Antimicrobial Effect of Biocompatible Silicon Nanoparticles Activated


Feb 17, 2017 - In this study, we report a method for the suppression of Escherichia coli (E. coli) vitality by means of therapeutic ultrasound irradia...
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Antimicrobial effect of biocompatible silicon nanoparticles activated by therapeutic ultrasound Svetlana N. Shevchenko, Markus Burkhardt, Eugene V. Sheval, Ulyana A. Natashina, Christina Große, Alexander L. Nikolaev, Alexander V. Gopin, Ute Neugebauer, Andrew A. Kudryavtsev, Vladimir Sivakov, and Liubov Andreevna Osminkina Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.6b04303 • Publication Date (Web): 17 Feb 2017 Downloaded from http://pubs.acs.org on February 21, 2017

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Antimicrobial effect of biocompatible silicon nanoparticles activated by therapeutic ultrasound Svetlana N. Shevchenko†, Markus Burkhardt‡, Eugene V. Sheval§, Ulyana A. Natashina†, Christina Grosse‡,⊥, Alexander L. Nikolaev║, Alexander V. Gopin║, Ute Neugebauer‡,⊥, Andrew A. Kudryavtsev∇, Vladimir Sivakov*,‡, and Liubov A. Osminkina*,†,O



Lomonosov Moscow State University, Department of Physics, Leninskie Gory 1, 119991 Moscow, Russian

Federation ‡ §

Leibniz Institute of Photonic Technology, Albert Einstein Str. 9, D-07745 Jena, Germany Lomonosov Moscow State University, A.N. Belozersky Institute of Physico-Chemical Biology, Leninskie

Gory 1, 119991, Moscow, Russian Federation ⊥ ║

Center for Sepsis Control and Care, Jena University Hospital, Erlanger Allee 101, 07747 Jena, Germany Lomonosov Moscow State University, Department of Chemistry, Leninskie Gory 1, 119991 Moscow,

Russian Federation ∇ Institute

of Theoretical and Experimental Biophysics, Russian Academy of Science, Pushino, 142290,

Russian Federation O

National Research Nuclear University MEPhI, Kashirskoe sh. 31, 115409 Moscow, Russian Federation

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ABSTRACT: In this study, we report on a method for suppressing of Escherichia coli (E.coli) vitality by means of therapeutic ultrasound irradiation (USI) using biocompatible silicon nanoparticles as cavitation sensitizers. Silicon nanoparticles without (SiNPs) and with polysaccharide (dextran) coating (DSiNPs) were used. Both types of nanoparticles were non-toxic to Hep 2 cells up to concentration of 2 mg/ml. The treatment of bacteria with nanoparticles and application of 1 W/cm2 USI resulted in reduction of their viability up to 35% and 72% for SiNPs and DSiNPs, respectively. The higher bacteria viability reduction for DSiNPs as compared to SiNPs can be explained by the fact that the biopolymer shell of polysaccharide provides a stronger adhesion of nanoparticles to the bacteria surface. Transmission electron microscopy (TEM) studies showed that the bacteria lipid shell is partially perforated after combined treatment of DSiNPs and USI, what can be explained by the lysis of bacterial membrane due the cavitation sensitized by the silicon nanoparticles. Furthermore, we have shown that 100% inhibition of E.coli bacteria colonies growth is possible by coupling treatments of DSiNPs and USI with increased intensity up to 3 W/cm2. The observed results reveal an application of SiNPs as promising antimicrobial agents.

Keywords: Silicon nanoparticles, Escherichia coli, antimicrobial effect, therapeutic ultrasound, sonosensitizer.

TOC image. Schematic representation of antimicrobial effect of silicon nanoparticles activated by therapeutic ultrasound.

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INTRODUCTION

The severity of bacterial disease, the lack of effective treatment and the potential for largescale outbreaks have propelled an intensive research focus in order to suppress the bacterial activity via novel methods1. As it is well-known, E.coli bacteria are mostly harmless. However, disease-associated serotypes such as O157:H7, O121, O104:H21 and O104:H4 are capable of producing lethal toxins. One of the most frequent diseases caused by E.coli bacteria toxins (Shiga toxins) is diarrhea associated with the hemolytic-uremic syndrome and neurologic complications2-5. This type of bacteria is gram-negative and has a thick cell wall containing peptidoglycan, lipoprotein, phospholipid and lipopolysaccharide for protection from the environment2-5. The growth of bacteria resistance to antibiotics and the lack of bacteria treatment possibilities lead to the development of completely new methods of suppressing bacteria activity, using different types of nanoparticles6. The antibacterial properties of silver nanoparticles (AgNPs) are well-known7-9. It has been shown that copper nanoparticles (CuNPs) have also a significant potential as bactericidal agents10. Silicon nanowires (SiNWs) decorated by metallic nanoparticles demonstrated antibacterial activity against E.coli bacteria (86% and 94% of bacteria were destroyed for СuNPs and AgNPs, respectively)11. Some other types of nanoparticles synthesized from gold, MgO, TiO2, ZnO, NiO and aluminum oxide can also inhibit various bacteria growth like E. coli, Staphylococcus aureus, Salmonella enteritidis, etc.12-16. A typical disadvantage of using nanoparticles is rather high cytotoxicity. In particular, it was found that metal and metal oxide nanoparticles, which had been implanted into mammalian cells, caused alteration of the normal function of mitochondria, the increase of membrane permeability and the generation of reactive oxygen species which resulted in oxidative stress and cellular damage7,17-21. However, nanoparticles, that are biocompatible and biodegradable (it strongly depends on their physical-chemical properties), can potentially be used for in vivo applications22. In the present study, we have examined antimicrobial properties of silicon nanoparticles (SiNPs) in coupled action with therapeutic ultrasound irradiation (USI). From previous in vitro and in vivo studies it is well-known that SiNPs have low cytotoxicity and have proved to be

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biodegradable

23-28

. The biodegradation of SiNPs is mainly caused by gradual dissolution in water

with the formation of non-toxic silicic acid25,27,28. Moreover, SiNPs low genotoxicity and teratogenicity have been shown in vivo

29

. As regards biomedical applications of SiNPs, they can

be used for bioimaging of cells and tissues

25,30-33

, for drug delivery applications33-35 and for

photodynamic and photothermal therapy36-38. Nowadays USI plays a key role in a wide range of novel applications for diagnosis and therapy of various diseases39. The penetration depth of ultrasound through the biological tissue is rather high in comparison to light, which opens treatment ability of deeply located diseases40 with USI. To enhance the therapeutic effect of USI at relatively low intensity in the range of 1-10 W/cm2, various types of nanoparticles-sonosensitizers are used40-42. Recently, it has been found that SiNPs could act as sonosensitizers for destroying cancer cells both in vitro and in vivo43-45. Thus, the sonosensitizing properties of SiNPs together with their high biocompatibility and biodegradability make them promising candidates for using as antimicrobial agents. In the present paper, we report on a method for lysis of E.coli cell wall by using a combined action of biocompatible SiNPs and therapeutic ultrasound irradiation. The proposed approach opens a new way of using biocompatible SiNPs as promising antimicrobial agents for the treatment of antibiotic resistant bacteria.

EXPERIMENTAL SECTION Silicon Nanostructures Synthesis. SiNPs were fabricated by ultrasound grinding of silicon nanowires (SiNWs) in distilled water. SiNWs were prepared by metal-assisted chemical etching (MACE) of heavily boron-doped (doping level 1020 cm−3; (100)-oriented single crystalline Si wafers). MACE method to produce SiNWs is based on a two-step process45. Firstly, thin (~100 nm) layers of Ag nanoparticles of different morphology were deposited on the Si substrates by immersing them in aqueous solution of 0.02M of silver nitrate (AgNO3) and 5M of HF in the volume ratio of 1:1 for 20 sec. Secondly, the Si substrates covered with Ag nanoparticles were immersed in the solution containing 5M of HF and 30% H2O2 in the volume ratio of 10:1 in a teflon vessel for 60 min. The etching was performed at room temperature. Then SiNW arrays were rinsed several times in de-ionized water and additionally immersed in concentrated (65%) nitric acid (HNO3) for ACS Paragon Plus Environment

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15 min to remove residual Ag nanoparticles from the SiNWs. Finally, the samples were rinsed several times in de-ionized water and dried at room temperature. To obtain SiNPs solution, the surface with SiNWs was placed into deionized water and treated in an ultrasonic bath at a frequency of 37 kHz for 4 hours. Afterward, the suspension was centrifuged for 3 min at 2000 rpm and the resulting supernatant was used. The SiNPs concentration of the final suspension was calculated by gravimetric method. Silicon Nanoparticles Coating with Dextran. Dextran-coated SiNPs (DSiNPs) were prepared by mixing of 1 ml of the aqueous suspension of SiNPs with concentration of 1 mg/ml and 1ml of aqueous dextran solution (30000–40000 mol. wt, 10% dextran, 0.9% NaCl) (EAST-PHARM, Russia). The mixture was homogenized by sonification in an ultrasonic bath at 37 kHz for 1 h and stored in the dark for 24 h before the in vitro experiments. Silicon Nanoparticles Characterization. Microstructural investigations of SiNPs and SiNWs were carried out by using a field emission scanning electron microscope (Carl Zeiss ULTRA 55, FE-SEM) and a transmission electron microscope (LEO912 AB OMEGA, TEM). N2 sorption (Micromeritics Tristar II 3020) at 77 K was used for the characterization of the porous structures of the SiNPs. BET (Brunauer–Emmett–Teller) theory46 was used to calculate surface areas while BJH (Barrett–Joyner–Halenda) analysis47 was applied to determine the pore size distributions and average pore diameters. Before measurements SiNPs suspension was dried by evacuation. A Malvern Zetasizer Nano ZS instrument was used to determine zeta potential (ZP) and the size of SiNPs from the dynamic light scattering (DLS) data. Mammalian Cell Viability. The cytotoxicity of SiNPs and DSiNPs was assessed through the use of Hep-2 human lung cancer cell line. The cells were cultured in culture flask of 25 cm2 in DMEM/F12 supplemented with 2 ml/l gentamicin, 10 mМ Hepes, 10% FBS Gibco®, and incubated at 37°C with 5% CO2. Then, the cells were seeded into 48 well plate at 0.3 ml per well at a density of 5 × 104 cells/ml. After 48h, the cell culture medium was replaced by culture medium which contained SiNPs or DSiNPs at different concentrations from 15 µg/ml to 2 mg/ml, and incubated for 2h. The reference cell group was incubated without nanoparticles. The wells then were washed twice with Hanks' balanced salt solution (HBSS) (Sigma, standard concentration, pH=7.3), and the cells were removed from the surface of the wells by trypsinization. Whereupon, 1 ml of HBSS ACS Paragon Plus Environment

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supplemented with 10% FBS Gibco®, containing 5 mM of 10(6) -Carboxyfluorescein diacetate Nsuccinimidyl ester (CFSE), (Sigma) and 50 µg/ml Propidium iodide (Biotium) were added to each well and then maintained for 15 minutes at 37°C. The number of cells in each well was determined on flow cytofluorimeter of Partec PAS III with the excitation wavelength of 488 nm. The results were statistically processed applying Student's t-test with certainty of 0.95. Antimicrobial Effect Assay. In the present paper we have used two different E.coli strains: ATCC® 25922TM and JM109. Bacterial culture strain ATCC® 25922TM was grown in tryptic soy bean broth at 37°C, while strain JM109 was grown in LB (Luria-Bertani) liquid medium at the same temperature. Then the diluted culture with the approximate 108 bacteria per milliliter (the optical

density (OD) was measured at 600nm (OD600) with an Agilent Cary 60 UV-Vis spectrometer to estimate the E.coli bacteria concentration) was mixed with SiNPs or DSiNPs and incubated at room temperature for one hour before USI treatment. To evaluate the effect of ultrasound irradiation and nanoparticles on the E.coli bacteria viability, the obtained suspension was exposed for 10 min with USI using Sonoplus 490 (Firma Enraf Nonius) equipment (1 MHz, 1 W/cm2) or with Albedo 037 equipment (0.88 MHz, 3 W/cm2) (for both ATCC® 25922TM and JM109 E.coli strains). Degassed distilled water (at 35°C) was used as contact medium between flat emitters with a radius of 2 cm and cuvette filled with the sample. Next, the samples were serially diluted and 100 µl aliquots of each suspension was spread-plated on solid tryptic soy bean/LB medium and kept for 18 hours at 37°C. Bacterial viability was evaluated by counting the number of colonies established on the plate. Bacteria not treated with USI and SiNPs were considered as negative controls in each experiment. Statistics and Data Analysis. Statistical data were collected and presented as mean ± standard deviation (SD). Statistical significance was calculated using a one-way ANOVA (GraphPad® software and OriginPro 2017® software). Transmission Electron Microscopy Studies. For the electron microscopy studies of E.coli (JM109 E.coli strain) the samples were fixed in 4% glutaraldehyde (Sigma) in 0.1M Sorensen buffer, postfixed in 1% OsO4, dehydrated (70% ethanol contained 2% uranyl acetate) and embedded in Epon 812 (Fluka). After Epon polymerization, the cells were cut into ultrathin sections using an LKB Ultratome III. The sections were stained with lead citrate and examined using ACS Paragon Plus Environment

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electron microscope JEM1400 (JEOL).

RESULTS AND DISCUSSION Figure 1a shows a cross-sectional SEM micrograph of SiNWs array. Typically, the length of SiNWs was about 10 µm after 20 min etching time. As it can be seen from the TEM image (inset in Figure 1a), the individual SiNW has highly porous microstructure.

Figure 1. Microscopy studies. Overview of SiNWs, SiNPs and DSiNPs. (a) Cross-sectional SEM micrograph of SiNWs array on c-Si, the inset shows TEM micrographs of the porous structure of the individual SiNW; (b) TEM micrograph of SiNP; (c) TEM micrograph of DSiNP; (d) size distribution of SiNPs (in blue) and DSiNPs (in red) obtained by DLS.

The TEM micrographs of SiNPs, fabricated by ultrasound grinding of SiNWs in water, is presented in Figure 1 b,c. The typical size of nanoparticles measured by TEM was about 80-100 nm and 100-150 nm for SiNPs and DSiNPs, respectively. The average nanoparticles size increased obviously due to the dextran coating. The porous microstructure of the nanoparticles is clearly visible from TEM studies as shown in Figure 1 b,c. According to BET and BJH data, SiNPs are characterized of average pore diameter of 13.4 nm, specific surface area of 200 m2/g and pore volume of 0.53 cm3/g. The size distributions of nanoparticles measured by DLS are shown in

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Figure 1d. The predominant diameter of SiNPs and DSiNPs was found to be 80 nm and 105 nm, respectively. These data fully correlated with the results obtained by transmission electron microscopy (see Figure 1 b,c). Polydispersity index (PDI) of the nanoparticles was determined by DLS48 and accounted 0.5 both for SiNPs and DSiNPs . ZP of SiNPs was negative and accounted –25±2 mV. It was found that the value of ZP for DSiNPs was –20±2 mV. The small difference between the ZP values of SiNP and DSiNP indicates a weak interaction between dextran and nanoparticles. This interaction can be covered by the Vander-Waals forces between adsorbed dextran and oxidized surface of SiNP44. Figure 2 shows the dependence of the viability of Hep2 cells, normalized to the control cell group, which was incubated without nanoparticles, on the nanoparticle concentration. It can be seen that the cytotoxic effect is nearly absent both for SiNPs (blue bars) and for DSiNPs (red bars) at concentrations up to 2 mg/mL at 2h of incubation. The uncoated SiNPs demonstrate slightly stronger cytotoxicity as compared to DSiNPs, what can be explained by the well-established biocompatibility of dextran49.

Figure 2. Cytotoxicity of silicon nanoparticles. In vitro toxicity of SiNPs (blue bars) and of DSiNPs (red bars) towards Hep2 cells after 2 hours of incubation. Error bars represent mean ± SD, n = 3.

The nanoparticles toxicity against E.coli (ATCC® 25922TM and JM109 strains) is shown in Figure 3a. This experiment has been performed by mixing bacteria culture E.coli (108 cells/ml) with ACS Paragon Plus Environment

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different concentration of SiNPs or of DSiNPs, and further incubation for 18h. Pure bacteria culture without silicon nanostructures was used as a control group, and the presented in the Figure 3a E.coli viability is normalized to the control group. As it is clearly visible, SiNPs were non-toxic to E.coli bacteria up to the concentration of 2 mg/ml. DSiNPs demonstrate here slightly stronger toxicity in comparison to SiNPs, what can be explained by better adhesion of dextran-coated nanoparticles on bacteria cell wall.

Figure 3. In vitro toxicity of SiNPs, DSiNPs or USI treatment towards E.coli bacteria (ATCC® 25922TM and JM109 strains). (a) In vitro toxicity of SiNPs (blue bars) and DSiNPs (red bars) towards E.coli bacteria; (b) E.coli bacteria viability in the control group (blue bar) after 10 min of 1 W/cm2 USI treatment and after 10 min of 3 W/cm2 USI treatment (cyan bars). Error bars represent mean ± SD, n = 3, *p˂0.05 compared to the control.

The experimental results of E.coli (for ATCC® 25922TM and JM109 E.coli strains, 108

cells/ml) viability after 10 min of USI treatment with intensities of 1 W/cm2 and 3 W/cm2 are presented in Figure 3b. It is clearly visible that the USI with intensity of 1 W/cm2 did not affect the E.coli bacteria colony viability in comparison with the control group without USI treatment (blue bar). On the other hand, the treatment of E.coli bacteria colonies with ultrasound of higher intensity (3 W/cm2) resulted in inhibition of the bacteria colonies growth, which corresponded to the viability lowered to 70%. This sonotoxicity effect at higher USI intensity can be explained by cavitation bubble formation50. 8

Figure 4a shows E.coli bacteria (ATCC® 25922TM and JM109 strains, 10 cells/ml) viability experimental results after bacteria were treated with 1 mg/ml SiNPs or DSiNPs followed by USI ACS Paragon Plus Environment

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irradiation with intensity of 1 W/cm2 and/or 3 W/cm2 and plating of the bacterial suspension afterwards. In these experiments E.coli without SiNPs and USI treatment were used as a control group (blue bar in Figure 4a). The 10 min USI treatment with 1 W/cm2 of the E.coli bacteria in the presence of nanoparticles resulted in an inhibition of the bacteria colonies growth, which corresponded to a drop of the viability up to 35% and 72% for SiNPs and DSiNPs, respectively (yellow and green bars in Figure 4a). The better results of DSiNPs in comparison to SiNPs can be explained by the fact that biopolymer shell of DSiNPs provides their stronger adhesion to the bacterial surface; therefore, the bacteria surface suffers more from cavitation processes. Moreover, the significant viability decrease of E.coli bacteria was observed after combined action of nanoparticles and higher USI intensity (3 W/cm2) (magenta and red bars in Figure 4a). The obtained results are confirmed by digital photos of Petri dishes of E.coli bacteria (ATCC® 25922TM stain) in the control group, after 1h exposure with 1 mg/ml DSiNPs without USI, after 10 min 3 W/cm2 USI treatment without SiNPs, and after 10 min combined treatment of 1 mg/ml DSiNPs and 3 W/cm2 USI, see Figure 4b. No significant changes in the number of bacteria colonies can be observed after treatment with 1 mg/ml DSiNPs, in comparison to the control group. A minor inhibition of E.coli bacteria growth was noticed after 10 min of 3 W/cm2 USI treatment. At the same time, 10 min of combined treatment of E.coli with 1 mg/ml DSiNPs and 3 W/cm2 USI provides 100% inhibition of bacteria colonies growth. The obtained results are in good agreement with the data presented in Figure 3 a,b and Figure 4a.

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Figure 4. In vitro toxicity of combined treatment of SiNPs or DSiNPs and USI towards E.coli bacteria. (a) E.coli (ATCC® 25922TM and JM109 strains) viability in control group (blue bar), after 10 min combined treatment of 1 mg/ml SiNPs and 1 W/cm2 USI (yellow bar), of 1 mg/ml DSiNPs and 1 W/cm2 USI (green bar), of 1 mg/ml SiNPs and 3 W/cm2 USI (magenta bar), of 1 mg/ml DSiNPs and 3 W/cm2 USI (red bar). The levels of significance between the control and treated group were set at probabilities: *p < 0.0001 compared to the control, **p < 0.001 compared to DSiNPs + USI 1W/cm2, #p < 0.001 compared to SiNPs + USI 3W/cm2,

##

p < 0.001 compared to

DSiNPs + USI 3W/cm2. Error bars represent mean ± SD, n = 3; b) digital photo of Petri dishes with E.coli bacteria (ATCC® 25922TM stain) in control group, after 1 h exposure with 1 mg/ml DSiNPs without USI, after 10 min 3 W/cm2 USI treatment without SiNPs, and after 10 min combined treatment of 1 mg/ml DSiNPs and 3 W/cm2 USI; (c) TEM cross-sectional view of E.coli (JM109 strain) after their mixing with 1 mg/ml DSiNPs; (d) TEM cross-sectional view of E.coli after combined 10 min treatment of 1 mg/ml DSiNPs and 1 W/cm2 USI. The yellow arrows show destruction (lysis) areas in bacteria membranes.

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Typical TEM images of E.coli bacteria (JM109 strain) after their mixing with 1 mg/ml DSiNPs and after combined 10 min treatment of 1 mg/ml DSiNPs and 1 W/cm2 USI are presented in Figure 4 c,d. No changes in E.coli bacteria cell wall structure have been noticed after the bacteria interaction with DSiNPs without USI (see Figure 4c). However, obvious changes in E.coli bacteria wall structure were observed after combined 10 min treatment of 1 mg/ml DSiNPs and 1 W/cm2 USI, as clearly visible in Figure 4d. The cell walls are partially perforated (noted by yellow arrows in Figure 4d) what can be explained by the lysis of bacterial membrane due the cavitation sensitized by nanoparticles. Therefore, in the present study for the first time the antimicrobial effect of biocompatible SiNPs activated by therapeutic ultrasound (1-3 W/cm2) has been studied. The proposed of lysis phenomenon of E.coli bacteria in presence of SiNPs and USI discussed above is schematically shown in Figure 5.

Figure 5. Schematic representation of antimicrobial effect of SiNPs activated by therapeutic ultrasound. Step I: Localization of SiNPs on the bacteria cell wall. Step II: SiNPs sensitized cavitation process, which consists of the bubble nucleation, growth and collapse (shown as blue bubbles and red stars, correspondently). Step III: perforation (lysis) of bacteria which appears after SiNPs and USI combined treatment.

We suggest that porous SiNPs, and especially DSiNPs can stick to the surface of E.coli bacteria. Under ultrasound irradiation the nanoparticles induce nucleation of cavitation bubbles, ACS Paragon Plus Environment

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their growth and collapse43. As a result, the perforation (lysis) of bacteria wall can take place. So, the full suppression of bacteria could be achieved after combined treatment of nanoparticles and USI.

CONCLUSIONS We reported here the method for suppressing of E.coli bacteria vitality using biocompatible SiNPs as sensitizers of the cavitation processes of therapeutic USI (0.88-1 MHz, 1-3 W/cm2). SiNPs with and without polysaccharide (dextran) coating were used. Both types of nanoparticles were found to be non-toxic to Hep-2 cells up to concentration of 2 mg/ml. Also, we found that in itself USI treatment of bacteria without silicon nanoparticles did not affect the bacteria proliferation. At the same time, the combined 10 min treatment of bacteria colonies with nanoparticles and 1 W/cm2 USI resulted in reduction of bacterial viability to 35% and 72% for SiNPs and DSiNPs, respectively. The stronger suppression of E.coli bacteria vitality in the presence of DSiNPs in comparison to SiNPs can be explained by the fact that the biopolymer shell of dextran provides a stronger adhesion of nanoparticles to the bacteria surface. TEM microphotographs showed that the bacteria's walls were partially perforated after combined treatment of DSiNPs and 1 W/cm2 USI, what can be explained by the lysis of the bacterial cell wall due to the cavitation sensitized by the nanoparticles. Moreover, the 100% inhibition of E.coli bacteria colonies growth was observed after combined treatment of the bacteria colonies with silicon nanoparticles and higher USI intensity (3 W/cm2). The discovered effect of full bacteria destruction after combined action of silicon nanoparticles and therapeutic USI is aimed to be universal, since the bacteria are hardly able to acquire any resistance to such type of treatment.

AUTHOR INFORMATION Corresponding Authors

*E-mail: [email protected], [email protected] (L.A.O.) *E-mail: [email protected] (V.S.) ORCID

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L.A. Osminkina: 0000-0001-7485-0495 Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS V.S. is gratefully acknowledges the German Federal Ministry of Education and Research (BMBF) in frame of Baltic Sea Network “NanoPhoto” under Grant No. 01DS14017 for the financial support. The study of nanoparticles cavitation was supported by the Russian Science Foundation (Grant No. №16-13-10145). S.N.S. gratefully acknowledges the financial support of “UMNIK-2013”. L.A.O. greatly acknowledges the financial support of the DAAD program. C.G. and U.N. acknowledge BMBF funding via the CSCC (FKZ 01EO1502). The authors thank Prof. Victor Yu. Timoshenko for the fruitful discussions, Dr. Sergej S. Abramchuk for TEM images, Mr. Konstantin P. Tamarov for BET/BJH analysis and Mr. Aleksej V. Lazarev for technical support. REFERENCES (1) Sengupta, S.; Chattopadhyay, M.K. Antibiotic resistance of bacteria: A global challenge. Resonance 2012, 17(2), 177-191. (2) Frank, C.; Werber, D.; Cramer, J.P.; Askar, M.; Faber, M.; an der Heiden, M.; Bernard, H.; Fruth, A.; Prager, R.; Spode, A. Wadl, M.; Zoufaly, A.; Jordan, S.; Kemper, M.J.; Follin, P.; Müller, L.; King, L.A.; Rosner, B.; Buchholz, U.; Stark, K.; Krause, G. Epidemic profile of Shiga-toxin–producing Escherichia coli O104: H4 outbreak in Germany. N. Engl. J. Med. 2011, 365(19), 1771-1780. (3) Smith, K.E.; Wilker, P.R.; Reiter, P.L.; Hedican, E.B.; Bender, J.B.; Hedberg, C.W. Antibiotic Treatment of Escherichia coli O157 Infection and the Risk of Hemolytic Uremic Syndrome. Pediatr. Infect. Dis. J. 2012, 31(1), 3741. (4) Croxen, M.A.; Law, R.J.; Scholz, R.; Keeney, K.M.; Wlodarska, M.; Finlay, B.B. Recent Advances in Understanding Enteric Pathogenic Escherichia coli. Clin. Microbiol. Rev. 2013, 26(4), 822-880. (5) Mellman, A.; Harmsen, D.; Cummings, C.A.; Zentz, E.B.; Leopold, S.R.; Rico, A.; Prior, K.; Szczepanowski, R.; Ji Y.; Zhang, W.; McLaughlin, S.F.; Henkhaus, J.K.; Leopold, B.; Bielaszewska, M.; Prager, R.; Brzoska, P.M.; Moore, R.L.; Guenther, S.; Rothberg, J.M.; Karch, H. Prospective Genomic Characterization of the German Enterohemorrhagic Escherichia coli O104:H4 Outbreak by Rapid Next Generation Sequencing Technology. PLoS ONE. 2011, 6(7), 22751.

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Figure 1 622x333mm (119 x 119 DPI)

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Figure 2 288x213mm (96 x 96 DPI)

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Figure 3 358x122mm (96 x 96 DPI)

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Figure 4 254x190mm (96 x 96 DPI)

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Figure 5 1166x451mm (72 x 72 DPI)

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