Materials. Fumed silica was purchased from Aladdin Industrial Corporation (Shanghai, China). Cloroplatinic acid (H2PtCl6 ·6H2O, 99.9%) and Co(NO3)2·6H2O were purchased from Changsha Chemical Reagents Company (Changsha, China). Gastric pepsin, 3, 3' 5, 5'-tetramethylbenzidine (TMB), horseradish peroxidase (HRP), 2, 2'-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), o-phenylenediamine (OPD), terephthalic acid (TA), and hydroethidine (HE) were purchased from Sigma-Aldrich China (Shanghai, China). 5-(Diethoxyphosphoryl)-5-methyl-1-pyrroline-N-oxide (DEPMPO) was purchased from Santa Cruz Biotechnology China (Shanghai, China). Helicobacter pylori (ATCC 43504) was purchased from American type culture collection (ATCC). BALB/c female mice (3–4 weeks old) were obtained from the Hunan SJA Laboratory Animal Co., Ltd (Changsha, China) and used under protocols approved by the Institutional Animal Care and Use Committee of Hunan University. All other chemicals of analytical reagent grade were obtained from Changsha Chemical Reagents Company (Changsha, China) and used as received without further purification. Ultrapure water (resistance > 18.2 MΩ cm− 1) was used throughout all experiments.
Characterization and instruments. All Raman measurements were performed in a Renishaw’s InVia Raman system with 633 nm laser excitation (Renishaw, UK). Ultraviolet-visible spectra were recorded by a UV-2450 UV-vis spectrophotometer (Shimadzu, Japan). Hydrodynamic diameters and Zeta potentials were measured by a DLS system (Malvern, UK). TEM images were taken with a JEM-2010 (JEOL, Japan). SEM images were measured by JSM-6700F (Japan). Fluorescence spectra were performed by HORIBA Fluoromax-4 (HORIBA, USA). Electron paramagnetic resonance (EPR) measurements were recorded by JES-FA200 electron spin resonance (JEOL, Japan). A Minispec MQ60 60 MHz TD NMR broadband spectrometer (Bruker, Germany) was used to measure T2 relaxivity. T2-weighted MR imaging was taken by the 1.5 T small animal MR scanner (MRI 1.5 T; Shining Global Science and Education Equipment Co., Ltd., Shanghai, China). Inductively-coupled plasma mass spectrometry (ICP-MS, Agilent, USA) was utilized to measure the concentrations of released Co2+ and Pt2+ in the solution.
Preparation and characterization of PtCo@G nanocrystals. Magnetic graphitic PtCo nanocrystals (PtCo@G) were prepared through a chemical vapor deposition (CVD) method. First, fumed silica (1.00 g) was impregnated with chloroplatinic acid (5.0 mL, 1.0%) and Co(NO3)2·6H2O (0.073 g) in methanol and sonicated for 1 h. Methanol was removed, and the mixture was dried at 45 °C. Then, the powder was placed into a methane chemical vapor deposition chamber for growth with a methane flow of 150 s.c.c.m. for 5 min at 1000 °C. After growth, hydrofluoric acid was used to etch the silica, followed by collecting the PtCo@G with a magnet. TEM (JEOL, Japan) was used to characterize the morphology of PtCo@G.
Comparison of stability of PtCo@G in H 2 O, PBS (pH 1.0), and SGF. Equal amounts of PtCo@G were added into H2O, PBS (pH 1.0), and SGF and incubated for 12 h. Then the digital photos, Hydrodynamic diameter, MR T2 relaxivity and T2-weighted phantom images of PtCo@G in H2O, PBS (pH 1.0), and SGF were measured by DLS (Malvern, UK), Minispec MQ60 60 MHz TD NMR broadband spectrometer (Bruker, Germany) and 1.5 T small animal MR scanner (MRI 1.5 T; Shining Global Science and Education Equipment Co., Ltd., Shanghai, China), respectively.
Dissolution experiments. To detect the release of Co2+ and Pt2+ from PtCo@G nanocrystals, dissolution experiments were carried out. Equal amounts of PtCo@G were added into H2O, PBS (pH 1.0), and SGF and incubated for different times (0, 0.5, 1, 2, 4, 6, 8, 10, 12 h) with constant shaking. The PtCo@G nanocrystals were removed from the solution by centrifugation (12000 rpm, 30 min), and the supernatant was stabilized to 5.0 mL. The concentrations of released Co2+ and Pt2+ in the solution were measured with inductively-coupled plasma mass spectrometry (ICP-MS, Agilent, USA).
Oxidase-like property of PtCo@G. The oxidation of TMB by PtCo@G in phosphate buffer produced a blue color with major absorbance peaks at 652 nm. In a typical test, certain amounts of TMB (final concentration 500 µM) and PtCo@G were added into buffer solution (25 mM PBS, pH 4.0), and then UV-Vis absorption spectra were recorded at different times using a UV-2450 UV-vis spectrophotometer (Shimadzu, Japan). Other organic dyes, such as ABTS and OPD, were also used to further confirm the oxidase-like property of PtCo@G in the same way.
Peroxidase-like property of PtCo@G. The oxidation of TMB by PtCo@G/H2O2 in 25 mM phosphate buffer (pH 4.0) produces a blue color with major peaks at 652 nm. Certain amounts of TMB (final concentration 500 µM), H2O2 (final concentration 100 µM) and PtCo@G were added into buffer solution, and then UV-Vis absorption spectra were recorded at different times using a UV-2450 UV-vis spectrophotometer (Shimadzu, Japan). Other organic dyes, such as ABTS and OPD, were also used to further confirm the peroxidase-like property of PtCo@G.
Oxidase-like and Peroxidase-like property of PtCo@G in PBS (pH 1.0). For assay of the product O2•−, dihydroethidium (HE) was used as a probe, which could be easily oxidized by O2•− to generate the highly fluorescent product ethidium (E+). In a typical procedure, HE was added into the reaction solution (PBS, pH 1.0) for 30 min, and then the fluorescence spectra of the samples were collected using fluorescence spectroscopy (HORIBA Fluoromax-4, USA).
For assay of the product •OH, TA and DEPMPO, which were used as probes, could easily react with •OH to form a highly fluorescent product (TAOH) and electron paramagnetic resonance (EPR) spin adduct (DEPMPO-OH). In experimental procedure, TA and DEPMPO were added into reaction solutions (PBS, pH 1.0). Then, the fluorescence spectra and electron paramagnetic resonance (EPR) of the samples were performed by fluorescence spectroscopy (HORIBA Fluoromax-4, USA) and electron paramagnetic resonance (EPR) spectroscopy (JEOL, Japan).
Oxidase-like property of PtCo@G in simulated gastric fluid (SGF). Chemicals were added into SGF in an order of certain amounts of PtCo@G and TMB (final concentration 500 µM). The oxidation of TMB by PtCo@G in SGF produces a yellow color, and UV-Vis absorption spectra were recorded using a UV-2450 UV-vis spectrophotometer (Shimadzu, Japan).
Bacterial culture and antibacterial experiments in vitro. H. pylori was cultured on a Columbia agar plate with 6.5% sterile Defibrinated Sheep Blood and multiple antibiotics (10 µg/mL vancomycin, 5 µg/mL trimethoprim lactate, 5 µg/mL cefsulodin sodium and 5 µg/mL amphotericin B) at 37 °C for 3–5 days and then transferred to Dulbecco’s phosphate-buffered saline (DPBS). The as-prepared bacterial solutions (20 µL) and certain amount of PtCo@G@CPB were added into SGF and incubated at 37 °C, 150 rpm, for 30 min. Then the solution was placed on the Columbia agar plate by the spread plate method and cultured for 3–5 days before observing the number of the bacterial colonies.
Preparation of bacterial samples for TEM/SEM. After incubation with PtCo@G@CPB at 37 °C for 30 min, the H. pylori solutions (106 CFU) were fixed in 2.5% glutaraldehyde overnight at 4 °C. Then, the above bacterial solutions were centrifuged (2000 rpm, 10 min) and dehydrated through treating with 50%, 70%, and 90% gradient ethanol for 8 min and 100% ethanol for 15 min. Finally the resulting samples were dropped on carbon film/silicon slice, and the morphology of H. pylori was observed on TEM/SEM.
Live/dead fluorescence staining. After treatment with PtCo@G@CPB, H. pylori solutions were incubated with AO and PI at 37 °C for 10 min, followed by washing with DPBS. Then, the live/dead bacterial cells were observed by confocal laser microscopy.
Establish H. pylori-infected mouse model. BALB/c female mice 3–4 weeks old were purchased from the Hunan SJA Laboratory Animal Co., Ltd (Changsha, China) and used under protocols approved by the Institutional Animal Care and Use Committee of Hunan University. Each BALB/c mouse received 300 µL of 5 × 107 CFU mL− 1 H. pylori in DPBS by oral gavage once a day for four days (on day 4, 5, 6 and 7, respectively), and the infection was allowed to develop for 2 weeks. Then, the established H. pylori-infected mice (n = 6) were sacrificed, and the stomachs were excised from the abdominal cavity. The stomach was cut longitudinally along the greater curvature. The reculture of gastric mucosa and Gram staining of gastric tissue section were used to demonstrate the successful colonization of H. pylori in the stomach of mice.
The retention of PtCo@G@CPB in vivo. The H. pylori-infected mice (6–7 weeks) were randomly divided into two groups (n = 6) and received DPBS or PtCo@G@CPB by intragastric administration. The groups of mice were administered with DPBS as negative control. 4 or 24 h after oral administration, the T2-weighted phantom imaging of PtCo@G@CPB in mouse stomach was measured by 1.5 T small animal MR scanner (MRI 1.5 T; Shining Global Science and Education Equipment Co., Ltd., Shanghai, China). After measuring T2-weighted phantom imaging of PtCo@G@CPB in mice stomach, the mice were sacrificed, and the stomachs were excised and cut open longitudinally along the greater curvature. The gastric tissue sections were prepared by the paraffin-embedded method. The D- and G-band of PtCo@G in the gastric tissue section were detected by Raman spectroscopy (Renishaw, UK) to certify the retention of PtCo@G@CPB in the mouse stomach.
PtCo@G@CPB selective treatment H. pylori in vivo. The established H. pylori-infected mice were randomly divided into three treatment groups (n = 6) to receive DPBS, PtCo@G@CPB or triple therapy OAC (omeprazole, amoxicillin, clarithromycin) by oral administration once daily for four consecutive days (on day 1, 2, 3 and 4, respectively). For the triple therapy group, the treatment mice were first administered omeprazole (a proton pump inhibitor) through oral gavage at 400 µmol/kg, followed by a lag time of 30 min before administration of the other two antibiotics, including 28.5 mg/kg of amoxicillin and 14.3 mg/kg of clarithromycin. The groups of mice were treated with DPBS as negative control. Two days (48 h) after the last administration, the three groups of treatment mice were sacrificed, and the stomachs were excised from the abdominal cavity. The stomach was cut longitudinally along the greater curvature, and the gastric tissues were used for bacterial colonization and tissue section staining study. At the same time, the intestine and feces of the mice were also collected. For H. pylori colonization, gastric tissue was suspended in 1 mL DPBS and homogenized for bacterial recovery. The homogenate was spotted on Columbia agar plate with 6.5% sterile Defibrinated Sheep Blood and multiple antibiotics (10 µg/mL vancomycin, 5 µg/mL trimethoprim lactate, 5 µg/mL cefsulodin sodium and 5 µg/mL amphotericin B). Then, the plates were incubated at 37 °C under microaerobic conditions for 5 days, and the H. pylori colonies were enumerated. For the effects of PtCo@G@CPB on the symbiotic bacteria, the bacterial load in the intestine and feces were determined by quantitative real-time PCR using the protocol in ref.44,45. The test results were normalized to intestine/feces weight.
The PtCo@G@CPB toxicity in vivo. During treatment, changes in mice weight were recorded daily for six consecutive days. Mice received daily gavage of DPBS, PtCo@G@CPB or triple therapy OAC for four consecutive days. Mice were sacrificed on the sixth day, and the stomachs were excised. The gastric tissue sections were prepared by the paraffin-embedded method for H&E staining. The inflammation and injury score of stomach were scored by Dr. Hu in a blind manner according to the H&E staining, and the specific scoring criteria used were detailed in the method in ref.8. Inflammation was graded on a 0–3 ordinal scale based on the Sydney System as follows: chronic inflammation (mononuclear cell infiltration independent of lymphoid follicles); grade 0, no inflammation; grade 1, mild inflammation (slight increase in mononuclear cells); grade 2, moderate inflammation (dense, but focal, mononuclear inflammatory cells); and grade 3, severe inflammation (dense and diffuse mononuclear inflammatory cells). The appearance of the mucosa was graded as follows: 0, normal; 1, spotty changes in cellular staining characteristics of some surface epithelial cells in an otherwise normal mucosa (mild injury); 2, more generalized changes and/or disruption of the surface epithelium in several areas (moderate injury); 3, extensive mucosal destruction (severe injury). The heart, liver, spleen, lung, and kidney of mice were gathered and nitrated with Lefort aqua regia (VHCl:VHNO3=1:3). The amounts of PtCo@G@CPB in the heart, liver, spleen, lung, and kidney of mice were determined by measuring Pt content in the organs’ nitrifying liquid with inductively coupled plasma series mass spectrometer (Agilent, USA).