Antimicrobial activity of a ferrocene-substituted carborane derivative targeting multidrug-resistant infection

Biomaterials 34 (2013) 902e911

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Antimicrobial activity of a ferrocene-substituted carborane derivative targeting multidrug-resistant infection Shuihong Li a, Zhaojin Wang b, Yuanfeng Wei c, Changyu Wu a, Shengping Gao a, Hui Jiang a, Xinqing Zhao d, Hong Yan b, Xuemei Wang a, * a

State Key Laboratory of Bioelectronics (Chien-Shiung Wu Lab), Southeast University, Nanjing 210096, China School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210093, China Institute of Traditional Chinese Medicine, China Pharmaceutical University, Nanjing 210009, China d School of Life Science and Biotechnology, Dalian University of Technology, Dalian 116024, China b c

a r t i c l e i n f o

a b s t r a c t

Article history: Received 31 August 2012 Accepted 30 October 2012 Available online 19 November 2012

Multidrug resistance (MDR) of bacteria is still an unsolved serious problem to threaten the health of human beings. Developing new antibacterial agents, therefore, are urgently needed. Herein, we have explored the possibility to design and synthesize some novel antibacterial agents including ferrocenesubstituted carborane derivative (Fc2SBCp1) and have evaluated the relevant antibacterial action against two clinical common MDR pathogens (i.e., Gram-positive Staphylococcus aureus and Gramnegative Pseudomonas aeruginosa) in vitro and in vivo. The results demonstrate that in vitro antimicrobial activity of Fc2SBCp1 could be gradually transformed into a bactericidal effect from a bacteriostatic effect with the increasing concentration of the active carborane derivative, which can also prevent biofilm formation at concentrations below MIC (i.e., minimal inhibitory concentration). Biocompatibility studies indicate that there exists no/or little toxic effect of Fc2SBCp1 on normal cells/tissues and leads to little hemolysis. In vivo studies illustrate that the new carborane derivative Fc2SBCp1 is highly effective in treating bacteremia caused by S. aureus and P. aeruginosa as well as interstitial pneumonia caused by S. aureus. This raises the possibility for the potential utilization of the new ferrocene-substituted carborane derivatives as promising antibacterial therapeutic agents against MDR bacterial infections in future clinical applications. Ó 2012 Elsevier Ltd. All rights reserved.

Keywords: Antibacterial Antibiofilm Multidrug-resistant infection Ferrocene-substituted carborane derivative Staphylococcus aureus Pseudomonas aeruginosa

1. Introduction Since antibiotics were widely employed for treating bacterial infections, the repeated emergence of antibiotic-resistant bacterial strains has been a serious problem that has long plagued public health. Development of new antimicrobial agents is more urgently needed than ever because of the unprecedented increase of multidrug resistance in common pathogens and the rapid emergence of new infections [1]. Staphylococcus aureus (S. aureus, Gram-positive bacteria) and Pseudomonas aeruginosa (P. aeruginosa, Gram-negative bacteria) are two of the major causes of fatal nosocomial infections as well as community-acquired infections [2]. The spread of these organisms in healthcare settings are often difficult to control due to the presence of multiple intrinsic and acquired mechanisms of

* Corresponding author. Tel./fax: þ86 25 83792177. E-mail address: [email protected] (X. Wang). 0142-9612/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biomaterials.2012.10.069

antimicrobial resistance. A biofilm is a structured consortium of bacteria embedded in a self-produced polymer matrix consisting of polysaccharide, protein and DNA [3]. Bacterial biofilms are notoriously difficult to eradicate and are a source of many recalcitrant infections. The biofilm-positive strains such as S. aureus and P. aeruginosa can form recalcitrant biofilms under antibiotic pressure. The survival of cells in a preformed biofilm is not easily eradicated by relevant antimicrobials, hence generating an increased resistance of cells and undoubtedly contributing to produce more drug resistance mutations [4]. Recently, enormous efforts have been directed to the design, synthesis and evaluation of biomedical activities of various metal complexes in medicinal organometallic chemistry, in which a typical metallocene, ferrocene derivative, has attracted special attention because of its small size, relative lipophilicity, ease of chemical modification, and accessible one-electron-oxidation potential [5e8]. Ferrocenyl derivatives exhibit promising bioactivities like antineoplastic [9], antimalarial [10,11], antifungal [12], antibacterial [13] and others. Currently, incorporation of ferrocenyl

S. Li et al. / Biomaterials 34 (2013) 902e911

moieties into the structures of the existing drug molecules has demonstrated an important strategy to increase their therapeutic properties. Meanwhile, as a promising and potential pharmophore, the three-dimensional carborane cage, with its structural integrity, ease of substitution, and delocalized bonding, allows the bioisosteric replacement for phenyl rings as rigid scaffolding in bioactive molecules and pharmacological agents, which has lately been attracting much attention in biomedical applications owing to its extraordinary characteristics [14]. Thus, in this contribution, we have explored the possibility to combine the ferrocenyl moieties with icosahedral carborane to design a ferrocene-substituted carborane derivative (designated as Fc2SBCp1, see Scheme 1) as a promising and effective antibacterial therapeutic agent against MDR infections in future clinical research. 2. Materials and methods

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effects and empirical absorption with the SADABS [17] program. The structures were solved by direct methods using the SHELXL-97 [17] program. 2.4. Time-kill studies For all of the in vitro tests, the solid powder of Fc2SBCp1 was dissolved in dimethylsulfoxide (DMSO) and diluted with bacterial culture medium. For all of the vivo experiments, it was dissolved in DMSO and diluted with phosphate buffer solution (PBS, pH ¼ 7.2). All of the DMSO concentration was controlled below 0.5% (v/v) to ensure that it has no physiological toxicity. Initially, the in vitro antimicrobial activity of Fc2SBCp1 against S. aureus and P. aeruginosa at a starting inoculum of ca. 2  105 cells/mL was determined following CLSI guidelines [18]. Then, bacterial suspensions supplemented with Fc2SBCp1 (0.5  MIC, 1.0  MIC, 1.0  MBC, 2.0  MBC) were incubated at 37  C (for S. aureus) or 30  C (for P. aeruginosa) for various time intervals (0, 2, 4, 6 and 24 h). At each time point, the viable colony counts were performed on TSA plates after incubating at 37  C or 30  C. 2.5. Confocal laser scanning microscopy (CLSM) assays

2.1. Reagents Acridine orange, crystal violet, fluorescein isothiocyanate isomer I (FITC-I), propidium iodide (PI) and pentobarbital sodium were purchased from SunShineBio Technology Co., Ltd. Cell alkaline phosphatase stain (cAKP stain) was purchased from Nanjing Jiancheng Technology Co., Ltd. Isoflurane was purchased from Shandong keyuan pharmaceutical Co., Ltd. Tryptic soy broth (TSB), tryptic soy agar (TSA) plates, Mueller-Hinton Broth (MHB) and Mueller-Hinton Agar (MHA) were purchased by OXOID (Basingstoke, UK). Ultrapure water (18.2 MU cm; Milli-Q, Millipore) was used for the preparation of all aqueous solutions. All chemicals are of analytical reagent grade and were used without further purification.

Overnight cultured strains incubated in TSBgluc 0.5% were diluted to a final density of 1.0  105 CFU/mL with fresh medium and dispensed into each well (preloaded with plastic cover slips) of a plastic 24-well plate. The plates were statically incubated at 37  C for 48 h. Afterwards, the cover slips were taken out and gently washed with 0.01 M phosphate-buffered saline (PBS) three times to wash off the non-adherent bacteria. After eliminating the moisture, the materials on the cover slips were stained with 20 mL acridine orange (0.02%, w/v) for 15 min at 4  C in the dark. Stained cover slips were gently washed twice with PBS and observed with CLSM (Zeiss LSM 710, Germany). 2.6. Scanning electron microscope (SEM) studies

2.2. Synthesis of Fc2SBCp1 All synthesis experiments were performed under an argon atmosphere using standard Schlenk techniques. Solvents were dried by refluxing over sodium (petroleum ether, ether) or calcium hydride (dichloromethane) under nitrogen and then distilled prior to use. The starting compounds CpCo(S2C2B10H10) [15] (Cp ¼ cyclopentadienyl) and ferrocenyl acetylenic ketone (HChCeC(O)Fc) [16] were prepared according to the literature methods. To a solution of CpCo(S2C2B10H10) (66.0 mg, 0.20 mmol) in tetrahydrofuran (THF, 25 mL) was added the HChCeC(O)Fc (71.4 mg, 0.30 mmol). The resulting mixture was stirred overnight at 65  C. After removal of solvent under vacuum, the residue was chromatographed on silica gel (100e200 mesh). Gradient elution with 10 mL petroleum ether/ CH2Cl2 (v/v) ¼ 1/1, 1/3, 1/5, and CH2Cl2 alone respectively. Then the silica gel column was eluted with ether. The concentrated solution of the eluate was purified using thin layer chromatography (TLC). Elution with petroleum ether/CH2Cl2 (1:1) gave the compound Fc2SBCp1 (Z isomer) 12 mg in 8% yield obtained (Scheme 1).. Here we only report the antibacterial properties of Fc2SBCp1 since it has better antibacterial activity than the other isomer. 2.3. Characterization of Fc2SBCp1 Elemental analysis was performed in an Elementar Vario EL III elemental analyzer. NMR data were recorded on a BrukerDRX-500 spectrometer. 1H NMR and 13 C NMR spectra were reported in ppm with respect to CHCl3/CDCl3 (d 1 H ¼ 7.24 ppm, d 13C ¼ 77.0 ppm) and 11B NMR spectra were reported in ppm with respect to external Et2OeBF3 (d 11B ¼ 0 ppm). The FTIR spectra were recorded on a Thermo Nicolet AVATAR 360 FTIR spectrophotometer with KBr pellets in the 4000e400 cm1 region. The mass spectra were recorded on Micromass GC-TOF for EI-MS (70 eV). X-ray crystallographic data (see Table S1) were collected on a Bruker SMART Apex II CCD diffractometer using graphite-monochromated Mo Ka (l ¼ 0.71073 Å) radiation. The intensities were corrected for Lorentz-polarization

Overnight cultured bacteria were diluted with fresh TSB medium to the cell density of 1.0  107 CFU/mL and dispensed into each well (pre-loaded with a silicon slice) of plastic 6-well plates. Treated groups were added 1.0  MBC or 2.0  MBC Fc2SBCp1, Untreated controls were added the same volume of 0.5% (v/v) DMSO aqueous solution. Then the 6-well plates were put into biochemistry incubators incubating at 37  C for 1 h. Prior to imaging, the bacteria were fixed and dehydrated. Briefly, the silicon slices were initially fixed by 2.5% glutaraldehyde for 2 h at 4  C. The surfaces were washed twice with 0.01 M PBS for 30 min. The silicon slices were post-fixed with 1% osmium tetraoxide in 0.1 M PBS for 30 min. The bacteria were then dehydrated with graded ethanol series (30%, 50%, 70%, 80%, 90%, 95% and 100%) for 15 min each. After critical point drying, a small amount of gold was sputtered on the samples to avoid charging in the microscope. The scanning electron microscopic (SEM) images were obtained on an ultra plus field emission SEM (Zeiss, Germany), with an acceleration potential at 10 kV. 2.7. Fluorescence assay The cell density of 5.0  107 CFU/mL of S. aureus or P. aeruginosa suspension was mixed with Fc2SBCp1 at a final concentration of their corresponding minimal bactericidal concentration (MBC) at 37  C for 4 h on an orbital shaker at 200 rpm. After filtration through nylon membrane filters (pore size 220 nm) to remove bacteria, the bacterial suspensions were centrifuged at 5000 rpm for 5 min to remove bacteria or bacterial debris. The control assay was performed without Fc2SBCp1. For testing the leakage of cytoplasmic contents of nucleic acids or proteins, the supernatants were cultured with an equal volume of PI (a red fluorescent dye) solution in PBS or a same volume of FITC-I (a green fluorescent dye) solution in PBS in the dark for 30 min at room temperature, washed them with PBS twice, and placed 20 mL of samples on a glass slide with a glass coverslip. We observed the fluorescence excited by a 535 nm (for PI-DNA complex) or 495 nm (for FITC-I-protein complex) laser with a CLSM.

Scheme 1. Synthesis procedure of Fc2SBCp1.

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2.8. Animals and procedures Animal experiments were reviewed, approved, and supervised by the Jiangsu province Institutional Animal Care and Use Committee of China. Pathogen-free 8week-old male Kunming mice (i.e. Swiss mice), of clean grade, weighing from 28 to 32 g, were obtained from Qinglongshan animal farm of Nanjing and were maintained in high efficiency particulate air-filtered barrier units kept inside biological safety cabinets for the duration of the experiments. Mice were given free access to tap water and pelleted rodent food and were kept at 25  C with alternating 12-h periods of light and dark. Animals were randomly assigned to three groups (unless otherwise noted), i.e., normal control group, untreated group, treated group. Before using, bacteria were grown in TSB, centrifuged at 5000 rpm for 5 min, washed and suspended in sterile PBS, and maintained at 37  C. Fc2SBCp1 suspension was prepared in accordance with the method mentioned in Section 2.4. Animals were clearly moribund or on the verge of death were killed with an overdose of pentobarbital sodium (150 mg/kg). 2.9. Bacteremia model and blood bacterial counts Mice were lightly anesthetized with isoflurane and inoculated by the tail vein injection with 50 mL of inoculum of S. aureus (1.8  105 CFU/mL) or P. aeruginosa (1.2  105 CFU/mL) suspension in PBS. Fc2SBCp1 suspension was immediately administered by tail vein injection at the designed dosage (60 mg/kg body weight/ day). 20 mL blood were obtained from the tail vein at various time points, diluted in sterile saline and determined by plating serial dilutions onto TSA plates for viable count (cfu/ml blood).

incubation of blood smears with naphthol ASeBI phosphate at 37  C for 15 min, the samples were stained with 1% hematoxylin for 3 min. The degree of the enzyme activity in each cell, as detected by red spots, was rated from 0 to V on the basis of the number of precipitated red granules in the cytoplasm. The sum of the rating of 100 cells was considered the NAP score in a given sample. The entire analysis of NAP activity was carried out by two specialists in their hematological laboratory. 2.11. S. aureus pneumonia model and lung bacterial counts The pneumonia model was a modification of that described by Lee MH et al [20]. Mice were intranasally infected with 50 mL inoculum of S. aureus (1.5  108 CFU/mL) suspension in PBS applied dropwise to the nares. The mice were hooked on a string by their front teeth and allowed to aspirate the inoculum for 10 min, before being returned to cages to recover. After administering 60 mg Fc2SBCp1/kg body weight/ day for different time, the mice were killed with pentobarbital sodium and removed lung tissue. The bacterial density in the lung tissue samples were homogenized, and plated onto TSA plates for bacterial colony counts. Results were expressed as the number of CFU per lung. 2.12. Histopathology The method was the same as the above section except the difference of handling lung. The experimental method of histopathology was performed according to the existing research [21] with appropriate modifications. Briefly, the fresh removed lungs were fixed in 10% formalin overnight before being embedded in paraffin. Fivemicrometer sections of tissue were stained with hematoxylin/eosin before being examined.

2.10. Analysis of neutrophil alkaline phosphatase (NAP) activity 2.13. Statistical methods Using the relevant venous blood samples of the above animal model, the cytochemistry analysis of NAP activity was evaluated according to the method described by Kaplow [19] and performed in accordance with the kit protocol. To investigate whether Fc2SBCp1 can interfere with the test results, a group of normal mice inject Fc2SBCp1 at the same dosage was supplemented. The blood smears were made of blood samples and fixed with formaldehyde/methanol (v/v) ¼ 1:9 after the

All the data are presented as the mean  SD. The statistical analysis was done using the statistical software “SAS 9.0”. Analysis of variance (ANOVA) and T-test were used for significance testing of between groups, and P < 0.05 is considered to be statistically significant difference. All experiments were performed in triplicate and repeated three times.

Fig. 1. Molecular structure of Fc2SBCp1. The single crystals were grown in petroleum ether/CH2Cl2 (v/v) ¼ 1:1. Thermal ellipsoids are depicted at 30% probability; all hydrogen atoms are omitted for clarity. Selected bond lengths (Å) and angles ( ): C(1)eC(2) 1.767(4), C(1)eS(1) 1.809(3), C(2)eS(2) 1.767(3), C(3)eC(4) 1.333(5), C(3)eS(1) 1.716(3), C(7)eC(8) 1.543(4), C(7)eC(14) 1.595(4), C(7)eS(2) 1.836(3), C(8)eC(11) 1.559(4), C(11)eC(12) 1.486(4), C(11)eC(15) 1.534(4), C(12)eC(13) 1.328(4), C(13)eC(14) 1.516(4), C(14)eC(15) 1.541(4), C(2)eC(1)eS(1) 115.65(19), S(2)eC(2)eC(1) 116.36(19), C(8)eC(7)eC(14) 104.6(2), C(8)eC(7)eS(2) 114.6(19), C(14)eC(7)eS(2) 107.85(18), C(9)eC(8)eC(7) 112.6(2), C(7)e C(8)eC(11) 100.9(2), C(13)eC(12)eC(11) 107.3(3), C(12)eC(13)eC(14) 108.6(3), C(13)eC(14)eC(15) 98.6(2), C(13)eC(14)eC(7) 101.7(2), C(15)eC(14)eC(7) 100.0(2), B(3)eC(14)eC(7) 107.2(2), C(11)eC(15)eC(14) 94.2(2), C(3)eS(1)eC(1) 100.16(15), C(2)eS(2)eC(7) 93.33(13).

S. Li et al. / Biomaterials 34 (2013) 902e911 Table 1 In vitro antibacterial activity and antibiofilm effect of Fc2SBCp1 against two clinical isolates of S. aureus and P. aeruginosa. Organism

MICa

MBCb

MBIC50c

MBIC90d

S. aureus P. aeruginosa

36 36

72 72

4 6

14 16

a

Minimum inhibitory concentration. Minimum bactericidal concentration. c Minimum biofilm inhibition concentration of Fc2SBCp1 that showed 50% inhibition on the biofilm formation. d Minimum biofilm inhibition concentration of Fc2SBCp1 that showed 90% inhibition on the biofilm formation. b

3. Results and discussion

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3.2. MIC, MBC, MBIC50 and MBIC90 determination The MIC, MBC, MBIC50 and MBIC90 distributions of Fc2SBCp1 against S. aureus and P. aeruginosa were shown in Table 1. It is observed that Fc2SBCp1 demonstrated nearly similar antimicrobial and antibiofilm activity against the two strains with the same MIC/ MBC values and similar MBIC50/MBIC90. For both strains, it exhibited bacteriostasis activity at 36 mg/mL (MIC value) and bactericidal activity at 72 mg/mL (MBC value). The MBIC50 values of Fc2SBCp1 were observed for S. aureus (4 mg/ml) and P. aeruginosa (6 mg/ml). Meanwhile, our results demonstrated that Fc2SBCp1 induced prevention of 90% of biofilm formation of S. aureus and P. aeruginosa when used at 14 and 16 mg/ml. All of the MBIC50 and MBIC90 values were lower than their MIC values, suggesting its strong biofilm inhibition efficiency.

3.1. Characterization of Fc2SBCp1 3.3. Time-kill assay Single crystals for X-ray diffraction of Fc2SBCp1 were grown in petroleum ether/CH2Cl2 (v/v) ¼ 1:1 and the solid structure is shown in Fig. 1. The synthetic compound Fc2SBCp1 is a reddish-brown solid, mp 240  C (dec.), 1H NMR (CDCl3) (Fig. S1): d 7.38 (d, 1H, J ¼ 10Hz, C(3)H), 6.75 (d, 1H, J ¼ 10Hz, C(4)H), 6.55 (d, 1H, J ¼ 5.5 Hz, C(13)H), 6.02 (dd, 1H, J ¼ 5.5, 3.0 Hz, C(12)H), 4.85(m, 1H, Fc), 4.83(m, 1H, Fc), 4.80(m, 1H, Fc), 4.76(m, 1H, Fc), 4.62 (m, 1H, Fc), 4.61 (m, 1H, Fc), 4.56 (dd, 1H, J ¼ 5.0, 1.5 Hz, C(7)H), 4.53 (m, 1H, Fc), 4.52 (m, 1H, Fc), 4.22 (s, 5H, Cp), 4.15 (s, 5H, Cp), 3.56 (dd, J ¼ 5.0, 3.0 Hz, 1H, C(8)H), 3.43 (m, 1H, C(11)H), 2.84 (d, 1H, J ¼ 9.0 Hz, C(15) H), 1.68 (d, 1H, J ¼ 9.0 Hz, C(15)H). 11B NMR (CDCl3): d 0.38 (3B), 0.60 (2B), 5.27 (4B), 7.82 (1B). 13C NMR (CDCl3) (Fig. S2): d 201.15 (CO), 192.99 (CO), 142.38 (C(3)), 140.42 (C(13)), 133.16(C(12)), 120.91 (C(4)), 99.06, 96.12 (carborane), 78.80 (Fc),78.17 (Fc),73.62 (C(7)), 73.28, 73.27, 72.60, 72.57 (Fc), 70.15 (Cp), 69.76 (Cp), 69.78, 69.60 ((Fc), 69.33, 68.92 (Fc), 55.12 (br, C(14)), 54.30 (C(11)), 52.49 (C(8)), 50.14 (C(15)). EI-MS (70 eV): m/z 748.3 (Mþ, 100%). Anal. calcd for C33H36B10O2S2Fe2: C, 52.95; H, 4.85. Found: C, 52.79; H, 4.95%. The 1 H NMR and 13C NMR spectra and the molecular structure were clearly identified by the two-dimensional NMR spectroscopy of 1 H-1H correlation spectroscopy (COSY) (Fig. S3) and 1H-13Cconnectivity (HMQC, HMBC) (Fig. S4). IR (KBr) (Fig. S5): n (cm1): 1660.5 and 1625.2 (C]O), 2591.8 (BeH). In UVeVisible absorption spectrum (in DMSO/PBS (v/v) ¼ 1/50 (pH ¼ 7.2), the absorption peak of compound Fc2SBCp1 appears at 484  1 nm and this signal was stable for at least 56 days (Fig. S6).

Time-kill assay is capable of detecting differences in the rate and extent of antibacterial agent activity over time and are better suited for assessing changes in the antibacterial agent activity [22,23]. A bactericidal effect is defined as a 3 log10 decrease in the CFU/mL or a 99.9% kill over a specified time [24,25]. As presented in Fig. 2, Fc2SBCp1 show a concentration- and time-dependent manner against S. aureus (Fig. 2A) and P. aeruginosa (Fig. 2B), whilst its antibiotic effects gradually change the effect from a bacteriostatic to a bactericidal action with the increasing of the Fc2SBCp1 concentration. The bactericidal activity of Fc2SBCp1 was fast-acting against S. aureus and P. aeruginosa at concentration of 1  MBC (72 mg/mL) and 2  MBC (144 mg/mL); the reduction in the CFU/mL was >3 log10 CFU/mL (99.99%). The bactericidal endpoints of Fc2SBCp1 against S. aureus were 2 h at 1  MBC and 4 h at 2  MBC, meanwhile its endpoint against P. aeruginosa was 6 h at 2  MBC. Thus, it is evident that Fc2SBCp1 has effective antibacterial activity against both S. aureus and P. aeruginosa. 3.4. CLSM visualization of biofilm formation Acridine orange is usually used as a fluorescent biofilm biomass indicator as this compound stains all cells in a biofilm, alive or dead [26,27]. In this study, the Leica confocal microscope and software was used for analysis of biofilm images, which allowed for collection of relevant three-dimensional (3D) reconstruction. Images were acquired from random positions of biofilms formed on the upper side

Fig. 2. Time-kill curves of Fc2SBCp1 against S. aureus (A) and P. aeruginosa (B) at different concentrations with different time.

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S. aureus only H=5.5µ µm

P. aeruginosa only H=5.8µm

+ 0.25 × MIC H=4.3µm

+ 0.25 × MIC H=3.9µm

+ 0.5 × MIC H=2.0µm

+ 0.5 × MIC H=2.8µm

Fig. 3. CLSM analysis of biofilms formed by S. aureus and P. aeruginosa incubated with 0, 0.25  MIC (9 mg/mL) and 0.5  MIC (18 mg/mL) of Fc2SBCp1 for 48 h. The images show the reconstructed 3D biofilm images at a magnification of 630. Biofilms were stained with acridine orange, resulting in all bacteria appearing green (including dead and live bacteria) as observed by CLSM. Scale bars ¼ 10 mm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

of the cover slips. Fig. 3 shows spatial biomass distributions of S. aureus and P. aeruginosa in 48-h-old biofilms in the absence or presence of Fc2SBCp1, as they appear by visual inspection of CSLM images characterizing the 3D structures. As shown in Fig. 3, CLSM zsection analyses can provide surface coverage of the biofilms as well

as the entire thickness of bacterial biofilms, where S. aureus and P. aeruginosa formed thick biofilms with compact architecture characterized by large clumps that were separated by water channels when grown in the absence of Fc2SBCp1. In contrast, the biofilm of S. aureus formed smaller aggregate of microorganisms and looser

Fig. 4. SEM micrographs of S. aureus and P. aeruginosa treated with different concentration of Fc2SBCp1. Scale bars ¼ 1 mm.

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S. aureus only

+ 1 × MBC

P. aeruginosa only

+ 1 × MBC

S. aureus only

+ 1 × MBC

P. aeruginosa only

+ 1 × MBC

Fig. 5. Monitoring Fc2SBCp1 induced permeability of cell membranes and leakage of nucleic acids and proteins via PI and FITC-I. The upper four fluorescence images show the nucleic acids leakage, while the bottom four fluorescence images show the proteins leakage.

structure than that of P. aeruginosa. Meanwhile, in the presence of Fc2SBCp1, at a concentration of 0.25  MIC, the biofilms of the both strains were much thinner than those of the respective strains incubated without Fc2SBCp1, with the biofilm thickness decreasing from 5.5 mm to 4.3 mm and 5.8 mm to 3.9 mm, respectively. Besides, the surface coverage and the density of bacteria of biofilms was significantly decreased. At concentrations of 0.5  MIC, the biofilm formation was almost completely inhibited by Fc2SBCp1 and thus only few biofilms were observed, indicating that it can effectively prevent biofilm formation on solid surfaces at very low concentration. 3.5. Characterization of bacterial cell damage Moreover, SEM was used to examine the ultrastructural changes in bacteria induced by Fc2SBCp1. It is noted that the untreated S. aureus and P. aeruginosa cells displayed a smooth and intact

surface (Fig. 4A1 and B1). After incubation with 1  MBC of Fc2SBCp1 for 1 h, some of S. aureus cells had single blisters and dents in their cell wall (Fig. 4A2). After treatment of S. aureus with 2  MBC of Fc2SBCp1, some bacteria had burst with deep destroy in their cell wall, with numerous lysed cells and cell debris observed (Fig. 4A3). Similar results were observed for P. aeruginosa cells (Fig. 4B1e3), indicating that Fc2SBCp1 can efficiently damage relevant bacterial cell wall and cause membrane damage. To further investigate whether the permeability of cell membranes dramatically changed in the presence of Fc2SBCp1, the relevant samples were treated with propidium iodide (PI) and FITCI. PI can bind DNA or RNA specifically to acquire enhanced fluorescence, but it cannot cross the membrane and is excluded from viable cells [28]. Thus, another general indication of drug-induced bacterial cell injury is the leakage of nucleic acid and protein from cells, where intracellular staining of PI can identify dead cells. Fluorescein Isothiocyanate (FITC) is widely used in biology and

Fig. 6. Blood bacterial counts of bacteremia mice infected with S. aureus (A) or P. aeruginosa (B). N: normal mice; I-1 and I-3: infected but untreated mice for 1 and 3 days respectively; T-1 and T-3: treated with Fc2SBCp1 mice for 1 and 3 days respectively.

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medicine as a fluorescent marker for labeling various proteins. After treating suspensions of S. aureus or P. aeruginosa with Fc2SBCp1 at 37  C for 4 h and staining them with PI and FITC-I, fluorescence images show that the permeability of treated bacteria increases (Fig. 5A and B). The diffused fluorescence clusters occurring beyond cells imply that some nucleic acids have leaked out of the cells. Nucleic acids are well known to absorb UV light at a wavelength of 260 nm. The amount of nucleic acids released into the cell suspension of the two bacterial strains was analyzed by measuring the absorbance at 260 nm (Fig. S7A). It is observed that the amount of leaked nucleic acid from the cells increased with the increasing of the Fc2SBCp1 concentration of the cell suspension, and the leakage of nucleic acid from S. aureus was higher than that from P. aeruginosa, implying that S. aureus may suffer greater membrane damage than P. aeruginosa. In agreement with the released nucleic acids, the amount of protein released into the cell suspension analyzed by Bradford assay in both strains also increased with the increasing of the Fc2SBCp1 concentration of the cell suspension (Fig. S7B), indicating that Fc2SBCp1 can damage to the cell membrane to effectively kill the relevant bacteria. 3.6. Preliminary biocompatibility evaluation of Fc2SBCp1

Fig. 8. Lung bacterial counts of mice suffered from S. aureus pneumonia. N: normal mice; I-1, I-3 and I-5: infected but untreated mice for 1, 3 and 5 days respectively; T-1, I-3 and T-5: treated with Fc2SBCp1 mice for 1, 3 and 5 days respectively.

A good antibiotic must be low toxic to human or animals, thus, the cytotoxicity and hemolysis of Fc2SBCp1 has been explored in

Fig. 7. Neutrophil alkaline phosphatase (NAP) activity of mice. The NAP positive rate (%) and score of bacteremia mice infected with S. aureus (A and B) and P. aeruginosa (C and D). N: normal mice; I-1 and I-3: infected but untreated mice for 1 and 3 days respectively; T-1 and T-3: treated with Fc2SBCp1 mice for 1 and 3 days respectively; Nt-3: uninfected but treated with Fc2SBCp1 mice for 3 days.

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this study. As shown in Fig. S8, the cytotoxicity assay was used to characterize the probability of Fc2SBCp1 induced cell death and the release of hemoglobin was used to quantify the membranedamaging properties [29]. It is observed that the CC50 values of Fc2SBCp1 to these cell lines are much higher than its MBCs against S. aureus and P. aeruginosa (Fig. S8). Moreover, the viability of L-929 cells and MRC-5 cells were 88.7% and 96.5% respectively at

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its MBCs (data not shown), indicating its low toxicity. Furthermore, from the hemolysis assay, it is observed that all of the hemolysis percentage was <5.0% (meet the England standard) [30]. Altogether, the above results of in vitro cytotoxicity assay and hemolysis analysis demonstrate that Fc2SBCp1 have low-level of cytotoxicity and hemolysis when it exert their antimicrobial effects at its MBCs.

Fig. 9. Histology of lungs after intranasal infection with S. aureus. Lungs were inflated and fixed with 10% formalin, and 5-mm sections were stained with hematoxylin/eosin. The paraffin section of lungs were observed by low-(A1-D1, 100) or high-powered (A2-D2, 400) optical microscope. A, healthy mice; B, infected but untreated mice for 5 days; C, infected and treated mice for 3 days; D, infected and treated mice for 5 days.

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3.7. In vivo antibacterial effect

Acknowledgments

Based on the above observations, the in vivo therapeutic effect of Fc2SBCp1 against bacteremia caused by S. aureus and P. aeruginosa was further investigated and evaluated by blood bacterial counts and NAP activity analysis. Fig. 6 shows the number of viable cells in the blood of the testing samples. The results indicate that the number of bacteria in the blood of mice was significantly decreased after 1-day treatment of Fc2SBCp1 for S. aureus bacteremia, and bacterial counts reach normal levels after 3-day treatment (Fig. 6A). The identical results were found for Fc2SBCp1 against P. aeruginosa bacteremia (Fig. 6B). Moreover, the activity of NAP was evaluated as another diagnosis marker of infection diseases. The NAP activity is usually significantly increased in acute bacterial infection, so the NAP positive rate and score generally reflect the severity of infection. The NAP positive rate and score of mice blood samples were examined by following routine medical pathology blood tests. As shown in Fig. 7, the NAP positive rate (Fig. 7A and C) and NAP score (Fig. 7B and D) was significantly increased after infected with S. aureus or P. aeruginosa for 1 and 3 days and they were reduced remarkably after treatment with Fc2SBCp1. After treatment for 3 days, the NAP positive rate and score returned back to normal levels, implying that the S. aureus bacteremia or P. aeruginosa bacteremia had been cured. Furthermore, the therapeutic effect of Fc2SBCp1 against S. aureus pneumonia was examined by lung bacterial counts and lung histopathology. By compared with untreated group, the number of bacteria in the lung tissue was significantly decreased with the increase of treatment time (Fig. 8). After 5 days of Fc2SBCp1 treatment, The number of bacteria in the lung tissue was almost reduced to normal levels. The results of lung histopathology also confirmed the therapeutic effect of Fc2SBCp1 against S. aureus pneumonia (Fig. 9). The infected mice (Fig. 9B1 and B2) show the symptoms of interstitial pneumonia compared with normal control group (Fig. 9A1 and A2). Interstitial pneumonias are a confusing and frustrating set of diseases both for the treating physician and for the diagnostic pathologist. The pathological features of infected 5 days mice (Fig. 9B1 and B2) showed alveolar interval widened, a larger number of lymphocytes infiltrated, pulmonary capillary hyperemia, etc. After 3 days treatment, inflammatory cells (mainly lymphocytes) were obviously reduced but the situation of alveolar interval and hyperemia has not improved (Fig. 9C1 and C2). After 5 days treatment (Fig. 9D1 and D2), inflammatory cells and alveolar interval has been almost completely returned to its normal state, but the hyperemia has not been improved.

This work is supported by National Key Basic Research Program (2010CB732404), National Nature Science Foundation of China (21175020, 20925104 and 21021062), Key Project of Science and Technology of SuZhou (ZXY2012028), Doctoral Fund of Ministry of Education of China (20090092110028), National High Technology Research and Development Program (2007AA02 2007), Graduate Research and Innovation Program of Jiangsu Province (CXLX_0145), and State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry Chinese Academy of Sciences.

4. Conclusion In summary, a ferrocene-substituted carborane derivative Fc2SBCp1 has been synthesized in this study and utilized as a promising antibacterial therapeutic agent against MDR bacterial infections. The results demonstrate the significant antibacterial effect of Fc2SBCp1 against two clinical common MDR pathogens (i.e., Gram-positive S. aureus and Gram-negative P. aeruginosa), both in vitro and in vivo, with no/or little toxicity to normal cells and tissues. It is evident that this ferrocene-substituted carborane derivative could act on bacteria via damaging the cell walls, destabilizing cell membranes and inducing the leakage of cellular contents including nucleic acids and proteins. Meanwhile, Fc2SBCp1 can effectively prevent biofilm formation at sub-MIC and quickly kill bacteria at MBC. This raises the possibility to utilize this ferrocene-substituted carborane derivative as a promising and effective antibacterial agent against MDR infections in future clinical practice.

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