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Bactericidal Effect of Gold-Chitosan Nanocomposites in Coculture Models of Pathogenic Bacteria and Human Macrophages Gracia Mendoza, Anna Regiel-Futyra, Vanesa Andreu, Victor Sebastian, Agnieszka Kyzio#, Grazyna Stochel, and Manuel Arruebo ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b15123 • Publication Date (Web): 22 Feb 2017 Downloaded from http://pubs.acs.org on February 23, 2017

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ACS Applied Materials & Interfaces

Bactericidal

Effect

of

Gold-Chitosan

Nanocomposites in Co-culture Models of Pathogenic

Bacteria

and

Human

Macrophages Gracia Mendoza†§*, Anna Regiel-Futyraǁ, Vanesa Andreu†§, Víctor Sebastián†‡§, Agnieszka Kyziołǁ, Grażyna Stochelǁ, Manuel Arruebo†‡§

†

Department of Chemical Engineering, Aragon Institute of Nanoscience (INA),

University of Zaragoza, Campus Río Ebro-Edificio I+D, C/ Mariano Esquillor S/N, 50018 Zaragoza, Spain ‡

Networking Research Center on Bioengineering, Biomaterials and Nanomedicine,

CIBER-BBN, Madrid 28029, Spain §Aragon Health Research Institute (IIS Aragón), 50009 Zaragoza, Spain ǁ

Faculty of Chemistry, Jagiellonian University, Ingardena 3, 30-060 Kraków, Poland

Keywords: Bactericidal nanomaterials, infection, nanocomposites, chitosan, gold nanoparticles

1 ACS Paragon Plus Environment


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Abstract Pathogenic bacteria are able to develop resistance mechanisms to avoid the antimicrobial potential of antibiotics becoming an increasing problem for the healthcare systems. The search for more effective and selective antimicrobial materials, though not harmful to mammalian cells, seems imperative. Herein we propose the use of goldchitosan nanocomposites as effective bactericidal materials avoiding the damage to human cells. Nanocomposites were obtained taking advantage of the reductive and stabilizing action of chitosan solutions on two different gold precursor concentrations. The resulting nanocomposites were added at different final concentrations to a coculture model formed by Gram-positive (Staphylococcus aureus) or Gram-negative (Escherichia coli) bacteria and human macrophages. Gold-chitosan colloids exhibited superior bactericidal ability against both bacterial models without showing cytotoxicity on human cells at the concentrations tested. Morphological and in vitro viability studies supported the feasibility of the infection model here described to test novel bactericidal nanomaterials. Flow cytometry and scanning electron microscopy analyses pointed to the disruption of the bacterial wall as lethal mechanism. Data obtained in the present study suggest that gold-chitosan nanocomposites are powerful and promising nanomaterials for reducing bacteria-associated infections respecting the integrity of mammalian cells and displaying a high selectivity against the studied bacteria.


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1. INTRODUCTION The discovery and development of antibiotics, since penicillin was introduced, underwent an explosion resulting in the mistaken belief that any bacterial infection can be treated with first-line antibiotics. However, the development of multidrug resistance by microorganisms has become a serious threat in the efficient treatment of bacteria induced infections1,2. In fact, some microorganisms such as Staphylococcus aureus1, Clostridium difficile3, Enterobacteriaceae4, Neisseria gonorrhoeae5 and some Gramnegative strains1 have developed resistance to a wide range of antibiotics making their successful treatment extremely difficult. In this sense, the last report of the Centers for Disease Control and Prevention of the US published in 2013 revealed that around two million people are affected annually by antibiotic resistant infections and 23,000 die this pathological situation6. The predictions say that by 2050 antimicrobial resistance would cause 10 million people deaths per year and a reduction of 2% to 3.5% in Gross Domestic Product (GDP)7. The risk of pan-drug resistance has been pointed out as a real threat as pathological bacteria are predicted to become increasingly resistant together with the clinical abuse of a broad spectrum antibiotic drugs8,9. With this scenario, the development of novel bactericidal approaches seems to be imperative. Antimicrobial nanomaterials have been postulated as a novel and effective approach in the development of bactericides, mainly metal nanoparticles (NPs), against both Grampositive and Gram-negative bacteria2,10–12 due to their multiple mechanisms of antibacterial activity13–15, though the potential toxicity exerted by copper and silver NPs has diverted the attention towards gold-based nanomaterials as safer and more biocompatible antimicrobials12,16,17. Gold NPs (AuNPs) have been generally regarded as non-bactericidal due to their biological inerticity although their surface modification has been shown as highly efficient in their acquisition of bactericidal ability even against



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multidrug resistant bacteria18. However, previous studies have pointed to bacterial wall disruption as the target of nanogold bactericidal activity while other authors have shown that the antibacterial effects of those metal NPs are mediated by the generation of reactive oxygen species12,19,20. Furthermore, AuNPs antimicrobial activity is related to their shape, size and surface, thus their modulation or modification is a valuable tool to increase their bactericidal effects12. One of the most attractive modifications is the coating of gold NPs with biocompatible polymers such as PEG, PAA or chitosan (CS), which also may be able to modulate the mechanical strength of the resulting nanocomposites in order to improve their features as biomedical scaffolds12,21–24. CS is a polycationic aminopolysaccharide obtained by the partial N-deacetylation of chitin, a natural biopolymer present in crustacean shells, insects and fungi2,25. CS molecular weight, deacetylation degree and water-solubility are important factors affecting its biological activity showing that high deacetylation degrees and medium molecular weights exert higher antimicrobial effects12,26–28. In addition, CS biocompatibility has also been demonstrated in vitro and in vivo through the absence of inflammatory or immune responses29, so its clinical and bactericidal use have been widely reported29–33. CS has been demonstrated to develop its bactericidal activity by the binding of CS amino groups to negatively charged bacteria wall or by the CS interplay with the lipid fraction of bacteria membrane. As a consequence, the affection at the level of genetic material occurs or even the formation of a surrounding barrier which does not permit the penetration of essential metals for bacteria growth. Possibly, all these events may concur simultaneously and have been postulated as potential antimicrobial mechanisms 34,35

. We previously demonstrated that the combination of CS with gold nanoparticles

produces a superior antimicrobial nanocomposite as films and as particles with no


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cytotoxicity against human cells due to its hindered internalization in the eukaryotic cells12, although producing disruptive effects on the bacterial wall. On the other hand, our main natural barrier against bacterial infections are macrophages which exist in every tissue and play a pivotal role in the host defense against pathogens36,37. This first-line sentinel cellular type shows a wide range of receptors and mediators in order to recognize, engulf and destroy bacterial cells. They also regulate other own cell populations and recruit other macrophages constituting the adaptive immune response38,39. Furthermore, macrophages exhibit an unusual heterogeneity showing a great adaptation to an specific tissue environment. These results in their tissue specialization being able to recruit other macrophages nevertheless all of them are derived from circulating monocytes37,40. The key event in host defense against bacteria is phagocytosis mediated by macrophages and monocytes. Once they recognize bacteria through different types of receptors and at different levels, the phagocytic process begins with the bind of these cells to the bacteria to conclude with the formation of the phagolysosome in which bacteria are eliminated through enzymatic activity mediated by low pH41. Regarding these events, the ideal environment to test novel bactericidal nanomaterials seems to be an infection model in which macrophages are not affected by nanomaterials while the antimicrobial material can exert its effects against pathogenic bacteria mimicking a real scenario during infection. In the present study, a comparative analysis of the bactericidal activity of Au-CS nanocomposites obtained with different initial gold precursor concentrations in an in vitro infection model composed of human macrophages and Gram-positive (S. aureus) or Gram-negative (Escherichia coli) bacteria was performed. The aim of this work was to evaluate the impact of this novel bactericidal approach in an infection model



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exploring the mechanism of the interaction between bacteria and Au-CS nanocomposites. 2. EXPERIMENTAL SECTION 2.1. Materials. CS with medium average molecular weight (Mw ~ 1278 ± 8 kDa) and gold(III) chloride trihydrate (≥99.9%) were purchased from Sigma-Aldrich (Germany). CS was obtained from chitin of shrimp shells. The molecular weight (1278 ± 8 kDa) and deacetylation degree (89 ± 2%) were determined experimentally in our previous work42. An aqueous solution of acetic acid (99.8% Sigma-Aldrich, Germany) was used as solvent. 2.2. Chitosan based gold nanoparticle synthesis. The synthesis was performed according to our previously described protocol12. Briefly, CS flakes were dissolved at 65 °C under stirring in 0.1 M acetic acid to obtain a 1 % (w/v) concentration. CS solution was then heated up at 60 °C and gold chloride solutions (1 and 2 mM; always in volume ratio CS:HAuCl4 = 5:2) were added drop wise. The synthesis was conducted under heating and stirring within 4 h. CS acts as reducing and stabilizing agent for the gold nanoparticles formation. To simplify further sample nomenclature, a system of abbreviation is here used (1 and 2 mM, stands for 1 mM or 2 mM initial gold precursor (tetrachlorouric(III) acid) concentration). 2.3. Gold nanoparticles characterization. AuNPs formation was monitored via UV–Vis spectroscopy. Measurements were carried out in a double beam UV–Vis spectrophotometer (Perkin Elmer Lambda 35), in the 400 and 800 nm range. Gold precursor concentration and resulting nanoparticles concentration after the synthesis was determined via inductively coupled plasma mass spectrometry (ICP-MS; Elan 6100 Spectrometer). The shape and average size of the resulting gold nanoparticles were analyzed through recording TEM images using a


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FEI™ Tecnai T20 Microscope operated at 200 kV and a FEI™ Tecnai F30 Microscope operating at 300 kV and in Scanning Transmission mode. The FEI™ Tecnai F30 microscope was equipped with a High Angle Annular Dark Field detector (STEMHAADF) an Energy-dispersive X-ray (EDS) analysis. The size distribution of colloidal nanoparticles was determined from the enlarged micrographs, using National Instruments IMAQ Vision Builder software, counting at least 800 particles. The surface charge of colloidal Au-CS NPs was determined by measuring the zeta potential at pH = 4.5 (Zetasizer Nano ZS, Malvern Instruments, Malvern, UK). Analysis was performed in triplicate. 2.4. Bacteria and macrophage cultures. E. coli S17 was kindly donated by Dr. Jose Antonio Ainsa and used as Gram-negative model while S. aureus (ATCC 25923; Ielab, Spain) was the Gram-positive bacteria model evaluated. Both strains were grown overnight in tryptone soy broth (TSB; Conda-Pronadisa, Spain) at 37 ºC under shaking (150 rpm) obtaining 108-109 colony forming units/mL (CFU/mL). Tryptone soy agar (TSA; Conda-Pronadisa, Spain) was used for seeding bacteria in an incubator at 37 ºC (Memmert, Germany) to evaluate the bactericidal effects of Au-CS nanocomposites quantifying the CFU/mL and CFU/cell. THP1 human monocytes (ATCC TIB-202; LGC Standards, Spain) were routinely cultured in RPMI 1640 with stable glutamine (Biowest, France) supplemented with 10% fetal bovine serum, 1% non-essential aminoacids, 1% HEPES, 0.1% 2mercaptoethanol 50 mM, 1% sodium pyruvate 100 mM, antibiotics (Penicilin 100 U/Streptomycin 100 µg/mL) and amphotericin B (1.5 µg/mL), all provided by Gibco (UK). The in vitro differentiation of monocytes to macrophages was developed by the addition of 1 µM phorbol 12-myristate 13-acetate (PMA) (Sigma Aldrich, US) to the



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supplemented culture medium resulting in the complete differentiation of cells after 72 h. Cells were cultured in a humidified atmosphere at 37 ºC and 5 % CO2. 2.5. Cytotoxicity study. Macrophages viability after treatment with Au-CS nanocomposites at concentrations between 31.25 and 250 µM obtained from the dilution of nanocomposites synthesized from the two different initial gold precursor concentrations (1 mM and 2 mM) was analyzed by the Alamar Blue™ assay (Invitrogen, US). After induction of macrophage differentiation as described above, cells were plated in a gelatinized 96-well plate (Nunc, ThermoFisher Scientific, Spain) and incubated with Au-CS nanocomposites for 24 h. Then, the Alamar Blue reagent was added following the manufacturer indications (10%; incubation of 4h at 37 ºC and 5 % CO2) and the fluorescence displayed by the reduction of the dye by metabolically active cells was recorded in a microplate reader (Multi-mode Synergy HT Microplate Reader; Biotek, US) at 530 nm excitation and 590 nm emission wavelengths. The viability was calculated by linear interpolation of the fluorescence data from the treated cells with Au-CS nanocomposites vs the non-treated ones (control sample, 100% viability). 2.6. In vitro model of infection of human macrophages. Once the complete differentiation of monocytes (THP1 cell line) into macrophages in a 24-well plate (Nunc, ThermoFisher Scientific, Spain) was achieved, supplemented medium was changed to medium without antibiotics. Then, Au-CS nanocomposites were added to the cells at two of the subcytotoxic concentrations determined by the Alamar Blue™ assay previously described (62.5 and 125 µM) in order to test the concentration-dependent bactericidal effect. Prior to the development of the co-culture model, the bactericidal activity of the Au-CS nanocomposites alone (without macrophages) was also tested against both microbial models at the bacteria and


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nanocomposites concentrations used in the infection experiments during the same timing as the co-culture was performed. Then, macrophages were subsequently infected with E. coli at a multiplicity of infection (MOI) of 5:1 while S. aureus infection was separately induced by the addition of a MOI of 10:1. Control samples were also run as biological control (not treated and not infected) and as infection control (not treated and infected). To ensure the adherence of bacteria to macrophages and thus the infection, plates were centrifuged at 200g for 5 minutes to be further incubated at 37 ºC for 30 minutes. After incubation, cells were washed with PBS and treated with 100 µg/mL of gentamycin sulfate (Calbiochem, Merck Millipore, Spain) at 37 ºC for 1 h to eliminate the extracellular bacteria2. Later, cells were washed with PBS and 0.5% Triton X100 (Bio-Rad, Spain) was added for 15 minutes at 37ºC to the macrophages to disrupt the cell membrane and obtain the intracellular bacteria. Bacterial suspensions were diluted in PBS and further seeded in agar plates to count after 24 h the bacteria colonies grown. 2.7. Macrophage viability and morphology after infection and treatment with Au-CS nanocomposites. The evaluation of the macrophages viability after co-culturing with bacteria and with the nanocomposites was carried out by flow cytometry and confocal microscopy by the Live/Dead® Viability/Cytotoxicity Kit for mammalian cells (Molecular Probes™, ThermoFisher Scientific, Spain). To test the viability of macrophages after infection and to choose the appropriate MOIs to obtain an infection though viable model, flow cytometry was performed. The addition of bacteria was performed as described above. Then, cells were collected and centrifuged (1500 rpm, 5 minutes). The pellet was resuspended in a solution containing 2 µl of 50 µM calcein AM working solution and 4 µl of 2 mM of ethidium homodimer



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as indicated by the manufacturer. After 20 minutes of incubation, the samples were analysed by flow cytometry (FACSARIA BD equipment and software, Cell Separation and Cytometry Unit, CIBA, IIS Aragon, Spain). Cell viability after infection and treatment with Au-CS nanocomposites was evaluated by confocal microscopy. As detailed in the previous section, cells seeded in poly-lysine coated µ-slide 8-well glass bottom plates (Ibidi, Spain) were treated with Au-CS nanocomposites and infected with E. coli or S. aureus. The calcein/ethidium homodimer working solution was added to the wells as indicated by the manufacturer’s protocol. Following an incubation of the cells of 30 minutes at room temperature, the samples were visualized by confocal microscopy (Leica TCS SP2 Laser Scanning Confocal Microscope, Microscopy Unit, CIBA, IIS Aragon, Spain). Furthermore, in order to check macrophages morphology after infection and to assure that all extracellular bacteria were discarded before the collection of the samples for intracellular colony counting, cells were seeded in gelatinized glass coverslips, infected as described above and fixed in paraformaldehyde 4% in PBS (Affymetrix, UK) for 30 minutes. After washing with PBS, samples were air-dried and coated with a thin Pt layer and images were recorded in a scanning electron microscope (SEM) Inspect™ F50 (FEI Company, LMA-INA, Spain) in an energy range between 10-15 keV. In addition, the ability of gentamycin sulfate (100 µg/mL) to eradicate bacteria in the conditions described in the co-culture was also tested following the methodology detailed above though without cells. 2.8. Evaluation of Au-CS nanocomposites bactericidal mechanism. To further elucidate the mechanism by which Au-CS nanocomposites eradicate bacteria, two methodologies were performed, flow cytometry to investigate the effect of


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nanocomposites on bacteria membrane permeability and SEM to study the changes in bacteria morphology after nanocomposites treatment. Samples of 107 CFU/mL of E. coli and S. aureus starting cultures were centrifuged at 4400g for 10 minutes and resuspended in PBS (control sample) or in Au-CS nanocomposites dispersions (125 µM) or in CS (5.5 µM) or in Au (125 µM) precursor solutions. After incubation for 2 h at 37 ºC, 25 µg/mL of propidium iodide (SigmaAldrich, Germany) were added to the samples and analyzed by flow cytometry (FACSARIA BD equipment and software, Cell Separation and Cytometry Unit, CIBA, IIS Aragon, Spain) as previously described 43. After an incubation of 2 h at 37 ºC with Au-CS nanocomposites (125 µM), bacteria samples (106 CFU/mL) were washed twice in PBS 0.1 M and fixed in 2.5% glutaraldehyde for 90 minutes. Then, samples were filtered (0.2 µm, pore size cut-off), subsequently dehydrated in a series of ethanol washes (30%, 50%, 70%, 80%, 90% and 100%; twice for 15 minutes) and evaporated the solvent at room temperature to be finally coated with a thin layer of metal (Pt, 15 nm) to allow observation. SEM images were acquired at different magnifications using a SEM Inspect™ F50 (FEI Company, LMA-INA, Spain) operating at an accelerating voltage of 10-15 keV. 2.9. Statistical analyses. All results are expressed as mean ± SEM. Normal distribution of the variables was analyzed by the Shapiro Wilk test followed by U-Man Whitney, ANOVA or t-Student tests (StataSE 12 statistical software, StataCorp LP, US). Statistically significant differences among groups were considered when p ≤ 0.05.



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3. RESULTS 3.1. Gold nanoparticles characterization. Common excitation of the conduction electrons in metal results in the localized Surface Plasmon Resonance (SPR) phenomenon observed as an absorption band in the visible region. The spectrum of colloidal AuNPs exhibited the maximum resonance absorption at ~525 nm characteristic of spherical AuNPs (Figure 1A)44. The SPR band exhibited an exponential-decay Mie scattering profile with decreasing photon energy. As the concentration of AuNPs increased, the SPR absorption intensity and colloid color intensity increased (Figure 1A). TEM micrographs confirmed the spherical shape and reduced size of AuNPs (Figure 1B). The presence of chitosan grafted to Au NPs was also observed by TEM (Figure 1C). The use of phosphotungstic acid to stain chitosan allowed visualization of the chitosan coating as a homogeneous (ca. 3-4 nm thick) halo around the AuNPs. The STEM-HAADF micrographs with EDX analysis confirmed the AuNPs chemical identity (Figure 1D-E).


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Figure 1. Physicochemical characterization of Au-CS nanocomposites: A) UVVis spectrum of an Au-CS colloid with a characteristic Surface Plasmon Resonance Band ~525 nm. Inset, optical image of Au-CS colloids; B) TEM micrograph of Au-CS NPs; C) TEM micrograph of Au-CS stained NPs. Phosphotungstic acid salt was used as a negative stain to allow electron observation of the chitosan coating. The halo around the Au nanoparticles corresponds to the tungsten-containing chitosan grafted on their surface. D) STEM-HAADF micrograph of Au-CS NPs. E) X-Ray elemental analysis at the area marked in figure 1-D, confirming gold presence.

The enlarged TEM micrographs were used for the determination of colloidal AuNPs size distributions. The average diameter of 1 mM AuNPs was 14 ± 5 nm while for 2



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mM was14 ± 3 nm. The stability of the obtained Au-CS colloids was assessed via Zetapotential (ζ) measurements. The surface charge of Au-CS NPs after synthesis was determined at pH = 4.5 (Zetasizer Nano ZS, Malvern Instruments, UK). For the tested Au-CS colloids, ζ-potential value was 27 ± 7 mV for 1 mM and 30 ± 6 mV for 2 mM, confirming the presence of a positively-charged polymeric layer on the NPs surface and a high colloidal stability. These results indicate that chitosan has a dual role as reducing and stabilizing agent thanks to the coordinative and reductive action of its amino groups12. 3.2. Au-CS nanocomposites cytotoxicity. Alamar Blue™ assay was carried out to evaluate the in vitro toxicity of Au-CS nanocomposites on human macrophages. Figure 2 shows the viability percentages obtained after 24 h of incubation with both Au-CS nanocomposites assayed (gold precursor concentration 1 and 2 mM).


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Figure 2. Macrophages viability after exposure to Au-CS nanocomposites with different gold precursor concentrations for 24 h. The possible interferences of the nanomaterials with the assay or the reagent were evaluated and discarded. Data are expressed as mean ± SEM (n = 5).

Both Au-CS nanocomposites showed a similar fashion exerting very low toxicity (viability higher than 77%) until the highest concentration assayed in which viability was depleted (4-16%). For further experiments, we considered Au-CS colloidal concentrations of 125 µM or lower as subcytotoxic as it is recommended in the ISO 10993-5 norm (Biological evaluation of medical devices - Part 5: Tests for in vitro cytotoxicity), which considers a material as non-cytotoxic when cellular viability is higher than 70%.



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3.3. Bactericidal activity of Au-CS nanocomposites in an in vitro infection model. The antibacterial activity of the nanocomposites obtained from different gold precursor concentrations (1 and 2 mM) was assayed at the two higher subcytotoxic concentrations (62.5 and 125 µM) in the colony counting assay in which the intracellular survival bacteria were grown in TSA plates after incubation with the nanocomposites. Furthermore, the bactericidal effects of these Au-CS nanocomposites concentrations were also tested in bacteria cultures alone (without macrophages). The resulting colonies after 24 h were compared to those obtained with the control samples (bactericidal or infection model non treated with nanocomposites). Figure 3 shows the results obtained regarding CFU/mL and CFU/cell in the co-culture model while Figure S1 exerts the significant reduction in bacteria growth (≥ 95%) after nanocomposites treatment without the presence of mammalian cells. The data showed statistically significant differences between the control samples and the treated ones, pointing to an inhibitory effect of the Au-CS colloids on bacteria growth (≥ 80% of bacteria mortality compared to the control sample) at the concentrations tested and in a dose-dependent manner into each group of nanocomposites (1 mM and 2 mM as initial gold precursor concentrations) being even more accentuated at 125 µM for S. aureus in which 100% of the bacteria were eliminated (Figure 3 C and D).


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Figure 3. Intracellular CFU per mL (A and C) and per cell (B and D) obtained after infection of macrophages with E. coli (A and B) or S. aureus (C and D) and after treatment with Au-CS nanocomposites at different initial gold precursor concentrations (1 and 2 mM) and at final colloid concentrations (62.5 and 125 µM). Control samples showed represent infected macrophages with E. coli (A and B) or S. aureus (C and D) no treated with Au-CS nanocomposites. Data are expressed as mean ± SEM of at least 4 independent experiments performed in triplicate and showed statistically significant differences between the control samples and the treated ones (p≤0.05).



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3.4. Cellular morphology and viability after bacterial infection and Au-CS nanocomposites treatment. The MOIs used in our infection model were set by the evaluation of the suitable dose at which infection was developed maintaining high cell viability. Macrophages viability after infection was determined with the Live/Dead® assay by flow cytometry. Cells were infected with E. coli and S. aureus at MOIs of 5:1 and 10:1, respectively. These MOIs were chosen due to the efficiency in the infection whereas maintaining a high viability of the macrophages which was, compared to the control sample (non-infected macrophages), 94.8% after E. coli infection and 97% in the case of S. aureus. At these infective doses we did not observe any apoptotic response as other authors have previously shown

36,45

. It should be noted that higher MOIs depleted cell viability and

drastically affected cell morphology and were excluded from the study. To further study the changes in cell viability by our infection model and Au-CS nanocomposites treatment, Live/Dead® assay by confocal microscopy was also performed in all the experimental groups developed (Figure 4), staining in green alive cells while death cells were dyed in red. Minimal cell death was observed in control samples, only infected (Figure 4A and 4B) or only treated with colloids (Figure 4C and 4D), though higher cell mortality was observed in the infected groups in which the initial concentration of the gold precursor was 1 mM (Figure 4E and 4G). This fact might contribute to a superior intracellular toxicity and leakage thus a consequent reduction in bacterial viability (as shown in Figure 3). The development of the infection model and the addition of the Au-CS nanocomposites implied the presence of a few more dead cells compared to the control samples, validating the in vitro treatment and infection model.


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Figure 4. Macrophages viability by confocal microscopy under the different experimental conditions assayed. Viable cells are stained in green, death cells in red. A) Co-culture of macrophages and E. coli; B) Co-culture of macrophages and S. aureus; C) Treated macrophages with Au-CS nanocomposites 125 µM, initial gold precursor concentration 1 mM; D) Treated macrophages with Au-CS nanocomposites 125 µM, initial gold precursor concentration 2 mM; E) Co-culture of E. coli with treated macrophages (Au-CS nanocomposites 125 µM, initial gold precursor concentration 1 mM); F) Co-culture of E. coli with treated macrophages (Au-CS nanocomposites 125 µM, initial gold precursor concentration 2 mM); G) Co-culture of S. aureus with treated macrophages (Au-CS nanocomposites 125 µM, initial gold precursor concentration 1 mM); H) Co-culture of S. aureus with treated macrophages (Au-CS nanocomposites 125 µM, initial gold precursor concentration 2 mM). Scale bars represent 500 µm in all the panels. Magnification 20X, insets 10X.

Figure 5 shows the morphology of macrophages before (Figure 5A and 5B) and after infection (Figure 5C-F). These images support the results explained above regarding the



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high viability and suitable morphology of human cells after co-culture with bacteria as well as the absence of extracellular bacteria. This was further confirmed with the incubation of bacteria alone with gentamycin not obtaining bacterial growth (results not shown).

Figure 5. SEM micrographs showing cell morphology: A and B) untreated macrophages; C and D) Macrophages infected with E. coli; E and F) Macrophages infected with S. aureus.


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3.5. Au-CS nanocomposites bactericidal mechanism. Flow cytometry and SEM were the methodologies used to elucidate the mechanism by which Au-CS colloids exerted their lethal effects against bacteria. Table 1 shows the viability of bacteria treated with colloids and precursors. These data displayed a high viability for bacteria treated with precursors at the concentrations used in the NPs formulations while the addition of the Au-CS nanocomposites (final concentration 125 µM) decreased viability significantly being more accentuated in S. aureus though precursors hardly reduced their viability. The depletion in viability percentages indicates an increase in the access of the dye, propidium iodide, to the bacteria nucleic acids, pointing to cell wall and membrane leakage as the colloids lethal mechanism in the microorganisms assayed.



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Table 1. Bacteria viability determined by flow cytometry after treatment for 2h with Au-CS nanocomposites and their precursors. Nanocomposites were assayed at a final concentration of 125 µM from both initial gold precursor concentrations (1 and 2 mM). Percentages are referred to control sample (bacteria non treated = 100% viability)

Sample

% Viability

E. coli + chitosan precursor

62.6

E. coli + gold precursor

70.0

E. coli + nanocomposites 1 mM

10.6

E. coli + nanocomposites 2 mM

14.4

S. aureus + chitosan precursor

84.5

S. aureus + gold precursor

83.0

S. aureus + nanocomposites 1 mM

4.5

S. aureus + nanocomposites 2 mM

4.3

To further confirm that cell disruption is the mechanism for which our Au-CS nanocomposites eliminate bacteria, SEM micrographs of both model microorganisms


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were acquired after treatment with the colloids (Figure 6). These images clearly show a smooth surface in the control samples (Figure 6A and C) and the disruption of the cell outer envelope in treated bacteria (Figure 6B and D) displaying also the elongation of E. coli cells (Figure 6B) and the complete destruction of S. aureus structure (Figure 6D), as well as the adhesion of bacterial debris to the filters surface, which is consistent with the results obtained in the CFUs assay described above (Figure 3).

Figure 6. Bacteria morphology observed by SEM before (A and C) and after (B and D) treatment with colloids (final concentration 125 µM, initial gold precursor concentration 1 mM): A) E. coli; B) E. coli treated with Au-CS nanocomposites; C) S. aureus; D) S. aureus treated with Au-CS nanocomposites.



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4. DISCUSSION Microorganism´s resistance to drugs has become a serious problem in the treatment of infections and results in the search of novel antibiotic-free approaches as a topic of substantial research. Our previous studies have shown the Au-CS nanocomposites antibacterial effects against S. aureus and Pseudomonas aeruginosa as well as the nontoxicity of colloids on A549 and HaCaT in vitro cell cultures12, showing also the effects in the bacterial wall after contact with nanocomposite-based films. In our previous work12, the biocompatible and biodegradable polymer CS was used as reducing and stabilizing agent for the optimized in situ synthesis of colloidal AuNPs. The influence of CS properties, such as average molecular weight and deacetylation degree, on the resulting AuNPs characteristics was studied for the first time. CS with medium molecular weight appeared to be the best stabilizing agent for AuNPs. Using that chitosan, the obtained spherical AuNPs showed the smallest diameter and the most uniform distribution compared to the other chitosans tested. The presence of numerous amino groups responsible for NPs formation resulted in the highest tetrachloroaurate ions reduction rate. Insufficient AuNPs stabilization and aggregation was observed when using low and high molecular weight chitosans. The resulting nanocomposites demonstrated a total bactericidal effect against biofilm forming strains of Pseudomonas aeruginosa and S. aureus. Importantly, the reduced or totally excluded cytotoxic effect was confirmed by in vitro cell viability studies. In the present work, we have focused our efforts on the effect of the colloids in an in vitro infection model. In this approach the human barrier against pathogens, macrophages, were involved, trying to simulate a natural occurring infection with intracellular pathogens. In our experimental system, Au-CS colloids exerted a low cytotoxicity and a high bactericidal effect, mainly in the Gram-positive model, showing that the lethal mechanism proceeded by the disruption of


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the bacterial wall. This finding is consistent with our previous studies in which the antibacterial activity of Au-CS films against S. aureus and P. aeruginosa was most prominent against the Gram-positive strain12, pointing to the presence of the additional outer cell membrane in Gram-negative bacteria as the potential mechanism to exert higher resistance to the bactericidal agent. Furthermore, we show the co-culture model as a useful tool to investigate the bactericidal potential of novel materials as well as for applications such as screening of intracellular and extracellular activity of pathogenic bacteria. Previous studies have hypothesized the bactericidal mechanisms of CS and AuNPs though the exact mechanism is not yet fully understood46,47. Antimicrobial effects of AuNPs have been attributed mainly to cell wall damage, due to the interaction of NPs with sulfur-containing proteins on the cell membrane, but also to the generation of reactive oxygen species, even though the NPs characteristics, such as shape, size or surface area, are crucial in the development of bactericidal activity12,19,20. In fact, smaller NPs displayed more harmful effects in bacteria though without damaging eukaryotic cells at the doses tested12,48. Moreover, CS has been postulated as a harmful polycationic biopolymer for bacteria cell wall due to charge interactions showing higher antimicrobial activity with higher CS deacetylation degree and number of amino groups though lower molecular weights27,49,50. Even though other bactericidal CS mechanisms have been also proposed, such as the binding to DNA or to essential microbial nutrients, cell wall damage and leakage of intracellular components seem to be the most prevalent antimicrobial effects but occurring the three mechanisms simultaneously35. The antimicrobial efficacy against Gram-negative or Gram-positive bacteria is a controversial issue pointing to CS as more effective against Gram-negative bacteria due to higher hydrophilicity of Gram-negative envelope favoring adhesion attributed to



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chemical interactions though other reports clearly showed a higher activity against Gram-positive2,27,51,52. The charge density on the bacteria surface determines CS adsorption, thus at higher amount of CS adsorbed, higher cell wall damage was observed, so the bactericidal mechanism depends really on the host bacteria and not in Gram staining2. Our results clearly showed the harmful activity of Au-CS colloids to bacteria wall fully changing cell morphology and favoring cell lysis principally in S. aureus. Gold and CS have been widely shown as bacteriostatic or bactericidal materials combined with different compounds though not displaying full antimicrobial activity as our Au-CS nanocomposites. Chitlac-nAg coating, a nanocomposite composed of modified CS and silver NPs, has been shown as an effective bacteria growth inhibitor against S. aureus, P. aeruginosa or Streptococcus mitis53,54, although not achieving a full bactericidal effect even at high silver concentrations (0.5 and 1 mM Ag vs. 0.125 mM Au in our studies). CS-based Ag NPs displayed inhibitory activity against S. aureus and other bacteria species achieving up to 75% of bacteria killing. However, these authors clearly also showed that the cell wall damage was the bactericidal mechanism. They also showed the intracellular killing ability of these nanomaterials in an infection model composed of Mycobacterium smegmatis and macrophages in which the pretreatment with CS-based Ag NPs was more effective than the post-treatment and the antimicrobial effect was found as concentration-dependent2, in agreement with our results. The use of other materials such as photoexcited quantum dots (QDs) that modify the cellular redox state eliminating bacteria without damaging HEK 293T cells have been shown as bactericidal for different bacteria cultures, including E. coli and S. aureus, showing a significant reduction of bacteria growth, though not complete, when materials were illuminated with light4.


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In summary, Au-CS colloids showed bactericidal ability without damaging human macrophages in an in vitro infection model, exerting bacteria wall damage as killing mechanism. These results point to the potential application of these nanomaterials for biomedical purposes avoiding antimicrobial infections and preserving tissue integrity. 5. CONCLUSION Au-CS nanocomposites successfully showed concentration-dependent antibacterial activities against E. coli as Gram-negative and S. aureus as Gram-positive bacteria model avoiding the damage to host mammalian cells. CFU assays together with morphological and viability studies highlighted the suitability of the in vitro treatment and the co-culture model for the evaluation of novel antibacterial nanomaterials. In addition, bacterial death mechanism mediated by our Au-CS colloids may be attributed to cell wall disruption and intracellular content leakage as flow cytometry and SEM studies have shown. The results of our research underline the importance of the search for novel efficient antibacterial materials in order to solve the multidrug resistances associated to antibiotics using a co-culture model which closely mimics a real infection scenario. AUTHOR INFORMATION Corresponding Author *

Email: [email protected] Tel.: +34 876 554 342.

Notes The authors declare no competing financial interests.

SUPPORTING

INFORMATION

AVAILABLE:

Figure

S1

including

the

antibacterial effects of Au-CS nanocomposites against E. coli and S. aureus alone. This material is available free of charge via the Internet at http://pubs.acs.org.



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ACKNOWLEDGEMENTS Financial support from the EU thanks to the ERC Consolidator Grant program (ERC2013-CoG-614715, NANOHEDONISM) is gratefully acknowledged. CIBER-BBN is an initiative funded by the VI National R&D&i Plan 2008–2011, Iniciativa Ingenio 2010, Consolider Program, CIBER Actions and financed by the Instituto de Salud Carlos III (Spain) with assistance from the European Regional Development Fund. We acknowledge the LMA-INA and Microscopy, Cytometry and Cell Culture Core Units from IACS/IIS Aragon for their instruments and expertise. The authors are grateful to Dr Jose Antonio Ainsa, Dr Ainhoa Lucia, Dr Liliana Rodrigues and Mrs Begoña Gracia for helpful advice and comments. REFERENCES (1)

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