In vitro anti-biofilm effect

Preformed MRSA biofilms were incubated with the saline solutions of MNZ (25 μg/mL), Van (50 μg/mL), HC (Ce6: 50 μg/mL), and HCM (Ce6: 50 μg/mL, MNZ: 25 μg/mL) for 6 h, respectively. The Calcein-AM was used to stain the viable bacteria inside MRSA biofilms after various treatments. To observe the structure of biofilm, MRSA biofilms were fixed with formalin for 10 min and washed with saline for three times. After natural drying, the fixed MRSA biofilms were stained with crystal violet solution (0.02%) for 0.5 h, and imaged by a fluorescence microscope (Olympus IX71). To calculate the colony numbers of MRSA in biofilms, the samples were sonicated in saline for 5 min and tested by plate counting method. To study the long-term inhibition by HCM NPs, MRSA biofilms were treated by Van (50 μg/mL), HC NPs (Ce6: 50 μg/mL), and HCM NPs (Ce6: 50 μg/mL, MNZ: 25 μg/mL) dispersed in chemically defined medium with or without laser irradiation, and further incubated for 72 h to evaluate the anti-biofilm efficacy by using the crystal violet staining method.

SEM imaging of MRSA biofilms

Following various treatments, MRSA biofilms were fixed by 2.5% glutaraldehyde solution for 30 min. Then, the samples were dehydrated by gradient ethanol solutions (15%, 30%, 50%, 75%, and 100%) for 15 min, respectively. The samples were sputter-coated with gold and imaged by using a scanning electron microscope (Hitachi S4800).

Assessment of the hypoxia level of MRSA biofilms

MRSA biofilms were grown on Petri dishes and incubated with HCM NPs in chemically defined medium (Ce6: 50 μg/mL, MNZ: 25 μg/mL) for 6 h. After 635 nm laser irradiation (20 mW/cm2, 30 min), MRSA biofilms were stained with [Ru(dpp)3]Cl2 (10 μg/mL) for 6 h. Fluorescence images of MRSA biofilms were obtained using a confocal laser scanning microscope (Olympus IX81).

In vivo fluorescence imaging of biofilm infections

Female Balb/c mice (20 g, 6–8 weeks old) were purchased from Nanjing Junke Biological Engineering Co., Ltd. All animal procedures were performed in accordance with the Guidelines for the Care and Use of Laboratory Animals of Nanjing Tech University and approved by the Animal Ethics Committee of Nanjing Tech University. All mice were raised in 25 ± 3 °C (temperature), 60–70% (humidity), and 12 h light/dark cycle conditions. LB medium dispersions of MRSA (109 CFU/mL, with 1% glucose, 50 μL) were subcutaneously injected into the right thigh of mice and infected for 48 h to form subcutaneous biofilm infections. Fluorescence images of mice were captured on an IVIS Lumina K Series III system (Perkin Elmer) post i.v. injection of HCM NPs (Ce6 = 4 mg/kg; MNZ = 2 mg/kg) at given time points.

Treatment of subcutaneous biofilm infected mice by HCM NPs

MRSA biofilm infected mice were i.v. injected with HCM NPs (Ce6 = 4 mg/kg; MNZ = 2 mg/kg) and irradiated with 635 nm laser (20 mW/cm2, 30 min) at 8th h post-injection. The area of infected tissue was calculated as follows: area = (width/2 × length/2) × π. The infected tissues from mice were captured following an 8 d treatment period, and the bacteria were separated by ultrasonication for 15 min. The number of bacteria in the infected tissues was measured by the plate counting method. For histological analysis, all samples were fixed with 10% formalin, embedded in paraffin, and sliced for H&E and Masson’s trichrome staining.

Treatment of biofilm infected wounds in diabetic mice by HCM gel

Male C57BL/6 mice (20 g, 6–8 weeks old) were intraperitoneally (i.p.) injected with streptozotocin (STZ) (60 mg/kg) every 3 days for 2 weeks. Blood glucose levels >16.7 mM indicated the establishment of diabetic mice58. A circular cut (8 mm in diameter) was excised from the back of diabetic mice, incubated with MRSA biofilm dispersions (107 CFU), and dressed with semiocclusive transparent films (Tegaderm, 3 M) for 24 h to form MRSA biofilm infected wounds. Then, the mixtures of different materials with fibrinogen and thrombin were sprayed on the wounds (Ce6 = 1 mg/kg; MNZ = 0.5 mg/kg) to form HCM gel. The wounds were irradiated with 635 nm laser (20 mW/cm2, 30 min) at 12 h post-incubation. The infected tissues were harvested from mice at 12 d post-treatment and processed for histological analysis.

Immunofluorescence imaging

After various treatments for 4 d, the biofilm-infected tissues were harvested from mice, fixed in 10% formalin, and then embedded in paraffin. After slicing, the tissue sections were incubated with 3% methanol for 10 min and 1% BSA for 20 min. The sections were then incubated with the primary antibodies, including anti-HIF-1α (Abcam, ab16066), anti-VEGF (Abcam, ab52917), anti-F4/80 (Abcam, ab6640), anti-CD80 (Abcam, ab254579), and anti-CD 206 (Abcam, ab64693), at room temperature for 2 h. Following incubation, the sections were incubated with fluorescently labeled secondary antibodies, including Alexa Fluor594-IgG (Abcam, ab150160), FITC-IgG (Jackson ImmunoResearch, 111-095-003), and tetramethylrhodamine (TRITC)-IgG (Jackson ImmunoResearch, 115-025-062) for 1 h. All antibodies were diluted 200 times before used. After further staining with 4′, 6-diamidino-2-phenylindole (DAPI), all slices were imaged by using a digital pathological section scanner (Olympus VS200).

Cytokine detection

The infected tissues and serums of mice were harvested at 4th d post-treatment. The cytokine level in tissues and serums was measured by ELISA kits (IL-6 (Abcam, ab222503), IL-4 (Abcam, ab100710), Arg-1 (Abcam, ab269541), TGF-β (Abcam, ab119557), TNF-α (Abcam, ab208348), and IL-12p70 (Abcam, ab119531)) according to the manufacturer’s instructions.

Western blotting

The proteins were isolated from biofilm-infected tissues at 4th d post-treatment. The content of protein was determined by using bicinchoninic acid protein assay (KeyGen BioTech). The gel electrophoresis and protein transformation were conducted by using western blotting kit (KeyGen BioTech) and the primary antibodies: anti-GADPH (Abcam, ab8245), anti-CD80 (Abcam, ab254579), and anti-CD 206 (Abcam, ab64693). All antibodies were diluted 200 times before used. The photographs were captured by G:BOX chemi-XR5 (Syngene) and relatively protein expression was quantified by using Gel-Pro Analyzer software.

Statistical analysis

All data are expressed as the mean ± standard deviation (SD). Inter-group and intra-group comparison analyses in each experiment were calculated by one- or two-way ANOVAs with a Tukey post-hoc test. All statistical analyses were carried out by using Graphpad Prism (version 8.4.0). Probability (p) values < 0.05 were considered statistically significant.

Reporting summary

Further information on research design is available in the Nature Research Reporting Summary linked to this article.


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