Materials
HAuCl4, PME, and LPS were purchased from Aladdin Biochemical Technology Co., Ltd (Shanghai, China). Sodium citrate and MnSO4·H2O were obtained from Zhiyuan Chemical Reagent Co., Ltd (Tianjin, China). Peptone, beef paste, and bismuth sulfite agar were purchased from Aoboxing Biotechnology Co., Ltd (Beijing, China). Endotoxin detection horseshoe crab reagent kit was acquired from Horseshoecrab Reagent Biotechnology Co., Ltd (Xiamen, China). The probiotic BC (BNCC192989) was obtained from BeiNa Chuang Lian Biotechnology Research Institute (Beijing, China). Escherichia coli (E. coli, ATCC25922), S. Tm (SL1344) and Staphylococcus aureus (S. aureus, ATCC25922) were purchased from Guangdong Microbial Culture Collection Center (Guangdong, China). IL-6, IL-1β, and TNF-α enzyme-linked immunosorbent assay (ELISA) kits were obtained from Shanghai Enzyme-linked Biotechnology Co., Ltd (Shanghai, China).
Preparation of SC-Au NPs
According to the literature [29], 25 µL HAuCl4 stock solution (0.01%, W/V) was diluted into 50 mL H2O for stiring and when the temperature rised to 90 ℃, 1.5 mL of 1% sodium citrate (SC) solution was added quickly. After heating for 15 min, the solution gradually turned into purple-red and was stirred only until it cooled to room temperature (RT).
Preparation of PME-Au NPs
PME-Au NPs were synthesized via a one-pot reaction between HAuCl4 and PME. 62 µL HAuCl4 stock solution (1 mM) was diluted into 30 mL H2O. Then PME was added (n(HAuCl4): n(PME) = 3:1) and stirred for 5 min. Subsequently, the pH was adjusted to 6–7 with NaOH, causing the solution color to change from pale yellow to clear. The mixture was stirred at 37 ℃ for over 0.5 h until the solution turned purple-red.
Extraction of BCs
After recovering BC, a 4% inoculation was used to obtain a second-generation bacterial solution. The sporulation medium was prepared by adding 1 g peptone, 0.3 g beef paste, 0.5 g NaCl, 0.5 mg MnSO4·H2O and 100 mL H2O and sterilizing at 121 ℃ for 15 min. Then, 4 mL of second-generation bacterial solution was added to the sporulation medium and cocultured at 37 ℃, 150 rpm for 48 h. Subsequently, the BCs solution was then heated at 80 ℃ for 30 min to kill any surviving bacteria and centrifuged three times at 5000 rpm for 10 min with 0.9% NaCl. The precipitation was resuspended in H2O and freeze-dried for further use.
Preparation of BCs@PME-Au
BCs@PME-Au was synthesized via a one-pot reaction using HAuCl4, PME, and freeze-dried BC powder. Firstly, 10 mg BCs was diluted into 30 mL H2O. Then 62 µL HAuCl4 stock solution (1 mM) was added and preheated for 5 min. Then PME was added in a molar ratio of HAuCl4: PME (3:1) and stirred for 5 min. Subsequently, the solution pH was adjusted to 6 ~ 7 with NaOH, accompanied by a change of the solution color from pale yellow to white with a suspended state. Then, the mixture was stirred at 37 ℃ for over 0.5 h until the solution turned purplish red. Finally, the BCs@PME-Au solution was cooled to RT for use.
Characterization of BCs@PME-Au
The morphology of PME-Au NPs and BCs@PME-Au was analyzed with transmission electron microscopy (TEM, JEOL, Japan) and scanning electron microscope (SEM, SU8010, Japan). The size distribution and zeta potential were characterized using a dynamic light scattering (DLS) analyzer (Zetaster, Malvern, UK). X-ray diffraction (XRD, Almelo, Netherlands) pattern and X-ray Photoelectron Spectroscopy (XPS, ThermoFisher, USA) were used to assess the properties of PME-Au NPs and BCs@PME-Au. Fourier transform infrared spectroscopy (FT-IR) was used to characterize the loading of PME. The system contents were determined by the ultraviolet-visible spectrum (UV-Vis, L-2489), and the drug loading capacity was measured by high-performance liquid chromatography (HPLC, Waters, USA).
In vitro stability of BCs@PME-Au
The in vitro stability of BCs@PME-Au was characterized by incubation with simulated gastric fluid (SGF) and simulated intestinal fluid (SIF) at 37 ℃. Then, OD600-monitoring and spread plate methods were used to characterize the BC’s vitality.
Measurement of encapsulation efficiency
According to the literature, the mobile phase for detecting PME by HPLC included an Agilent SB-Aq analysis column (250 mm x 4.6 mm, 5 μm), UV detector at 205 nm, mobile phase of 0.01 M trifluoroacetic acid: acetonitrile, binary gradient elution (77:23 (0–6.5 min), 76:24 (6.5–10 min), 75.5:24.5 (10–15 min)), flow rate of 1.0 mL/min, column temperature of 30 ℃, and injection volume of 20 µL. During the preparation of PME-Au NPs and BCs@PME-Au, supernatants were obtained by centrifugation. Encapsulation efficiency was calculated as [(original PME content − free PME content)/original PME content] × 100%.
The ability of formulations to neutralize LPS
According to the endotoxin detection instructions from the limulus test kit, a standard curve was established firstly. Then PME and PME-Au NPs solutions with different concentrations were mixed with an equal volume of 30 µg/mL of LPS working solution to a final concentration of 20 µg/mL in a endotoxins-removal test tube. Furtherly, the mixed solution was incubated at 37 ℃ for 1 h. The optical density value at 545 nm was measured to calculate the neutralizing LPS content of different formulations based on the standard curve.
The culture of bacteria
1 g tryptone, 0.5 g yeast powder, 1 g NaCl and 100 mL ddH2O were placed in a conical flask and sterilized at 121 ℃ for 15 min to obtain the LB medium. Then the E. coli (ATCC25922), S. Tm (SL1344) and S. aureus (ATCC25922) were recovered and cultured in LB medium for 6–8 h at 37 ℃, 150 rpm to obtain the first-generation bacterial solution. By analogy, the second-generation bacterial solution was stored at 4 ℃ for future use.
Measurement of minimal inhibit concentration
The minimal inhibit concentration (MIC) of PME, SC-Au NPs and PME-Au NPs was measured using the broth dilution method. Firstly, the gut pathogenic strain E. coli and S. Tm were tested as representatives of Gram-negative bacteria, and antibacterial sensitive strains BC and S. aureus were tested as representatives of Gram-positive bacteria. Firstly, 180 µL bacteria solution per well was mixed with 20 µL antibacterial agents with different concentrations to final concentrations of 256, 128, 64, 32, 16, 8, 4, 2, 1, 0.5, 0.25, 0.125 µg/mL and incubated overnight at 37 ℃, 150 rpm. Meanwhile, blank culture medium group and bacteria group were set as negative and positive control, respectively. The concentration corresponding to the well without precipitation was determined as MIC.
Determination of bactericidal kinetics curves
The bacterial survival curves under different concentrations of PME were investigated. Specifically, 500 µL bacterial solution was added to shaking tube, and PME were added with final concentrations of 2, 4, and 8 µg/mL, respectively. The bacterial solution at each time point (0, 0.5, 1, 2, 4, 8, 12, 18, and 24 h) was plated to determine the colony forming units (CFU). The procedure was repeated to determine the bactericidal kinetics curves of PME and PME-Au NPs.
In vitro bacterial competitive colonization
A selective solid medium with bismuth sulfite agar was used specifically for S. Tm growth to investigate the antibacterial efficacy of BCs. S. Tm and BC bacterial solutions (1.0 × 105 CFU/mL) were mixed and incubated at 37 ℃ and 150 rpm for 16 h. Then the colony counts were determined using the spread plate method with selective culture medium.
Cell biocompatibility assessment of BCs@PME-Au
Firstly, Caco-2 cells were seeded into a 96-well plate (7 × 103/well) for 24 h and incubated with different concentrations of PME, BCs, PME-Au NPs, and BCs@PME-Au. Then, the absorbance values (λ = 450 nm) were recorded according to the instructions of the CCK-8 assay. The survival rate (%) = (OD experimental group / OD control group) × 100% was used to calculate the survival rate of each group.
In vivo distribution of BCs@PME-Au
An appropriate amount of IR780 was mixed with BCs@PME-Au and stirred overnight to obtain IR780/BCs@PME-Au (IR780: 10 µg/mL). 30 mice were randomly assigned into IR780 and IR780/BCs@PME-Au group. Then IR780 and IR780/BCs@PME-Au were orally administered to mice, respectively. At 2, 4, 8, 12, and 24 h post-administration, the entire gastrointestinal tract (GIT) of mice was collected for real-time imaging to observe the GIT distribution.
Induction of colitis induced by S. Tm
Mice were given streptomycin sulfate (200 mg/kg) for 2 days to enhance the sensitivity to S. Tm. After 24 h of pre-treatment, mice were orally administered 200 µL S. Tm (1 × 109 CFU/mL). If symptoms such as mental lethargy, a sudden drop in weight and a significant decrease in vitality were observed in mice, it preliminarily indicated the successfully establishment of S. Tm-induced colitis mouse model.
For in vivo pharmacological experiments, mice were randomly divided into the following 6 groups: (1) NC; (2) S. Tm; (3) BCs; (4) PME; (5) PME-Au NPs and (6) BCs@PME-Au. Continuous administration via oral gavage (PME: 1 mg/kg, 200 µL; BCs: 1 × 109 CFUs/mL, 200 µL) was conducted once daily for 5 days. Body weight was recorded daily during the treatment period. After treatment, the colon length of different treatment groups were recorded. Then, the colon and other main organs were collected for hematoxylin and eosin (H&E) and periodic acid-schiff (PAS) staining. The colon tissues were also collected for immunofluorescence of tight junction proteins (ZO-1, Occludin, and Claudin-1) and inflammatory pathway related proteins (TLR4, MyD88 and NF-κB p65). Serum was collected for inflammatory cytokines analysis (TNF-α, IL-1β and IL-6).
Detection of bacterial counts in tissues
Bacterial counts in tissues were measured to evaluate the therapeutic effects of formulations on S. Tm-induced colitis. After treatment, the liver, spleen, colon, and feces from each group were collected. Each tissue with the same weight was placed in a 2 mL sterile tube for high-speed homogenization at 4 ℃. Subsequently, the suspensions were subjected to S. Tm selective culture medium for spread plate count.
Measurement of myeloperoxidase
Myeloperoxidase (MPO) is a pro-inflammatory lysosomal protein highly expressed in monocytes and neutrophils [51], which can be used to evaluate the inflammation severity induced by S. Tm. The colon tissues from each group were processed according to the instructions of the MPO detection kit, and MPO activity was calculated using the following formula: MPO activity (U × g − 1 wet weight of colon tissue) = (Measured OD value – Control OD value)/(11.3 × sample size (g)).
Western blot
The colon tissues were lysed in RIPA buffer. The protein concentration was quantified by using BCA Protein Assay Kit (Epizyme ZJ101). Equal proteins were loading on SDS-PAGE gel and transferred to nitrocellulose membranes. After blocking with 5% non-fat milk for 1 h, the membranes were probed with primary antibodies (TLR4 (D8L5W) Rabbit mAb, 14358, Cell Signaling Technology; MyD88 (D80F5) Rabbit mAb, 4283, Cell Signaling Technology; NF-κB p65 (D14E12) Rabbit mAb, 8242, Cell Signaling Technology; β-Actin (8H10D10) Mouse mAb, 3700, Cell Signaling Technology) overnight at 4 ℃ and then incubated with the secondary antibodies (Anti-Rabbit IgG, HRP-linked, 7074, Cell Signaling Technology). The immunoblots were recorded with the BioSpectrum imaging system (UVP, USA).
16 S sequencing and Microbiome analysis
To evaluate the potential effect of BCs@PME-Au on intestinal microbiota modulation, faecal samples from each group were collected for 16 S rDNA gene sequencing post-administration (Meiji Co. Ltd., Shanghai, China). The sample libraries were constructed and analyzed for alpha diversity, beta diversity, and other indices to illustrate the impact of different treatments on gut microbiota.
Biosafety assessment
Mice were randomly divided into the following 5 groups: (1) NC; (2) BCs; (3) PME; (4) PME-Au NPs and (5) BCs@PME-Au. Continuous administration via oral gavage (PME: 1 mg/kg, 200 µL; BCs: 1 × 109 CFUs/mL, 200 µL) was conducted once daily for 5 days. After the completion of the dosing cycle, whole blood was collected for hematological parameters and biochemical indicators testing. Among them, the hematological parameters: red blood cell (RBC), white blood cell (WBC), hemoglobin (HGB) and platelet (PLT); the liver function indicators tested include: glutamate pyruvate transaminase (ALT), aspartate aminotransferase (AST); the renal function indicators include urea nitrogen (BUN) and creatinine (CRE).
Statistical analysis
All data were expressed as mean ± SD. Differences considered statistically significant were performed by the Student’s t-test of GraphPad Prism 9 (*P < 0.05, **P < 0.01, and ***P < 0.001).