Materials
H. pylori NCTC 11637, which was kindly provided by the H. pylori Strain Pool, Beijing, China, and two H. pylori clinical strains (27054 and L2) stored in our laboratory were used in this study. The following chemical materials were bought from Solarbio (China): Cell Count Kit-8, Dulbecco’s Modified Eagle Medium (DMEM), Crystal Violet Stain solution (1%), Hoechst 33342, fetal bovine serum (FBS), prussian blue iron stain kit, phosphate buffered saline (PBS), broth medium, trypsin, and penicillin/streptomycin (10000 U/mL). Campylobacter Agar Base were purchased from OXOID.
Synthesis of Zn0.3Fe2.7O4 NPs
Zn0.3Fe2.7O4 NPs were synthesis by the thermal decomposition method [22]. First, 2.7 mmol Iron(III) acetylacetonate (Fe(acac)3), 0.3 mmol Zinc(II) acetylacetonate hydrate (Zn(acac)2⋅nH2O), 2 mmol sodium oleate, 4.4 mL oleic acid and 20 mL benzyl ether were added in a four neck flask and mixed by magnetically stirring. The mixture was heated to 120 °C for 30 min with nitrgon flow. Under nitrigon blanketing, the mixture was heated to 295 °C (reflux temperature), and kept refluxing for 2 h. Finally, the mixture was cooled down to room temperature, and was treated by ethanol to precipitated out the NPs.
Nanocluster Preparation
Nanoclusters (NCs) loaded with Zn0.3Fe2.7O4 NPs were prepared by the solvent evaporation method. 20 mg Zn0.3Fe2.7O4 NPs and 50 mg m-PEG-PCL were added in 8 mL of tetrahydrofuran (THF) then magnetically stirred for 20 min. Next, the mixture solution was ultrasonicated for 20 min. THF was then removed by rotary evaporation. The prepared aqueous solution was centrifuged at 3000 rpm for 5 min and filtered through a 0.2 μM cellulose acetate filter.
Characterization of NPs and NCs
The morphology of NPs was observed by a transmission electron microscope (Hitachi H-7650 ). The hydrodynamic particle size was measured using a dynamic light scattering instrument (Malvern ZS90 Red). Optical absorption spectra of the samples were recorded by using a UV-visible spectrophotometer (CARY 300 Conc.) with the wavelength range of 500-900 nm.
Photothermal efficiency
To assess the photothermal efficiency of NPs and/or NCs, a diode laser with a power of 1000 mW and a wavelength of 808 nm was used to irradiate the dispersions of NPs or NCs. Different concentrations of NPs or NCs (0, 25, 50 μg/mL) dispersions diluted in 1 mL PBS in McBurney turbidimetric tubes were exposed to the laser light with a power density of 1.0 W/cm2. PBS was selected as a control. The temperature rising of dispersions was recorded by thermometer. The temperature rising of dispersions was recorded by thermometer. The thermometer probe was placed vertically in the centre of the samples. The initial distance between laser source and PTAs liquids was 50 cm. When the temperature of the system reached 40.5 ℃, the distance was adjusted to around 80 cm to keep the temperature at 41 ℃ constantly.
Cell culture
BGC-823 cells stored in our laboratory were used and maintained in DMEM//HIGH GLUCOSE medium containing 10% FBS and penicillin/streptomycin (100 µg/mL penicillin and 100 µg/mL streptomycin) and cultured in a humidified atmosphere of 5 % CO2 at 37 °C.
H. pylori culture
H. pylori was cultivated on Campylobacter Agar Base with 7% sheep blood in a microaerobic condition (5% O2, 10% CO2, 85% N2) at 37 °C. For liquid culture, the medium consisted of brucella broth contained 10% FBS under agitating conditions (120 r/min) at 37 °C in a microaerobic environment.
Cytotoxicity of Zn0.3Fe2.7O4 NCs on BGC-823 cells
Cell Counting Kit-8 test was used to assess the cytotoxicity of Zn0.3Fe2.7O4 NCs to BGC-823 cells. For the CCK-8 test, the BGC-823 cells were cultured in 96-well plates at a density of 1 × 104 cells per well and were grown 10 h to stick to the wall (n = 5 per group). Then, they were co-incubated with different concentrations (0, 25, 50, 100, 200, 250 µg/mL) of Zn0.3Fe2.7O4 NCs at 37°C for 24 h and 48 h. After this step, wells were washed three times with PBS. Afterward, cells were incubated in media with 10% CCK-8 solution (150 uL) at 37 °C for 1 h in the dark. Following this period, the supernatant (100 uL) was transferred to a new plate to avoid the affect of NCs on optical density (OD) measurement. And finally, the absorbance was measured at 450 nm to quantify the cell growth.
Prussian blue staining
Prussian blue staining was used to assess the cellular uptake of Zn0.3Fe2.7O4 NCs to BGC-823 cells. Different concentrations (25, 50, 100 µg/mL) of Zn0.3Fe2.7O4 NCs were incubated with BGC-823 cells in 24- well plates at a density of 105 cells per well (n = 4 per group). After 12 h or 24 h incubation, the cells were washed with PBS for three times, and then fixed with 4% paraformaldehyde. To stain the iron in cell, prussian blue solution, 2% hydrochloric acid aqueous solution and 2% potassium ferrocyanide (II) trihydrate were mixed, and then incubated with the fixed cells for 30 mins at 37 °C. Then, the cells were washed three times, and counterstained with nuclear eosin for 20 s. Finally, the cells were observed by a microscope after washing three times with ultrapure water.
Inductively coupled plasma mass spectrometry (ICP-MS)
Zn0.3Fe2.7O4 NPs or NCs were incubated with BGC-823 cells for 12 h or 24 h in 6-well plates (3×105 cells per well). After this, cells were washed five times with PBS, and cells in all wells were collected as one sample (n=3 per group). Samples were digested with HNO3 and heated to 80℃ for 3 h for ICP-MS analysis. The Fe content was measured using an Agilent Technologies 7700x inductively coupled plasma mass spectrometer (Agilent Technologies, Santa Clara,CA).
Effect of Zn0.3Fe2.7O4 NCs heating on H. pylori growth
H. pylori in exponential growth phase was collected by PBS. 1 ml mixture of NCs (50 µg/mL) and H. pylori (1 x108 CFU/mL) was prepared in a McBurney turbidimetric tube. The tube was heated by with an 808 nm laser and the temperature of the mixture was recorded by a thermometer. 10 uL of the mixture after the heating process was added to 3 mL broth medium and was cultured at 37 °C for 96 h. Then, the absorbance of each group was measured with a spectrophotometer (UV-2000, China) at OD600, which quantified the H. pylori survival rate. After heat treatment the mixture was diluted to 4 x 105 CFU/mL and each culturing agar medium was full of 100 uL of the diluted mixture and cultured at 37 °C for 96 h. Finally, each group was counted to quantify the H. pylori survival rate.
Transmission electron microscopy
H. pylori in exponential growth phase was collected in PBS, and 1 ml mixture of NCs (50 µg/mL) and H. pylori (1 x108 CFU/mL) were prepared using McBurney turbidimetric tube and the tube was exposed to an 808 nm laserto heat the mixture to 41°C for 20 min. After centrifugation for 4 min at 1500 r/min, discarding the supernatant. Then, the bacterial was resuspended using a 2.5 % glutaraldehyde fixation solution, and the bacteria was fixed at 4 °C. After that, 10 μL of bacterial liquid was dropped on the amorphous carbon-coated copper grids and allowed to dry. Then, one drop of 3 % phosphotungstic acid dye solution was added for negative staining. Finally, samples were observed by TEM.
Evaluation of H. pylori adhesion ability and vacuolating cytotoxin
H. pylori was collected in PBS and heated to 41 °C for 20 min. The bacteria were dissolved in DMEM/HIGH GLUCOSE medium without antibiotics and serum, to form H. pylori- DMEM/HIGH GLUCOSE solution (OD600 = 0.1) for standby. For adhesion ability, BGC-823 cells were planked in a 96-well plate with 1 × 104 cells/well, and cultured overnight. The above-mentioned bacteria liquid was added for co-cultivation with a ratio of bacteria: cells of 100: 1 for 2 h. Then, discarding the supernatant and the cells were washed with PBS three times. Adding 100 μL urea reagent into each well, and incubated for 2 h at room temperature. Finally, the absorbance of each group was measured with a spectrophotometer at a wavelength of 540 nm. For vacuolating cytotoxin, BGC-823 cells were planked in 96-well plate with 5 × 103 cells per well, and incubated at 37 °C overnight. The above-mentioned bacteria liquid was added for co-cultivation for 24 h with a ratio of bacteria: cells of 200: 1. The supernatant was discarded and 100 μL neutral red (0.005%) was added into each well for 5 minutes. Then, discarding the dye, and washing the cells with PBS three times, and 100 μL hydrochloric acid alcohol (0.04 %) was added into each well. The absorbance of each group was measured with a spectrophotometer at a wavelength of 550 nm.
Assessment of H. pylori susceptibility to antibiotics
MIC of various antibiotics for NCTC 11637 and all the clinical strains were measured by the Epsilometer test (E-test) using an E-strip (Liofilchem, USA). All the strains cultured in agar medium, and the third-generation colonies were selected and suspended in PBS to an OD600 of 1 for standby. For control groups, above-mentioned bacteria solution were diluted to OD600 = 0.1 and 100 μL of the bacterial solution was coated on Karmali Agar Base evenly. After each agar plate was left to dry, E-strip was affixed, and then the plates were incubated at 37 °C under microaerobic conditions, and MIC values were determined after 72 h. The method used to determine the MICs for heated groups was similar to that used for the control groups, 100 μL of the bacterial solution (OD600 = 1) was exposed to an 808 nm laser to heat to 41 °C for 20 min, then was evenly coated on Karmali Agar Baseto determine the MIC values.
Crystal violet staining
Biofilm formation was assessed under agitating conditions (120 r/min) in 12-well plates. H. pylori was collected by PBS and resuspended to an OD600 of 1, and further diluted to 5 × 105 CFU/mL in broth medium and each was filled with 3 mL of the diluted bacterial solution and incubated at 37 °C for 4 days. The plates were washed with PBS three times gently, and then stained with 500 μL of 1% (w/v) crystal violet for 30 min. The crystal violet stain was solubilized with 80% ethanol–20% acetone solution, and then measuring the absorbance at OD580. For experimental groups, the samples were heated with NIR to 41 °C last for 20 min, and the remaining steps were the same as above after a generation of cultivation.
Confocal laser scanning microscopy (CLSM)
CLSM was aimed at observing the biofilm as described with slight modifications [23]. A biofilm model of H. pylori was constructed in 6-well cell culture plate. Sterile cover glass with a diameter of 1 cm was put into 6-well plates, and 3 mL broth medium was added into each well. Then 10 μL H. pylori solution with an OD600 of 1 was added into each well. The bacteria were cultured for 4 days in an incubator at 37 ℃. The cover glass was removed, washed 3 times in PBS buffer to remove excess planktonic bacteria, and then moved into a new 6-well plate and fixed in 2.5% glutaraldehyde at 4 °C for 1.5 h. After washed with PBS, the fixed cover was added 300 μL FITC-ConA (100 mg/mL) for 30 mins at 4 ℃ away from light, and PBS buffer was used to rinse slowly twice. Then, equal PI staining was performed in the same way and the cover glass was dried at room temperature away from light. Afterwards, the cover glass was sealed with anti-fade mounting medium, then observed under a laser confocal microscope. Five fields of the slides were randomly selected for shooting.
Hoechst 33342 accumulation assay
Accumulation assay was performed as described previously [24]. H. pylori was collected by PBS and resuspended to an OD600 of 1, Then, 180 μL of this bacteria solution was added to 96-well plate. The excitation and emission wavelengths were 355 and 460 nm respectively, using SpectraMax M5/M5e (USA). After adding Hoechst 33342 (25μM, 20μL) 5 min, recordings were started. Readings were taken every 75 s for 30 cycles, and each experiment was repeated three times. For experimental groups, the samples were heated with NIR to 41 °C for 20 min, and the remaining steps were the same as above after a generation of cultivation.
Statistical analyses
Statistical analysis was carried out using GraphPad Prism (GraphPad Software, Inc., San Diego, CA, USA). All data were present as mean ± standard errors of the means and determined using Student’s t-test. For all analyses, p values <0.05 were considered significant, and the level of significance was described as *p < 0.05, **p < 0.01, ***p < 0.001.