Enhancing antibiofilm activity with functional chitosan nanoparticles targeting biofilm cells and biofilm matrix

Enhancing antibiofilm activity with functional chitosan nanoparticles targeting biofilm cells and biofilm matrix

Accepted Manuscript Title: Enhancing antibiofilm activity with functional chitosan nanoparticles targeting biofilm cells and biofilm matrix Authors: Y...

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Accepted Manuscript Title: Enhancing antibiofilm activity with functional chitosan nanoparticles targeting biofilm cells and biofilm matrix Authors: Yulong Tan, Su Ma, Matthias Leonhard, Doris Moser, Greta M. Haselmann, Jia Wang, Dominik Eder, Berit Schneider-Stickler PII: DOI: Reference:

S0144-8617(18)30864-6 https://doi.org/10.1016/j.carbpol.2018.07.072 CARP 13872

To appear in: Received date: Revised date: Accepted date:

20-3-2018 19-6-2018 24-7-2018

Please cite this article as: Tan Y, Ma S, Leonhard M, Moser D, Haselmann GM, Wang J, Eder D, Schneider-Stickler B, Enhancing antibiofilm activity with functional chitosan nanoparticles targeting biofilm cells and biofilm matrix, Carbohydrate Polymers (2018), https://doi.org/10.1016/j.carbpol.2018.07.072 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Enhancing antibiofilm activity with functional chitosan nanoparticles targeting biofilm cells and biofilm matrix

Yulong Tan1#*, Su Ma2#, Matthias Leonhard1, Doris Moser3, Greta M. Haselmann4,

1 Department

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Jia Wang4, Dominik Eder4, Berit Schneider-Stickler1

of Otorhinolaryngology and Head and Neck Surgery, Medical University

2 Food

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of Vienna, Vienna, Austria.

Biotechnology Laboratory, Department of Food Sciences and Technology,

of Cranio-Maxillofacial and Oral Surgery, Medical University of Vienna,

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3 Department

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BOKU-University of Natural Resources and Life Sciences, Vienna, Austria.

of Materials Chemistry, Vienna University of Technology

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4 Institute

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Vienna, Austria.

authors contributed equally to this manuscript

author: Yulong Tan

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*Corresponding

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#Both

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Email address: [email protected] Tel.: +43-1-40400 28910

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Fax.: +43-1-40400 42840

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Graphical abstract

CS nanoparticles loaded with oxacillin and DNase (CSNP-DNase-Oxa) were

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Highlights

fabricated.

CSNP-DNase-Oxa exhibited the best antibiofilm activity in all in-vitro tests.



CSNP-DNase-Oxa reduced the biofilm matrix, thickness and the amount of viable

CSNP-DNase-Oxa showed the highest eradication of clinical isolates biofilms.

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cells.

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Abstract

Bacterial biofilms play a key role during infections, which are associated with an increased morbidity and mortality. The classical antibiotic therapy cannot eradicate biofilm-related infections because biofilm bacteria display high drug resistance due to 2

biofilm matrix. Thus, novel drug delivery to overcome biofilm resistance and eliminate biofilm-protected bacteria is needed to be developed. In this study, positively charged chitosan nanoparticles (CSNP) loaded with oxacillin (Oxa) and Deoxyribonuclease I (CSNP-DNase-Oxa) were fabricated. The antibiofilm activity was evaluated against Staphylococcus aureus biofilms. Biofilm architecture on silicone surfaces was

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investigated by scanning electron microscopy (SEM). Confocal laser scanning

microscopy (CLSM) was used to examine live/dead organisms within biofilm. CSNP-

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DNase-Oxa exhibited higher antibiofilm activity than Oxa-loaded nanoparticles without DNase (CSNP-Oxa) and free Oxa (Oxa and Oxa+DNase) at each

concentration in all in-vitro tests. CSNP-DNase-Oxa inhibited biofilm formation in-vitro

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and eradicated mature biofilm effectively. CSNP-DNase-Oxa could disrupt the biofilm

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formation through degradation of eDNA, reduced biofilm thickness and the amount of

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viable cells on silicone. Repeated treatment with CSNP-DNase-Oxa for two days

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resulted in 98.4% biofilm reduction. Moreover, CSNP-DNase-Oxa was not only able

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to affect the biofilm of a standard S. aureus strain, but also showed the highest eradication of biofilms of clinical isolates compared with control groups. These results

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suggest the potential applicability of NPs for the treatment of biofilm-related infections

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and provide a platform for designing novel drug delivery with more functions.

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Key words: antibiofilm; nanoparticle; oxacillin; Staphylococcus aureus

1. Introduction Staphylococcus aureus is one of the well-known gram-positive pathogens with strong adaptation to the human host. Serious infections are associated with S. aureus because of its ability to adhere to surfaces and form a biofilm on medical 3

devices. (Hall-Stoodley, Costerton, & Stoodley, 2004) Biofilm can be defined as a structured community of bacterial cells in which microbes are embedded in a selfproduced extracellular polymeric matrix. Biofilms play a key role during infections by protecting the embedded microbes against drug and immune response. In fact, once a biofilm is established, bacteria in biofilms can show an up to 1000-fold greater

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resistance to antibiotic treatment than as the planktonic cell form. (Davies, 2003)

New strategies and agents for inhibition of biofilm formation or eradication of mature

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biofilms have been proposed.(Goswami, Thiyagarajan, Das, & Ramesh, 2014; Mu,

Tang, Liu, Sun, Wang, & Duan, 2016; Tan, Leonhard, Moser, Ma, & SchneiderStickler, 2016a) There has been an increasing interest to enhance the efficacy of

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antibiofilm drugs by the development of new drug carriers. For example, polymeric

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nanoparticle (NP), prepared with biocompatible polymers, are attractive due to the

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ability of delivering drugs with a sustained release(Baelo, Levato, Julian, Crespo,

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Astola, Gavalda, et al., 2015; Klinger-Strobel, Ernst, Lautenschlager, Pletz, Fischer,

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& Makarewicz, 2016; Tan, Ma, Liu, Yu, & Han, 2015) into the biofilm matrix.(Fei, Jia, Du, Yang, Zhang, Shao, et al., 2013; Hetrick, Shin, Paul, & Schoenfisch, 2009;

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Peulen & Wilkinson, 2011; Slomberg, Lu, Broadnax, Hunter, Carpenter, & Schoenfisch, 2013) Chitosan (CS) is the N-deacetylated derivative of chitin, which

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has been widely used in pharmaceutical and medical applications, owing to its biocompatibility, biodegradability and non-toxicity.(Jayakumar, Menon, Manzoor,

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Nair, & Tamura, 2010) Additionally, antibiofilm activities of chitosan itself and some of its derivatives have also been reported recently.(Luis R Martinez, Mihu, Han, Frases, Cordero, Casadevall, et al., 2010; L. R. Martinez, Mihu, Tar, Cordero, Han, Friedman, et al., 2010; Tan, Leonhard, Moser, & Schneider-Stickler, 2016) CS nanoparticle (CSNP) is one of the most suitable carriers for drug delivery.(Jonassen, Kjøniksen, & Hiorth, 2012; Maruyama, Guilger, Pascoli, Bileshy-José, Abhilash, Fraceto, et al., 4

2016) In our previous study, CSNP as carrier has been evaluated for treatment of biofilm.(Klinger-Strobel, Ernst, Lautenschlager, Pletz, Fischer, & Makarewicz, 2016) The increased drug resistance of microbes in biofilms compared to their planktonic form is mainly due to the secreted biofilm matrix, which functions as a scaffold and protects the cells in biofilms from drug attack.(Chandra, Kuhn, Mukherjee, Hoyer,

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McCormick, & Ghannoum, 2001) Moreover, even if antimicrobial drugs can kill cells

in biofilms effectively without detachment of the biofilm structure, the dead biofilm

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could still promote other microbial cells adhesion and biofilm regrowth.(Flemming & Wingender, 2010) Therefore, besides the bacterial cells, the biofilm matrix itself should also be a target for the treatments of biofilms. The degradation of the biofilm

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matrix to enhance the drug effectiveness has been proposed as a new

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strategy.(Klinger-Strobel, Ernst, Lautenschlager, Pletz, Fischer, & Makarewicz, 2016;

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Martins, Henriques, Lopez-Ribot, & Oliveira, 2012) The biofilm matrix is mainly

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composed of polysaccharides, proteins, extracellular DNA (eDNA), and other

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components. Recently, eDNA has been proven to be a key component in biofilm structural stabilization, protection of cells and pathogenicity.(Montanaro, Poggi, Visai,

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Ravaioli, Campoccia, Speziale, et al., 2011; Mira Okshevsky & Meyer, 2015) The combination of antimicrobial drugs and Deoxyribonuclease I (DNase) can

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disassemble the biofilm matrix and enhance the killing of microbial cells by the drug in synergy.(Baelo, et al., 2015; Martins, Henriques, Lopez-Ribot, & Oliveira, 2012;

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Swartjes, Das, Sharifi, Subbiahdoss, Sharma, Krom, et al., 2013) Therefore, development of new antibiotic delivery combined with an enzyme which can target both the bacterial cells and biofilm matrix is an urgent necessity. In this study, CSNP prepared by ion gelation method were loaded with oxacillin (Oxa) and DNase (CSNP-DNase-Oxa). The aim of this in-vitro study is to assess the inhibition and eradication activity of CSNP-DNase-Oxa on S. aureus biofilms in 965

well microplate and on medical silicone surface, which is compared with that of CSNP loaded with Oxa (CSPN-Oxa) only and free Oxa. 2. Materials and methods 2.1. Bacterial strains, media and reagents S. aureus ATCC 6538 was used in this study. Three clinical bacterial isolates S.

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aureus, named S. aureus BF 1, BF 2 and BF 3, were obtained from Department of Otorhinolaryngology and Head and Neck Surgery at Medical University of Vienna. All

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of them are capable of forming strong biofilms.The strains were cultured in Tryptic Soy Broth (TSB) medium at 37 °C.

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CS (low molecular weight, degree of deacetylation 75-85%), DNase (≥2,000 Kunitz

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units/mg protein), Oxa (~95%, TLC) and other reagents were all purchased from

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2.2. Preparation of nanoparticles

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Sigma-Aldrich (Austria).

CSNP were prepared by ion gelation method with polyanionic sodium triphosphate

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(TPP). In brief, CS was dissolved in acetic acid (5 mg/ml) and kept under agitation overnight. TPP solution was added dropwise to the chitosan solution with stirring

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vigorously for 2 h. The mass ratio of CS to TPP was 4:1. The CSNPs were separated

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by ultracentrifugation (14000 rpm, 30 min) and then resuspended in PBS. Purification of CSNPs was carried out by subsequent dialysis against deionized water through a dialysis tubes (MWCO 12 kDa; Sigma-Aldrich) for 48 h.

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For CSNP-Oxa preparation, CSNP suspension (1 mg/ml) was mixed with 100 μg/ml

of the antibiotic under stirring overnight. CSNP-DNase-Oxa (1 mg/ml) was prepared with the same method using the PBS containing 100 μg/ml of antibiotic and 100 μg/ml DNase while stirring overnight. The by-products and unreacted chemicals were removed by ultracentrifugation. The NP suspensions were freeze-dried eventually and stored at −20 °C before usage. 6

2.3. Nanoparticle characterization The size and surface charge of NPs were measured by ZetaSizer NanoZS (Malvern Instruments, UK). NP suspensions were analyzed by Dynamic Light Scattering (DLS) for size determination. Laser Doppler Velocimetry assays were used to measure the zeta potential of the particles. NP size distribution is reported by a polydispersity

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index (PDI). The morphological characterization was confirmed with Transmission

2.4. Quantification of DNase activity containing NPs

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Electron Microscopy (TEM, FEI Tecnai F20, 200 kV).

20 μg of DNase-containing NPs was added to a 500 ng known DNA plasmid pGEM-

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T (Promega) in water. As a control, DNA alone (500 ng in water) and NPs alone (20

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μg in water) were also tested. After 60 min of incubation at 37 °C, the mixtures were

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loaded in a 0.8% TAE agarose gel, stained with ethidium bromide and visualized

2.5. Drug encapsulation

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under UV light (Gel DocTM XR+, Bio-Rad Laboratories).

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An Oxa standard curve was performed with UV–spectroscopy at 220 nm to detect the quantification. The amounts of Oxa encapsulated in the NPs were determined

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using the centrifugation method. Immediately after the CSNP-Oxa or CSNP-DNase-

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Oxa preparation, CSNP-Oxa or CSNP-DNase-Oxa solution was centrifuged (14000 rpm, 30 min) and the supernatant solution was analyzed using UV–spectroscopy to quantify the amount of non-loaded Oxa. The Oxa loading capacity (LC) was

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calculated according to: LC = (A-B)/C

A = Total amount of Oxa; B = Total amount of non-loaded Oxa; C = Weight of the nanoparticles. 2.6. In-vitro release

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In-vitro release kinetic of Oxa was assessed as follows: 4 mL CSNP-Oxa or CSNPDNase-Oxa solution (1 mg/ml) was added in a dialysis bag and placed in the 40 mL of PBS. The mixture was stirred at 100 rpm. At predetermined time intervals, 4 mL PBS were withdrawn and replaced with 4 mL fresh PBS. The content of Oxa in the PBS was determined by UV–spectroscopy at 220 nm.

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2.7. Minimum inhibitory concentration (MIC) assay

100 μl of bacteria at a final concentration of 1×10 5 CFU/ml in TSB was added into

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the wells of 96-well microplates. Free Oxa and loaded Oxa with same concentrations

(1, 0.5, 0.25, 0.125, 0.0625 or 0 μg/ml) were then added to each well. The microplate was incubated at 37 °C for 24 h at 150 rpm. The MIC was defined as the lowest

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concentration of Oxa at which no visible growth was detected.

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2.8. Growth of biofilm

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Biofilm formation in 96-well microplates were conducted and assayed as described

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previously.(Klinger-Strobel, Ernst, Lautenschlager, Pletz, Fischer, & Makarewicz,

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2016) In brief, bacteria from an overnight culture were diluted to 1×10 5 CFU/ml with TSB containing 0.25% glucose. The microplates were incubated at 37°C for 24 h

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without shaking.

2.9. Penetration of NPs into biofilms

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2.9.1. Synthesis of fluorescently-labeled NPs 5 mg of rhodamine B isothiocyanate (RBITC) was dissolved in 1 ml of methanol.

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100 µl of RBITC solution was added to 1 ml of 5 mg/ml CSNPs and the mixture was shaken for 24 h at 4°C under dark condition. The RBITC-NPs were dialyzed against distilled water to remove the free RBITC. 2.9.2. Examination of NPs penetration An autoclaved medical grade silicone platelet (3-mm-diameter, Websinger, Austria) was placed into each well of the 96-well microplate. Biofilms were formed as 8

described above in the 96-well microplate. RBITC-NPs (100 µg/mL) were mixed with biofilm samples in PBS for 2 h. The samples were washed with PBS for three times and the penetration was examined by confocal laser scanning microscopy (CLSM). 2.10. Effect of NPs on the biofilm formation Biofilm formation in 96-well microplates was conducted as described above in 2.8.

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In order to quantify the effect of NPs on the biofilm formation, the microplates with bacteria were incubated in the presence of free soluble Oxa, a combination of Oxa

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and DNase (5 μg/ml), CSNP-Oxa and CSNP-DNase-Oxa with different drug concentrations from 0.0625 to 1 μg/ml. Also, the CSNPs alone were added to test the influence of CSNP without drug on biofilm formation. Biofilms were washed with PBS

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and stained with 100 μl of 0.1% (w/v) crystal violet solution for 30 min. 100 μl of 30%

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(v/v) acetic acid was used to extract the crystal violet retained by the cells. The A 570

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of the extract was measured with a microtiter plate reader to determine the amount of

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biofilm.

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2.11. Effect of NPs on the mature biofilm

The activity of the NPs against 24 h-biofilms was also tested. Biofilms were

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prepared as described above without Oxa or NPs. After 24 h, the microplates were rinsed with PBS and fresh TSB containing different concentrations of free soluble

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Oxa, Oxa-loaded NPs (CSNP-Oxa or CSNP-DNase-Oxa) and a combination of Oxa and DNase (5 μg/ml, both free soluble) was added. The microplates were incubated

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at 37 °C for another 24 h or 48 h (once treatment per day). The biofilms were quantified as previously described. 2.12. Effect on biofilms on medical silicone surface Biofilms formed on silicone platelets were treated with free Oxa (Oxa or Oxa+DNase) and the Oxa-loaded NPs (CSNP-Oxa or CSNP-DNase-Oxa) as described above. 9

Biofilm architecture on medical grade silicone was investigated by scanning electron microscopy (SEM). Biofilms on silicon platelets were fixed in 3 vol.% glutaraldehyde in PBS solution overnight at 4°C, and then subjected to serial dehydration with 25%, 50%, 75%, 100% ethanol for 10 min each. The biofilms were coated with gold and examined with SEM (JSM 6310, Jeol Ltd, Akishima, Tokyo, Japan).

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The cells of biofilms were stained with a LIVE/DEAD® BacLight™ Bacterial Viability and Counting Kit (L34856, Invitrogen) following the manufacturer’s instructions. The

2.13. Antimicrobial activity against clinical isolates

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cell viability was observed with CLSM.

The antimicrobial effect of Oxa, CSNP-Oxa or CSNP-DNase-Oxa on several clinical was

tested.

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obtained

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isolates

from

Department

of

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Otorhinolaryngology and Head and Neck Surgery at Medical University of Vienna.

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Biofilm of each clinical strain formed in the wells of a 96-well microplate and

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evaluated using crystal violet assay as described above.

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2.14. Determination of mammalian cytotoxicity To determine the cytotoxic effects of NPs in vitro, HaCaT (human immortalized

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keratinocytes) was used to test the cell viability assays with CCK-8 assay. Cells were seeded with 3 × 103 cells per well into 96-well plates and incubated for 24 h. Then

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cells were treated with CSNP, CSNP-Oxa and CSNP-DNase-Oxa with the same concentration for antibiofilm. After 72 h of incubation cell viability was measured by

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CCK-8 according to manufacturer’s protocol. 2.15. Statistical analysis All the experiments were done in triplicate. Statistical analyses were performed using GraphPad Prism software program (GraphPad Software, CA, USA). For each experiment, means±standard deviations (SD) were calculated. Statistical significance was determined by t-test analysis with p<0.05 found to be significantly different. 10

3. Results 3.1. Characterization of nanoparticles TEM image shows surface appearance and morphological properties of NPs. CSNP-DNase-Oxa have smooth surface and nearly spherical shape (Fig. 1). The physical characterization, including size, zeta potential and drug loading capacity, of

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the NPs are summarized in Table 1. The average diameter of the CSNP and CSNPDNase-Oxa was estimated to be 158.1and 166.7 nm. The surface zeta potential of

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NPs changed from +11.4 mV to +8.3 mV. The average amount of Oxa in the carriers

was 6.65%. DNase loaded NPs retained DNase activity, as assessed by agarose gel electrophoresis (Fig. S1), with 1 μg of CSNP-DNase-Oxa being able to degrade 23

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ng of DNA in 60 min.

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3.2. In-vitro release studies

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Fig. 2 shows the in-vitro release profiles of Oxa from NPs. Both CSNP-Oxa and

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CSNP-DNase-Oxa presented an initial burst release phase in the first 1 h: 28.6% and

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35.8% of the loaded Oxa were released. Afterwards, the drug release rate was slower. Over 24 h, CSNP-Oxa and CSNP-DNase-Oxa released 76.3% and 88.4% of

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the total Oxa, respectively.

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3.3. Antibacterial activity of Oxa loaded NPs According to the MIC assay, Oxa alone and CSNP-DNase-Oxa were effective

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against planktonic bacteria (Table 2). Against S. aureus ATCC 6538, the MIC of free Oxa was 0.5 μg/ml and the MIC of CSNP-DNase-Oxa was 1 μg/ml. Moreover, both free Oxa and CSNP-DNase-Oxa were effective against clinical isolates growth.

3.4. Penetration of NPs into biofilms 11

As shown in Fig. S2, after 2 h treatment, red color (RBITC-NPs) can be observed on the surface of as well as inside biofilm, which indicates the penetration of NPs into biofilms. 3.5. Inhibition activity on biofilm formation As seen in Fig. 3, biofilm reduction was achieved with all the concentrations tested

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in this work. Both free Oxa and loaded Oxa showed a good extent of biofilm inhibition. The activity of both free Oxa and loaded Oxa increased with Oxa concentrations,

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showing that biofilm inhibition with free Oxa or loaded Oxa is concentration dependent. Even at the lowest concentration (0.0625 μg/ml), about 60% biofilm formation was inhibited. Almost no biofilm formation was seen at concentrations

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higher than 0.5 μg/ml. Loaded Oxa (CSNP-Oxa or CSNP-DNase-Oxa) showed

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over the same concentration range.

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slightly decreased inhibition activity relative to the free Oxa (Oxa or Oxa+DNase)

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3.6. Antibiofilm activity on mature biofilm Fig. 4 shows the antibiofilm activity of the free Oxa (Oxa or Oxa+DNase) and the

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Oxa-loaded NPs (CSNP-Oxa or CSNP-DNase-Oxa) on mature biofilm at various Oxa concentrations. Free Oxa and the Oxa-loaded NPs at the highest concentration (2

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μg/ml) resulted in more than 80% biofilm reduction. Furthermore, NPs with DNase (CSNP-DNase-Oxa) exhibited higher activity than NPs without DNase (CSNP-Oxa)

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and free Oxa (Oxa and Oxa+DNase) at each concentration. At the lowest concentration (0.0625 μg/ml), CSNP-DNase-Oxa increased detachment by 70 %. No biofilm formation was observed at 2 μg/ml Oxa concentration with CSNP-DNase-Oxa, indicating the best antibiofilm activity.

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3.7. Antibiofilm effect with repeated treatment In order to evaluate a longer effect of NP loaded Oxa on an established biofilm, we assessed the antibiofilm effect with repeated treatment for two days (Fig. 5). After 48 h treatment, the free Oxa and CSNP-Oxa reduced the biofilm by 92.3% and 97.2% at the highest concentration. Addition of DNase enhanced the antibiofilm effects. There

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is no biofilm observed with treatment of Oxa+DNase and CSNP-DNase-Oxa at the concentration of 2 μg/ml. Moreover, CSNP-DNase-Oxa exhibited the best antibiofilm

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activity at all of the concentrations tested except using the Oxa concentration of 2 μg/ml.

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3.8. Antibiofilm effect of NPs on silicone

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The biofilm grown on silicone platelets was observed using SEM (Fig. 6).

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aureus biofilm grown without Oxa treatment exhibit the typical 3D morphology with

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dense structures and water channels (Fig. 6 A). Under the same growth conditions

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but treated with free Oxa (Oxa or Oxa+DNase) and the Oxa-loaded NPs (CSNP-Oxa or CSNP-DNase-Oxa), biofilm showed structural changes (Fig. 6 B-E): Oxa (Fig. 6 B)

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and CSNP-Oxa (Fig. 6 C) reduced bacterial biofilm formation on the silicone surfaces. The combination with DNase improved the antibacterial and antibiofilm effects (Fig. 6

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D and E). The silicone surface displayed more regions with only single cells adhered or even free of cells.

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CLSM images confirmed the results above and revealed changes in the cell viability

of biofilm treated with free Oxa and the Oxa-loaded NPs (Fig. 7). A large number of green clusters (live cells) were observed in the control group (Fig. 7 A), which indicates a mature biofilm structure with multiple layers of active cells. With treatment of Oxa and CSNP-Oxa (Fig. 7 B and C), biofilms consisted of less biomass and biofilm thickness, and more biofilms were stained red (dead cells). Addition of DNase 13

led to a drastic reduction of the green color (live cells) (Fig. 7 D and E). CSNPDNase-Oxa (Fig. 7 E) resulted in an almost complete killing of S. aureus (red stain) and almost full destruction of the 3D architecture, which indicates the effect of CSNP-

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DNase-Oxa in killing the cells and disrupting the biofilm formation.

3.9. Antibiofilm activity against clinical isolates

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Fig. 8 shows the antibiofilm activity of free Oxa and the Oxa-loaded NPs against clinical isolates. Biofilm formation by all tested strains was significantly reduced in the presence of CSNP-DNase-Oxa. CSNP-DNase-Oxa caused 83.7%, 61.8% and 81.0%

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reduction of clinical isolates biofilms using the 1 μg/ml Oxa concentrations.

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3.10. Cytotoxicity

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The effect of NPs on cell viability was investigated using mammalian cell HaCaT. As

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shown in Fig. S3, the NPs did not affect the growth of mammalian cells at the concentrations used in this study.

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4. Discussion

The bacterial biofilm associated infections have been a huge challenge in health

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care because of the increased resistance to antimicrobials due to the protection by biofilm matrix. The development of a novel drug delivery to target the bacterial cells in

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the biofilm and disperse the biofilm structure is an urgent necessity. More attentions have been attracted to the use of NPs for the inhibition of biofilm formation and the detachment of mature biofilms recently. NPs for biofilm treatment have several benefits. The antimicrobial agent loaded on the NPs can be protected from sequestering drugs by biofilm matrix. Furthermore, NPs can penetrate the biofilm matrix and deliver a high concentration of antimicrobial agents directly to the bacterial 14

cells in the biofilm,(Guo, Zhao, Dai, Zhang, Yu, Zhang, et al., 2017; Jorgensen, Wassermann, Jensen, Hengzuang, Molin, Hoiby, et al., 2013; Nafee, Husari, Maurer, Lu, de Rossi, Steinbach, et al., 2014) so that the antimicrobial agents can maximize therapeutic benefit and decrease the side effects. Moreover, positively charged NPs, such as chitosan NPs, could bind to negatively charged biofilm components, thereby

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providing high local doses of antimicrobial agent.(Forier, Messiaen, Raemdonck,

Deschout, Rejman, De Baets, et al., 2013; Hou, Wang, Zhang, Bai, Sun, Duan, et al.,

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2017; Shrestha, Hamblin, & Kishen, 2014) For this purpose, CSNP loaded with Oxa and DNase was developed to incorporate the strengths of two strategies: to target for disruption of biofilm matrix and killing the bacteria.

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In this work, both of Oxa and DNase were loaded onto the CSNP simultaneously.

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The concentration of drug and enzyme used is lower than the saturated

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concentrations (data not shown) in order to provide more area of CSNP for both of

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them loading. In the follow-up work, the influence of the different loading capacities

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on the antibiofilm activity will be further evaluated, such as the ratio of drug and enzyme and different drug loading contents.

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As a drug delivery, in-vitro release profiles of Oxa from NPs were evaluated. Both CSNP-Oxa and CSNP-DNase-Oxa showed the typical release profiles, a fast burst

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release at beginning followed by a sustained release, which are similar to other reports. (Anitha, et al., 2011; Jain, Thakur, Sharma, Kush & Jain, 2016) The initial

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burst release might be due to the drug entrapped near the surface and then the sustained pattern happens as a result of the drug releases from the NPs matrix. This release profile is preferred for the treatment of biofilm. In the case of antibiotic effect on biofilms, the initial burst is more effective because the higher initial drug doses can reduce the antibiotic tolerance of the surviving bacteria in biofilm.(Cheow, Chang, & Hadinoto, 2010; Forier, Raemdonck, De Smedt, Demeester, Coenye, & Braeckmans, 15

2014) The MIC of Oxa loaded NPs are higher than free drug because the controlled antibiotic release of NPs meant S. aureus was exposed to less of Oxa. CSNP showed the penetration of NPs into biofilm, which can bind on the surface of biofilm and cells inside biofilm. It has been reported that polymeric nanoparticle delivery prepared with biocompatible polymers can deliver drugs into the biofilm

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matrix.(Baelo, et al., 2015; Fei, et al., 2013) Especially, NPs with positive charge

could bind to the negatively charged biofilm components, such as eDNA.(Nafee, et

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al., 2014) Moreover, chitosan has been shown to be able to penetrate into biofilms

and facilitate drug diffusion.(Lu, Slomberg, & Schoenfisch, 2014; Zhang, Mu, Zhang, Cui, Zhu, & Duan, 2013) In this work, CSNP as a drug carrier can deliver the Oxa into

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biofilms and then disrupt the biofilm inside.

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For the biofilm formation, both free Oxa and loaded Oxa were able to inhibit the

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biofilm formation. The effect of loaded Oxa is slightly lower than free Oxa, because

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Oxa loading on the CSNP was sustained continuously released which had a lower

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concentration and reduced the antibacterial activity. The DNase alone or the system with DNase didn’t exhibit any obvious inhibition or enhanced effect, which is because

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that the biofilm hadn’t formed and the drug could interact with the planktonic bacteria directly. In our previous work, we showed that chitosan can effectively inhibit surface

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colonization and biofilm formation, due to the interaction between the cationic charge of chitosan and the negative charge of the microbial membrane.(Goswami,

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Thiyagarajan, Das, & Ramesh, 2014; Tan, Leonhard, Moser, & Schneider-Stickler, 2016) Although the CSNP alone didn’t effect on the biofilm formation at the concentration used in this work (data not shown), positively charged NPs loaded with Oxa were able to penetrate into biofilms and interfere with microbial cells inside biofilm.

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For the mature biofilm, DNase alone didn’t show any antibiofilm activity in this work (data not shown), because the dispersed microbial cells without being killed by drug can form biofilms again. However, the mixture of free Oxa and DNase, and NPs with DNase (CSNP-DNase-Oxa) showed higher antibiofilm activity than free Oxa and CSNP-Oxa, which indicates a synergistic effect of DNase. This could be due to

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degradation of the eDNA in the biofilm matrix by the DNase, which improved mobility

of NPs. The similar results also were reported by other groups. Baelo et al. reported

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that charged polymeric NPs with antibiotic exhibited higher antibiofilm effect in the

presence of DNase.(Baelo, et al., 2015) Moreover, CSNP-DNase-Oxa exhibited the best antibiofilm activity, which suggested NPs bearing both DNase and Oxa at the

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same time may penetrate into the biofilm and kill the bacterial cell better. It is well

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described that DNase could degrade the eDNA and disassemble the structure of

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biofilm, so as to improve antibiotic efficacy.(Martins, Henriques, Lopez-Ribot, &

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Oliveira, 2012; M. Okshevsky, Regina, & Meyer, 2015) This interpretation could be

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further strengthened by the CLSM images. Fig. 8 showed that CSNP-DNase-Oxa was more effective at killing the S. aureus cells and disrupting the biofilm formation,

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which indicated the CSNP-DNase-Oxa disassembled better biofilm matrix and increased killing the bacterial cell.

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After two days of repeated treatment, loaded Oxa showed better activity than free

Oxa. As discussed above, with increasing time and repeated treatment of CSNP-

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DNase-Oxa, biofilm matrix might be disrupted steadily and more Oxa-loaded NPs can penetrate into the biofilm to interfere with bacterial cells. However, free Oxa hardly diffused into the biofilm and was therefore inactive. Thus, CSNP-DNase-Oxa showed higher biofilm detachment activity at all of concentrations tested for two days than all other groups.

17

Silicone, as one of the most widely used biomaterials, suffers from biofilm formation, which leads to dysfunction and replacement of medical devices eventually.(Pavlovic, Bozic, Milovanovic, Jotic, Djukic, Djukic, et al., 2016; Tan, Leonhard, Moser, Ma, & Schneider-Stickler, 2016b) Thus, we assessed the antibiofilm activity on the silicone surface. The results suggest that CSNP-DNase-Oxa can effectively eradicate biofilm

of CSNP-DNase-Oxa to treat biofilm related medical device infections.

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formation on medical grade silicone surface, which pave the way for the application

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It has been shown that the microbe can rapidly adapt to in-vitro conditions.(Fux, Shirtliff, Stoodley, & Costerton, 2005) The strain in standardized and idealized laboratory conditions might lose some important pathophysiological characteristics

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after being sub-cultured for decades. Therefore, we used clinical isolated specimens

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to mirror the clinical effect. Our results demonstrate that CSNP-DNase-Oxa was not

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only able to effect the biofilm of standard S. aureus strain, but also showed the

5. Conclusions

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highest eradication of biofilms of clinical isolates.

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In this work, DNase functionalized NPs, CSNP-DNase-Oxa, were prepared in order to disrupt the biofilm matrix and to kill the bacterial cells embedded in the biofilm. The

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positively charged NPs showed spherical shape and narrow size distribution, and displayed a sustained drug release profile for biofilm treatment application. CSNP-

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DNase-Oxa exhibited the best antibiofilm activity against standard S. aureus strain and clinical isolates in microplates or on the silicone surface. Moreover, the CSNP can be employed as a platform for designing more chemical modification or load other antibiofilm agents to obtain more functions as drug delivery system. Although further assessment of CSNP-DNase-Oxa on Gram-negative bacteria and effect or biocompatibility in vivo are still needed to be evaluated, these studies suggest the 18

potential applicability of CSNP-DNase-Oxa to the biomedical and pharmaceutical fields, especially to the delivery of antibiofilm agents to treatment of biofilm related

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infections.

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Funding This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Conflict of interest

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The authors claim no conflict of interest.

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Submission declaration

The work described above has not been published previously or under consideration

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for publication elsewhere.

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Acknowledgement

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We are very grateful to Prof. Erik Reimhult and Behzad Shirmardi Shaghasemi

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(BOKU-University of Natural Resources and Life Sciences) for technical assistance.

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Figure legends

Figure 1 TEM image of CSNP-DNase-Oxa. Figure 2 In vitro release kinetic from CSNP-DNase-Oxa.

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Figure 3 Inhibition effects of NPs on biofilm in 96-well microplate. The results shown represent the means and standard deviations (error bars) of three independent

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experiments, *p<0.05 for comparison between the untreated and treated groups.

Figure 4 Disruption effects of NPs on mature biofilm in 96-well microplate. The results shown represent the means and standard deviations (error bars) of three

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independent experiments, *p<0.05 for comparison between the untreated and treated

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groups.

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Figure 5 Mature biofilm responses to two consecutive NPs treatments. The results

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shown represent the means and standard deviations (error bars) of three

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independent experiments, *p<0.05 for comparison between the untreated and treated groups.

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Figure 6 SEM images of S. aureus biofilm formations on medical grade silicone surface with media supplemented without treatment (A) or with Oxa (B), CSNP-Oxa

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(C), Oxa+DNase (D) and CSNP-DNase-Oxa (E) treatment. Figure 7 CLSM images of S. aureus biofilm formations on medical grade silicone

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surface with media supplemented without treatment (A) or with Oxa (B), CSNP-Oxa (C), Oxa+DNase (D) and CSNP-DNase-Oxa (E) treatment on polymicrobial biofilm. Biofilms were stained with the Live/Dead® BacLight™ Bacterial Viability and Counting Kit. CLSM reconstructions show the three-dimensional staining pattern for live cells (SYTO-9, green) and dead cells (propidium iodide, red). Magnification, ×10.

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Figure 8 Antibiofilm activities of NPs against clinical isolates. The results shown represent the means and standard deviations (error bars) of three independent

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experiments, *p<0.05 for comparison between the untreated and treated groups.

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Figure 1 TEM image of CSNP-DNase-Oxa.

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Figure 2 In-vitro release kinetic from CSNP-DNase-Oxa.

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Figure 3 Inhibition effects of NPs on biofilm in 96-well microplate. The results shown represent the means and standard deviations (error bars) of three independent

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experiments, *p<0.05 for comparison between the untreated and treated groups.

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Figure 4 Disruption effects of NPs on mature biofilm in 96-well microplate. The

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results shown represent the means and standard deviations (error bars) of three

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independent experiments, *p<0.05 for comparison between the untreated and treated

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groups.

Figure 5 Mature biofilm response to two consecutive NPs treatments. The results shown represent the means and standard deviations (error bars) of three independent experiments, *p<0.05 for comparison between the untreated and treated 30

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groups.

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Figure 6 SEM images of mixed species biofilm formations on medical grade silicone

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surface with media supplemented without treatment (A) or with Oxa (B), CSNP-Oxa

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(C), Oxa+DNase (D) and CSNP-DNase-Oxa (E) treatment.

Figure 7 CLSM images of monomicrobial and polymicrobial biofilm formations on medical grade silicone surface with media supplemented without treatment (A) or with Oxa (B), CSNP-Oxa (C), Oxa+DNase (D) and CSNP-DNase-Oxa (E) treatment on polymicrobial biofilm. Biofilms were stained with the Live/Dead® BacLight™ Bacterial Viability and Counting Kit. CLSM reconstructions show the three31

dimensional staining pattern for live cells (SYTO-9, green) and dead cells (propidium

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iodide, red). Magnification, ×10.

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Figure 8 Antibiofilm activity of NPs against clinical isolates. The results shown

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experiments, *p<0.05 for comparison between the untreated and treated groups.

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Table legends

Table 1 Encapsulation efficiency and overall properties of NPs.

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Table 2 Minimal inhibitory concentrations (MICs) of free Oxa and CSNP-DNase-Oxa.

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Table 1 Encapsulation efficiency and overall properties of NPs.

Size (nm)

Size PDI

zeta potential (mV)

CSNP

-

158.1

0.193

+11.4

CSNP-Oxa

6.95%

158.3

0.208

CSNP-DNaseOxa

6.65%

166.7

0.179

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LC (%)

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+10.5 +8.3

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Table 2 Minimal inhibitory concentrations (MICs) of free Oxa and CSNP-DNase-Oxa.

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Oxa MIC (μg/ml)

1

1

4

BF 2

4

8

BF 3

1

4

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BF 1

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CSNP-DNase-Oxa

0.5

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ATCC 6538

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Oxa

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