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Nanocluster-mediated photothermia improves eradication efficiency and antibiotic sensitivity of Helicobacter pylori

Helicobacter pylori (H. pylori) eradication plays a crucial role in gastric cancer prevention, but the antimicrobial resistance of H. pylori is obstructing this elimination process. In this study, we developed nanoclusters (NCs) from Zn0.3Fe2.7O4 nanoparticles using a poly(ethylene glycol)-b-poly(ε-caprolactone)-based nanocarrier as an innovative antibiotic-independent H. pylori management. The nanocluster showed minimal toxicity and maximal biocompatibility. With a low concentration (50 µg/mL) of NCs under a short time period (~ 2 min) of near-infrared (808 nm) irradiation, we kept the culture medium temperature to 41 °C for 20 min with continuous irradiation. The heated NCs exhibited efficient photothermal effects and resulted in an excellent inhibition of H. pylori growth, adhesion and ability to induce vacuolization in eukaryotic cells in in vitro investigation. Transmission electron microscopy showed a dramatic morphologic change after NCs photothermia on H. pylori, including cell wall and membrane rupture, as well as ribosome damage. Besides, levofloxacin and clarithromycin resistance was decreased after photothermal treatment in H. pylori NCTC 11637 and/or clinical strains, however metronidazole resistance was unchanged. We also discovered a significant decrease in the biofilm formation of H. pylori under the NCs-based photothermal application, while efflux pump function was unchanged. Based on this novel NCs-based photothermal approach, we were able to demonstrate in vitro a significant inhibition of both H. pylori growth and molecular toxicity, and its improvement in antibiotic sensitivity alone with the eradication of H. pylori biofilms previously believed to be tolerant to conventional antibiotics.

gastric pathologies, such as chronic gastritis, peptic ulcers, and gastric cancer/mucosa associated lymphoid tissue lymphoma (Kusters et al. 2006;McColl. 2010;Parkin. 2006;Rocha et al. 2015;Uemura et al. 2001). Some studies have stated that H. pylori eradication may be the most practical way to reduce occurrence of gastric cancer and peptic ulcer disease and ultimately save lives, showing the importance of reliable eradication methods (Forman and Graham. 2004). The rising rate of antibiotic resistance and the emergence of multidrug-resistant (MDR) H. pylori strains during the last decade has reduced the success of eradication therapy, and this can be partially attributed to extended and inappropriate use of antibiotics (Boyanova et al. 2019;Kwon et al. 2003;Peake et al. 2016). The World Health Organization (WHO) has published a list of bacteria that urgently requires new antibiotics to overcome their current antibiotic resistance issues, and H. pylori is a high priority on the list (Tacconelli et al. 2018). Extensive research is being carried out on any innovative treatment.
One of these new antibiotics-independent approaches to eradicate H. pylori is the usage of nanotechnology. Nanoparticle-based drug delivery systems for targeted treatment of tumor and H. pylori have been described (de Souza et al. 2021;Zeng et al. 2017). Among them, light-based treatment has been used to utilize nanomaterials and their composites. Photothermal therapy (PTT) has exhibited great potentials in dealing with drug-resistant bacteria and bacterial biofilms. In recent years, photothermal effect has been reported to possess high light-thermal conversion efficiency under the irradiation of near-infrared (NIR) light (Cheng et al. 2019;Jaque et al. 2014). PTT has several advantages, such as being minimally invasive, remotely controllable and efficient. PTT is regarded as a safe and efficient strategy to manage bacterial infections (Han et al. 2020;Liu et al. 2017). Besides, nanoplatforms with synergistic effects of PTT and chemotherapy in tumor therapy (Li et al. 2021), as well as PTT and light-triggered nitro oxide release in antibacterial and antifungal application (Liu et al. 2021) have been developed to exhibit potential clinical significance. Various mechanisms have been shown to contribute to antimicrobial resistance in H. pylori, such as genetic mutations, altered efflux pump activity, and the formation of bacterial biofilms (Cammarota et al. 2012). While the field of H. pylori biofilm research is fairly new, groups have already begun to explore alternative therapeutic approaches that may target and eradicate bacterial biofilms including PTT (Gurunathan et al. 2015). PTT was reported to be efficient in treating biofilm-related infections and delayed the development of drug-resistant bacteria (Şen Karaman et al. 2020;Teng et al. 2016;Yu et al. 2019;Yuan et al. 2019). However, the effect of PTT on H. pylori biofilms remains to be elucidated.
In this study, we synthetized nanoclusters (NCs) from Zn 0.3 Fe 2.7 O 4 nanoparticles (NPs) using a PEG-PCL-based nanocarrier, and compared its photothermal effects on H. pylori inhibition with NPs. We have also assessed their biological safety, as well as any alteration on antibiotic resistance of H. pylori after photothermal therapy. We have also assessed the underlying mechanisms of antimicrobial resistance, including changes in biofilm and efflux pump activity. As the first study of its kind, we have confirmed that the photothermal effect of NCs restrict the growth of H. pylori and improve antibiotic sensitivity, providing new ideas and methods for future H. pylori treatment.

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 (10,000 U/mL). Campylobacter agar base was purchased from OXOID.  (He et al. 2018). First, 2.7 mmol Iron(III) acetylacetonate (Fe(acac) 3 ), 0.3 mmol Zinc(II) acetylacetonate hydrate (Zn(acac) 2 ⋅nH 2 O), 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 nitrogen flow. Under nitrogen 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 Zn 0.3 Fe 2.7 O 4 NPs were prepared by the solvent evaporation method. 20 mg Zn 0.3 Fe 2.7 O 4 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/cm 2 . 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 center of the samples. The initial distance between laser source and PTAs liquids was 50 cm. When the temperature of the system reached 40.5 °C, the distance was adjusted to around 80 cm to keep the temperature at 41 °C 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% CO 2 at 37 °C.

Cytotoxicity of Zn 0.3 Fe 2.7 O 4 NCs on BGC-823 cells
Cell Counting Kit-8 test was used to assess the cytotoxicity of Zn 0.3 Fe 2.7 O 4 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 × 10 4 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 Zn 0.3 Fe 2.7 O 4 NCs at 37 °C for 24 h and 48 h. After this step, wells were washed th ree 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 Zn 0.3 Fe 2.7 O 4 NCs to BGC-823 cells. Different concentrations (25, 50, 100 µg/mL) of Zn 0.3 Fe 2.7 O 4 NCs were incubated with BGC-823 cells in 24-well plates at a density of 10 5 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 min 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)
Zn 0.3 Fe 2.7 O 4 NPs or NCs were incubated with BGC-823 cells for 12 h or 24 h in 6-well plates (3 × 10 5 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 HNO 3 and heated to 80 °C for 3 h for ICP-MS analysis. The Fe content was measured using an Agilent Technologies 7700 × inductively coupled plasma mass spectrometer (Agilent Technologies, Santa Clara, CA).

Effect of Zn 0.3 Fe 2.7 O 4 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 × 10 8 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 μL 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 OD 600 , which quantified the H. pylori survival rate. After heat treatment the mixture was diluted to 4 × 10 5 CFU/mL and each culturing agar medium was full of 100 μL 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 × 10 8 CFU/mL) were prepared using McBurney turbidimetric tube and the tube was exposed to an 808 nm laser to 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 (OD 600 = 0.1) for standby. For adhesion ability, BGC-823 cells were planked in a 96-well plate with 1 × 10 4 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 × 10 3 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 min. 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 OD 600 of 1 for standby. For control groups, above-mentioned bacteria solution were diluted to OD 600 = 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 (OD 600 = 1) was exposed to an 808 nm laser to heat to 41 °C for 20 min, then was evenly coated on Karmali agar base to 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 OD 600 of 1, and further diluted to 5 × 10 5 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 OD 580 . 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 (Takenaka et al. 2001). 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 OD 600 of 1 was added into each well. The bacteria were cultured for 4 days in an incubator at 37 °C. 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 min at 4 °C 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 (Coldham et al. 2010). H. pylori was collected by PBS and resuspended to an OD 600 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 33,342 (25 μM, 20 μL) 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.

Synthesis and characterization of nanoclusters
The NPs were synthesized by the one-pot thermal decomposition method, and the nanoclusters were prepared by a solvent evaporation method (Additional file 1: Fig. S1). NPs or NCs in phosphate buffered saline (PBS) solution were exposed to 808 nm lasers to study the photothermal efficiency. The time required for the solution to rise to 41 °C Fig. 1  of NCs (50 μg/mL) and NPs (50 μg/mL) was 240 s and 300 s, respectively. It took longer at lower concentrations (25 μg/mL) to reach to 41 °C, and PBS alone has no appreciable temperature rise (Fig. 1f ). These results indicate that the photothermal converting efficiency of NPs or NCs dispersed in PBS solution is dose-dependent.

Biosafety evaluation of NCs and its heating effects on H. pylori growth
Cell Counting Kit-8 (CCK-8) assay indicated that when human gastric adenocarcinoma epithelial cells BGC-823 cells were cultured with various concentrations of NCs (range: 0-250 μg/mL) for 24 h or 48 h, cell viability was not affected until the concentration reached 200 μg/mL. When the concentration reached 250 μg/mL, an inhibitory effect on cell viability was observed (Fig. 2a). After co-culturing with NCs (300 μg/mL) for 48 h, the BGC-823 cell counts decreased to 50% of that in the control group (Additional file 1: Fig. S2).
Mammalian cell uptake and penetration of NCs and NPs were investigated: BGC-823 cells were cultured with different concentrations of NPs and NCs (0, 25, 50, 100 µg/mL) for 12 h and 24 h. In the NPs group, the cytoplasm was stained blue (indicating endocytosis) at 50 µg/mL at 12 h, and at 25 µg/ml at 24 h. The NCs group showed no cytoplasm staining at 100 µg/mL at 24 h. The results demonstrated that, compared with NPs, NCs showed no internalization into gastric cancer cells even at a high concentration and long incubation time (Fig. 2b), which illustrated the biosafety aspect of NCs as a photothermal hyperthermia agent. In addition to this experiment, we have incubated Prussian blue with NPs and NCs for 12 h, after the washing process, both samples still demonstrated blue color compared to the control group. This result proved that Prussian blue could penetrate PEG membrane and enter inside NCs, therefore when NCs infiltrated into cells they could be identified by dyed blue stainings (Additional file 1: Fig. S3).
We further quantified the NCs and NPs cellular uptakes in BGC-823 cells by measuring iron (Fe) content, through inductively coupled plasma mass spectrometry (ICP-MS). After 24 h, no significant difference in Fe content was observed between control group and NCs co-cultured group. In contrast, the ICP-MS showed a significant increase in the uptake of NPs in BGC-823 cells after 24 h of incubation, and Fe concentration was timedependent (Additional file 1: Fig. S4).
We then investigated the photothermal effects of NCs on H. pylori growth and bacteria toxicity. A NIR laser at 808 nm was applied to NPs and NCs to heat the solution. The real-time monitoring of temperature in the culture solution during the laser irradiation was performed using a thermometer placed in the solution. Bacterial colony counts and OD 600 values measured by a spectrophotometer were used for representing the growth of bacteria. The growth inhibition rate of H. pylori after NCs photothermia increased monotonically in a temperature-dependent and time-dependent way, as shown in Additional file 1: Fig. S5. Based on the photothermal efficiency and cell uptake test results, we chose a concentration of 50 µg/mL NPs or NCs in the planktonic H. pylori cultures which were then applied with laser irritation to increase the temperature to 41 °C for 20 min. NCs heat treatment group showed a significant inhibition to growth of H. pylori, with the cell growth of 6.7% that of the control group (n = 3, p < 0.001) (Fig. 3a). NCs without NIR irradiation group and the 41 °C water bath group had no influence on bacterial growth (Fig. 3a). The inhibitory efficiency of NCs group was superior to the NPs groups (Additional file 1: Fig. S6). Two clinical H. pylori stains (No. 27054 and No. L2) treated by laser irradiated NCs heating also demonstrated similar results (Fig. 3b and c). It is also notable that H. pylori growth inhibition after NCs heating was both concentration-dependent and time-dependent: the 50 µg/mL group showed a better inhibitory effect than the 25 µg/mL group, and the inhibitory effect in 20 min group was higher than in the 10 min group (Fig. 3d-f ). While H. pylori cells growth was inhibited, the bacterium itself experienced several changes limiting its toxicity. In H. pylori 11637, the NCs photothermia group showed significantly lower adhesion ability of H. pylori cells compared with the control group H. pylori amount was measured by both colony count (CFU/ml) and absorbance of OD 600 . NCs plus heating group demonstrated significant inhibition effect on H. pylori growth in all strains, *** p < 0.001, compared with control. In contrast, interventions such as NIR, NCs without NIR irradiation, water bath at 41 °C, showed no growth inhibition. Right panel: growth of d NCTC 11637, e clinical strain 27054, f clinical strain L2 after different heating time and concentration of NCs. In equal heating time, NCs in higher concentration illustrated better H. pylori inhibition effects. When the NCs concentrations were equivalent, more heating time equaled better effects. The group of 50 ug/mL NCs photothermia at 41 °C for 20 min exhibited the best efficiency (n = 3), ** p < 0.01 (n = 3, p < 0.001). NC photothermia groups also exhibited less cell vacuolization, with a 29.2% reduction compared with the control group (n = 3, p < 0.001) (Additional file 1: Fig. S7).

Effect of NCs photothermia on the morphology of H. pylori
The effects on the morphology of H. pylori by NCs photothermia can be visualized through TEM. In the control sample, typical appearances of the H. pylori bacteria can be seen, with most having intact outer and inner membrane, complete ribosomes, and uniform cytoplasm (Fig. 4, control). In the NCs photothermia sample, the addition of heat resulted in a partial disappearance and thinning and shrinking of the outer membranes, abnormal distribution or disappearance of internal structures such as ribosome, and leakage of cytoplasmic contents (Fig. 4, NCs + heating).

Induced changes in antimicrobial susceptibility of H. pylori
We tested NPs and NCs heating on H. pylori strains using the E-test strip, in order to test its susceptibility to antibiotics (levofloxacin, clarithromycin and metronidazole). The minimum inhibitory concentration (MIC) for levofloxacin of 11637 was 1.4 µg/mL (control group). After photothermal heating at 41 °C for 20 min, MIC of H. pylori to levofloxacin decreased to 0.3 µg/mL in the NPs group and 0.8 µg/mL in the NCs group, which were below the breakpoint of levofloxacin resistance (1.0 µg/mL) (European-Committee. 2021). Two levofloxacin-resistant clinical strains were also tested (27054 and L2), they were no longer resistant to levofloxacin after photothermia process. The MIC for clarithromycin of a clarithromycin-resistant clinical stain (27054) decreased after photothermia, but the value was still above the breakpoint of resistance. H. pylori NCTC 11637 strain and clinical strain (L2) were sensitive to clarithromycin before the heating, and this did not change after photothermia (Table 1, Additional file 1: Fig. S8). All of these clinical strains remained metronidazole-resistant before and after heating.

Biofilm and efflux pump function test of H. pylori after NCs photothermia
We therefore investigated whether biofilms alteration played a role in the decrease of H. pylori antimicrobial sensitivity in our NC experiments.
Crystal violet staining ( Fig. 5a and b) and confocal laser scanning microscopy (CLSM) (Fig. 5c) were used to observe the changes of biofilms produced by NIR heating. Figure 5a shows that biofilms were produced at the air-liquid interface on 12-well plates after 4 days of shaking culture in broth medium supplemented with 7% fetal bovine serum (FBS). Compared with the control group, the bacteria heated by NCs-based NIR produced less biofilms, and the two clinical strains were consistent with NCTC 11637. Biofilms were quantified using a spectrophotometry-based approach, as in Fig. 5b, and the ratio of biofilm to biomass was significantly reduced in H. pylori treated with NIR heating. We also compared the control group with the treatment group by CLSM (Fig. 5c). We added fluorescein isothioyanate concanavalin A (FITC-ConA) and propidium iodide (PI) to both groups, and FITC-ConA would show green fluorescence where biofilm were formed, while PI would display red fluorescence when H. pylori was detected. As can be seen in Fig. 5c, the control group has showed strong green fluorescence and formed large mature biofilms with compact structures. In the treatment group, the green fluorescence was weaker and the biofilms were discontinuous with larger apertures than the control group. Therefore, CLSM also indicated the photothermal effect of NCs exerted on H. pylori biofilms, and this might be a significant factor in the change of drug sensitivity of H. pylori in the experimental group.
In addition, we explored whether the photothermal effect of NCs could affect the efflux pumps of H. pylori. As shown in Fig. 6, we did not find any difference in accumulation of Hoechst 33342 between the experimental (NCs + heating) group and the control group, indicating that the photothermal effect of NCs has no effect on the function of efflux pumps, in contrast to the high fluorescence in CCCP group caused by suppressed efflux pump function.

Discussion
As the essential elements in PTT, photothermal agents can transform light energy into heat to cause membrane rupture, protein denaturation and irreversible bacterial destruction . Metallic compound nanocomposites (metal sulfide/ oxide) have been investigated as bactericidal agents, due to their source variety, high stability, semiconductor characteristics, good biocompatibility, easy preparation,  L2). b Changes of biofilm-to-biomass ratios of H. pylori NCTC 11637, clinical strain 27054 and L2. Bar graph represents mean biofilm-to-biomass ratios (A 580 /A 600 ± standard error of the mean). ** p < 0.01, vs. the control group, n = 3. c Confocal laser scanning microscopy (CLSM) images of H. pylori NCTC 11637 strain biofilms. Cells stained with FITC-ConA (green) which represented the biofilm and PI (red) which represented H. pylori were visualized by confocal microscopy (400 ×), scale bars 25 μm low cost, and photothermal conversion efficiency. It is reported that low concentrations (10 mg/mL) of ZnFe 2 O 4 -rGO nanostructures under NIR irradiation for a short time period (~ 1 min) exhibited efficient photothermal effects and resulted in effective destruction of cancer cells in in vitro investigation (Akhavan et al. 2014). This highlights the high thermal conversion efficiency of zinc-iron oxide nanostructures, reports have also stated its excellent magnetic thermal conversion efficiency compared to ordinary Fe 3 O 4 (He et al. 2018). In this study, the photothermal effect of NCs tended to be more effective and efficient than that of NPs of Zn 0.3 Fe 2.7 O 4 . Zinc oxide nanoparticles have been researched for clinical applications, it demonstrated antiproliferation efficacy in A549 (human lung adenocarcinoma) cells, and with hyperthermia, they revealed cytotoxicity against breast cancer cells and bone cancer cells (Kim et al. 2018;Vimala et al. 2017). In addition, in vivo study to evaluate the efficacy of zinc oxide nanoparticles as an anti-tumor agent demonstrated that rats with hepatocellular cancer treated with zinc oxide had a significant reduction in serum tumor markers such as alpha-fetoprotein, relative to the control group (Hassan et al. 2017). The physiological gastric environment may be hostile to NCs due to its acidic nature, however, with the prescription of proton pump inhibitors (PPI) the environment can be adjusted to a neutral pH value (6-7) (Freedberg et al. 2014). These results indicate the promising possibilities of photothermal therapy in clinical application. By clustering the nanoparticles, we believe that there will be an increased absorption of the indicated radiation, leading to a more efficient conversion of energy into heat than individual nanoparticles. Our results in this study demonstrated that Zn 0.3 Fe 2.7 O 4 NCs exhibited better photothermal performance than NPs did. Similar biocompatible nanoclusters were developed, with CoMn-iron oxide nanoparticles clustered inside a poly(ethylene glycol)-b-poly(ε-caprolactone) (PEG-PCL)-based nanocarrier. The nanoclusters were found to be safe and exhibited high heating efficiency, which can elevate the intratumoral temperature to 44 °C in the presence of a safe alternating magnetic field (Albarqi et al. 2019). Reliable biosafety in the human body is essential for any treatment. Good PTAs should have minimal toxicity and maximal biocompatibility. Iron oxide nanoparticles (IONs) stand out as suitable for PTT because of their biocompatibility, biodegradability, simple synthesis, and the ease with which they may be adapted and functionalized for specific applications (Revia and Zhang. 2016). Despite the potential of PTT in antibacterial use, its non-specific heat damage to nearby tissues is potentially concerning. Unlike intracellular hyperthermia of tumors when internalization of nanoparticles into cells is crucial, it is important to avoid adjacent tissue damage during antibacterial applications. This will require prevention of individual nanoparticles being endocytosed by gastrointestinal mucosa cells. In this study, we evaluated biosafety by its invasion on mammalian cell viability and penetration. In order to minimize non-specific heating of healthy tissues, we proposed a controlled clustering of nanoparticles, which will increase the hydrodynamic size of the particles and in turn reduce the chance of internalization into adjacent cells. As studies have successfully demonstrated nanoparticles up to 5-10 µm entering human tissue, there does not seem to be an absolute limit in size that prevents internalization (Gratton et al. 2008). That being said, a smaller nanoparticle is generally considered to have a faster internalization than larger particles with the same charge and clinical composition, one study stated that nanoparticle will encounter difficulty entering cells when the size is greater than 60 nm, due to cellular receptor shortage and entropic penalty (Debbage and Jaschke. 2008;Hoshyar et al. 2016;Sahay et al. 2010). Focusing specifically on gastric cells, smaller polysterene nanoparticles (44 nm) showed a faster and higher proportion of internalization than larger polysterene nanoparticles (100 nm) (Forte et al. 2016). Therefore, by clustering the nanoparticles, we theorized a reduced internalization into adjacent tissues, thus leading to a lower risk of non-specific heating. The ICP-MS results confirmed that NCs could not penetrate into cells easily and demonstrated good biosafety, and this was consistent with our Prussian blue staining findings. These findings could be explained by the size of NCs (ours is 130.1 ± 5.2 nm) which made it difficult to penetrate mammalian cell membrane.
Declining eradication rates of H. pylori worldwide related to antimicrobial resistance have inspired a switch from antibiotic-based regimens to novel therapeutic strategies such as nanoparticle-based approaches. In recent years, nanomaterials have exhibited great potential in the growth inhibition of H. pylori (Yang et al. 2020;Zhang et al. 2020). Besides, the photothermal anti-H. pylori effect of nanomaterials was evaluated effectively in vitro and in vivo (Zhi et al. 2019). In this study, H. pylori growth was significantly inhibited after NCs photothermia, while TEM visualized outer wall rupture and ribosomal damage of H. pylori cells, which can be indicative of the antibacterial effects of this therapy. H. pylori adhesion ability is particularly crucial for its virulence, since adhesion molecules on its surface are responsible for mucin binding, leading to gastric mucosa invasion (Huang et al. 2016). Vacuolization is also significant in H. pylori's toxicity, vacuolating cytotoxin (VacA) itself is a major virulence factor (Palframan et al. 2012). In our study, the NCs photothermia groups showed significantly lower adhesion ability of H. pylori cells and reduced cell vacuolization compared with the blank control group. This shows nanoparticle photothermia could be an efficient method to combat H. pylori for potential clinical use, both on its growth restriction and on molecular toxicity inhibition.
Alteration of antimicrobial susceptibility was reported after hyperthermia on Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, and our previous study found that NPs magnetic local heating could not only disrupt H. pylori cell directly, but could also enhance its susceptibility to amoxicillin, of which could be adopted for clinic applications readily (Alumutairi et al. 2020;Fang et al. 2017;Ma et al. 2019;Nguyen et al. 2016;Wu et al. 2019). One crucial mechanism of drug resistance is the formation of biofilms. Biofilms can be described as adherent aggregates of microorganisms encased in an extracellular polymeric substance (EPS), which contains proteomannans, LPS-related structures, extracellular DNA, proteins, and outer membrane vesicles (Flemming et al. 2016;Hathroubi et al. 2018). In addition, biofilms are associated with increasing mutations (to interfere with the antimicrobial activities), quorum-sensing-regulated mechanisms and the activation of the general stress response. This activity makes it difficult to achieve effective antibiotic activity (Høiby et al. 2010). H. pylori is a biofilm-producing bacteria: it forms biofilms on the surface of the gastric mucosa (adopting the biofilm mode of growth to the mucosa and glands and can cover up to 97.3% of cell surface) and it displays resistance to antibiotics including clarithromycin (Hathroubi et al. 2018). Specifically, the MIC increased 16-fold and the minimum bactericidal concentration increased by up to fourfold in biofilm cells compared to planktonic ones (Alumutairi et al. 2020). In general, relatively little is known in the field of H. pylori's biofilm structure and genes, and the therapies aiming to dissolve biofilms are still in their infancy (Yonezawa et al. 2015). Our findings provide a novel way to target H. pylori antibiotic resistance by the inhibition of biofilm formation.
Carbonyl cyanide m-chlorophenylhydrazone (CCCP), as a transmembrane proton gradient-collapsing agent, is a frequently used proton-transporting ionophore that transmits protons to disrupt transmembrane electrochemical gradient and inhibits the efflux pumps driven by hydrogen ion gradients, and thus restoring the antibiotic sensitivity of bacteria, including H. pylori (Fanelus and Desrosiers. 2013;Zhang et al. 2010). Hoechst 33342, a fluorescent probe, is a substrate for a wide range of bacterial multidrug resistance transporters (Coldham et al. 2010;van den Berg van Saparoea et al. 2005), it can penetrate into H. pylori and can be extruded by efflux pump, thus its fluorescence represents not only the amount of Hoechst 33342 in the bacteria, but also the efficiency of efflux pump too. The involvement of efflux pump in multidrug resistance of H. pylori has been identified (Bina et al. 2000;Cai et al. 2020;Liu et al. 2008;van Amsterdam et al. 2005). There is no evidence in this study that the function of efflux pumps was affected by NCs photothermia, however, the thinning of the outer membrane may be indicative of the mechanism behind increased antimicrobial sensitivity and the growth inhibition of H. pylori.
There was potential limitation to this measurement: although the temperature of the mixture was kept at 41 °C, the interior of NCs and NPs were likely to be much hotter than the macroscopic solution. Studies showed that the temperature around nanoparticles could be detected by using real-time molecular temperature probes and found to decrease exponentially with the increase of distance from the surface of nanoparticles (Riedinger et al. 2013). Acute temperature monitoring of the immediate nanoenvironment around nanoparticles may help to optimize local temperature controls for physical and biomedical applications. As the cooperative effects of antibiotics and NCs heating may reduce the dose of antibiotics required in the eradication of H. pylori greatly, it will be of great interest to couple antibiotics with NCs which can be localized to gastric area and then optimize the synergistic effects. Besides, for the envision that this approach can be applied to clinic applications, in vivo studies are needed to investigate both the anti-H. pylori effects of NCs photothermia and its biosafety.

Conclusions
In this study, we successfully designed NCs formed by PEG-PCL-based NPs and demonstrated antibacterial effect of NCs/NPs under NIR laser heating for the first time. We evaluated the biosafety of NCs and NPs, finding that NCs have higher biosafety compared with NPs. We found that NCs could inhibit the growth of H. pylori significantly under NIR laser heating. In addition, after NCs local heating, we found MIC of H. pylori to levofloxacin and clarithromycin to be decreased. Investigating further, we demonstrated for the first time that the photothermal effect of NCs disrupted H. pylori biofilm formation in vitro. Considering the current data, our research provides a new method for treating H. pylori infection, especially for drug-resistant strains.
Additional file 1: Fig. S1. The schematic illustration of synthesis of Zn0.3Fe2.7O4 NCs. Fig. S2. Effects of NCs on BGC-823 cells growth. NCs were added into the culture medium and cells were observed by inverted microscope (40×) after co-culturing for 48 h. (A) Control group; (B) NCs (300 μg/mL) group exhibited significant growth inhibition of BGC-823 cells. Fig. S3. Prussian blue staining of NPs and NCs. NPs and NCs were incubated with Prussian blue for 12h, then washed and observed. NPs/NCs in the control group (no incubation) showed brownish grey. After incubation with Prussian blue, both NPs and NCs were stained blue (400×). Fig. S4. Cellular uptake of NPs/NCs in co-cultured BGC-823 cells as quantitatively determined by ICP-MS.*** p < 0.001, vs. the control group, n = 3. When co-cultured with NCs, no significant difference was found compared to the control group in Fe content. However, when co-cultured with NPs, a significant increase in Fe content was measured for all exposure time. Fig. S5. Effect of Zn0.3Fe2.7O4 NCs (50 μg/mL) photothermia on H. pylori growth inhibition. Percentage of H. pylori growth inhibition (A) as a function of temperature, t = 20 min; (B) as a function of time, temperature = 41 °C. Fig. S6. Comparison of H. pylori NCTC 11637 growth inhibition between NPs and NCs heating. NCs (50 µg/mL) group showed higher growth inhibition rate (%) than NPs (50 µg/mL) group after heating induced by NIR laser to 41℃ for 20 min (n = 3). Fig. S7. Effects of NCs heating on H. pylori NCTC 11637 vacuolization and adhesion ability. Under the near-infrared (808nm) irradiation to 41℃ for 20 min, the NCs+heating group showed significant inhibition of vacuolization and adhesion ability, compared with control group (*** P<0.001). The near infrared group, NCs no heating group and water bath (41℃) group showed no difference (n = 5). Fig. S8. Changes of H. pylori NCTC 11637 antibiotic susceptibility to levofloxacin. The minimum inhibitory concentration (MIC) for levofloxacin was 1.4 µg/ml in control group (A), 0.3 µg/ mL in the NPs heating group (B), and 0.8 µg/mL in the NCs heating group (C).