at SciVerse ScienceDirect

Biomaterials 34 (2013) 902e911

Contents lists available

Biomaterials

journal homepage: www.elsevier .com/locate/biomateria ls

Antimicrobial activity of a ferrocene-substituted carborane derivative targetingmultidrug-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, Chinab School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210093, Chinac Institute of Traditional Chinese Medicine, China Pharmaceutical University, Nanjing 210009, Chinad School of Life Science and Biotechnology, Dalian University of Technology, Dalian 116024, China

a r t i c l e i n f o

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

Keywords:AntibacterialAntibiofilmMultidrug-resistant infectionFerrocene-substituted carborane derivativeStaphylococcus aureusPseudomonas aeruginosa

* Corresponding author. Tel./fax: þ86 25 83792177E-mail address: xuewang@seu.edu.cn (X. Wang).

0142-9612/$ e see front matter � 2012 Elsevier Ltd.http://dx.doi.org/10.1016/j.biomaterials.2012.10.069

a b s t r a c t

Multidrug resistance (MDR) of bacteria is still an unsolved serious problem to threaten the health ofhuman beings. Developing new antibacterial agents, therefore, are urgently needed. Herein, we haveexplored the possibility to design and synthesize some novel antibacterial agents including ferrocene-substituted carborane derivative (Fc2SBCp1) and have evaluated the relevant antibacterial actionagainst two clinical common MDR pathogens (i.e., Gram-positive Staphylococcus aureus and Gram-negative Pseudomonas aeruginosa) in vitro and in vivo. The results demonstrate that in vitro antimicro-bial activity of Fc2SBCp1 could be gradually transformed into a bactericidal effect from a bacteriostaticeffect with the increasing concentration of the active carborane derivative, which can also preventbiofilm formation at concentrations below MIC (i.e., minimal inhibitory concentration). Biocompatibilitystudies indicate that there exists no/or little toxic effect of Fc2SBCp1 on normal cells/tissues and leads tolittle hemolysis. In vivo studies illustrate that the new carborane derivative Fc2SBCp1 is highly effective intreating 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 carboranederivatives as promising antibacterial therapeutic agents against MDR bacterial infections in futureclinical applications.

� 2012 Elsevier Ltd. All rights reserved.

1. Introduction

Since antibiotics were widely employed for treating bacterialinfections, the repeated emergence of antibiotic-resistant bacterialstrains has been a serious problem that has long plagued publichealth. Development of new antimicrobial agents is more urgentlyneeded than ever because of the unprecedented increase ofmultidrug resistance in common pathogens and the rapid emer-gence of new infections [1].

Staphylococcus aureus (S. aureus, Gram-positive bacteria) andPseudomonas aeruginosa (P. aeruginosa, Gram-negative bacteria) aretwo of the major causes of fatal nosocomial infections as well ascommunity-acquired infections [2]. The spread of these organismsin healthcare settings are often difficult to control due to thepresence of multiple intrinsic and acquired mechanisms of

.

All rights reserved.

antimicrobial resistance. A biofilm is a structured consortium ofbacteria embedded in a self-produced polymer matrix consisting ofpolysaccharide, protein and DNA [3]. Bacterial biofilms are notori-ously difficult to eradicate and are a source of many recalcitrantinfections. The biofilm-positive strains such as S. aureus andP. aeruginosa can form recalcitrant biofilms under antibiotic pres-sure. The survival of cells in a preformed biofilm is not easilyeradicated by relevant antimicrobials, hence generating anincreased resistance of cells and undoubtedly contributing toproduce more drug resistance mutations [4].

Recently, enormous efforts have been directed to the design,synthesis and evaluation of biomedical activities of various metalcomplexes in medicinal organometallic chemistry, in whicha typical metallocene, ferrocene derivative, has attracted specialattention because of its small size, relative lipophilicity, ease ofchemical modification, and accessible one-electron-oxidationpotential [5e8]. Ferrocenyl derivatives exhibit promising bioactiv-ities like antineoplastic [9], antimalarial [10,11], antifungal [12],antibacterial [13] and others. Currently, incorporation of ferrocenyl

S. Li et al. / Biomaterials 34 (2013) 902e911 903

moieties into the structures of the existing drug molecules hasdemonstrated an important strategy to increase their therapeuticproperties. Meanwhile, as a promising and potential pharmophore,the three-dimensional carborane cage, with its structural integrity,ease of substitution, and delocalized bonding, allows the bio-isosteric replacement for phenyl rings as rigid scaffolding inbioactive molecules and pharmacological agents, which has latelybeen attracting much attention in biomedical applications owing toits extraordinary characteristics [14]. Thus, in this contribution, wehave explored the possibility to combine the ferrocenyl moietieswith icosahedral carborane to design a ferrocene-substituted car-borane derivative (designated as Fc2SBCp1, see Scheme 1) asa promising and effective antibacterial therapeutic agent againstMDR infections in future clinical research.

2. Materials and methods

2.1. Reagents

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

2.2. Synthesis of Fc2SBCp1

All synthesis experiments were performed under an argon atmosphere usingstandard Schlenk techniques. Solvents were dried by refluxing over sodium(petroleum ether, ether) or calcium hydride (dichloromethane) under nitrogen andthen 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 ofCpCo(S2C2B10H10) (66.0 mg, 0.20 mmol) in tetrahydrofuran (THF, 25 mL) was addedthe HChCeC(O)Fc (71.4 mg, 0.30 mmol). The resulting mixture was stirred over-night at 65 �C. After removal of solvent under vacuum, the residue was chromato-graphed on silica gel (100e200mesh). Gradient elutionwith 10mL petroleum ether/CH2Cl2 (v/v)¼ 1/1, 1/3, 1/5, and CH2Cl2 alone respectively. Then the silica gel columnwas eluted with ether. The concentrated solution of the eluate was purified usingthin layer chromatography (TLC). Elution with petroleum ether/CH2Cl2 (1:1) gavethe compound Fc2SBCp1 (Z isomer) 12 mg in 8% yield obtained (Scheme 1).. Here weonly report the antibacterial properties of Fc2SBCp1 since it has better antibacterialactivity than the other isomer.

2.3. Characterization of Fc2SBCp1

Elemental analysis was performed in an Elementar Vario EL III elementalanalyzer. NMR data were recorded on a BrukerDRX-500 spectrometer. 1H NMR and13C NMR spectra were reported in ppm with respect to CHCl3/CDCl3 (d1H ¼ 7.24 ppm, d 13C ¼ 77.0 ppm) and 11B NMR spectra were reported in ppm withrespect to external Et2OeBF3 (d 11B ¼ 0 ppm). The FTIR spectra were recorded ona Thermo Nicolet AVATAR 360 FTIR spectrophotometer with KBr pellets in the4000e400 cm�1 region. The mass spectra were recorded on Micromass GC-TOF forEI-MS (70 eV). X-ray crystallographic data (see Table S1) were collected on a BrukerSMART Apex II CCD diffractometer using graphite-monochromated Mo Ka(l ¼ 0.71073 Å) radiation. The intensities were corrected for Lorentz-polarization

Scheme 1. Synthesis pro

effects and empirical absorption with the SADABS [17] program. The structureswere 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 indimethylsulfoxide (DMSO) and diluted with bacterial culture medium. For all of thevivo experiments, it was dissolved in DMSO and diluted with phosphate buffersolution (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 andP. aeruginosa at a starting inoculum of ca. 2� 105 cells/mL was determined followingCLSI 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 timepoint, the viable colony counts were performed on TSA plates after incubating at37 �C or 30 �C.

2.5. Confocal laser scanning microscopy (CLSM) assays

Overnight cultured strains incubated in TSBgluc 0.5% were diluted to a finaldensity of 1.0 � 105 CFU/mL with fresh medium and dispensed into each well (pre-loaded with plastic cover slips) of a plastic 24-well plate. The plates were staticallyincubated at 37 �C for 48 h. Afterwards, the cover slips were taken out and gentlywashed with 0.01 M phosphate-buffered saline (PBS) three times to wash off thenon-adherent bacteria. After eliminating the moisture, the materials on the coverslips were stained with 20 mL acridine orange (0.02%, w/v) for 15 min at 4 �C in thedark. Stained cover slips were gently washed twice with PBS and observed withCLSM (Zeiss LSM 710, Germany).

2.6. Scanning electron microscope (SEM) studies

Overnight cultured bacteria were diluted with fresh TSB medium to the celldensity of 1.0 � 107 CFU/mL and dispensed into each well (pre-loaded with a siliconslice) of plastic 6-well plates. Treated groups were added 1.0 � MBC or 2.0 � MBCFc2SBCp1, Untreated controls were added the same volume of 0.5% (v/v) DMSOaqueous solution. Then the 6-well plates were put into biochemistry incubatorsincubating 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 werepost-fixed with 1% osmium tetraoxide in 0.1 M PBS for 30 min. The bacteria werethen 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 onthe 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 wasmixed with Fc2SBCp1 at a final concentration of their corresponding minimalbactericidal 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 removebacteria, the bacterial suspensions were centrifuged at 5000 rpm for 5 min toremove bacteria or bacterial debris. The control assay was performed withoutFc2SBCp1. 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 PBSin the dark for 30 min at room temperature, washed them with PBS twice, andplaced 20 mL of samples on a glass slide with a glass coverslip. We observed thefluorescence excited by a 535 nm (for PI-DNA complex) or 495 nm (for FITC-I-proteincomplex) laser with a CLSM.

cedure of Fc2SBCp1.

S. Li et al. / Biomaterials 34 (2013) 902e911904

2.8. Animals and procedures

Animal experiments were reviewed, approved, and supervised by the Jiangsuprovince Institutional Animal Care and Use Committee of China. Pathogen-free 8-week-old male Kunming mice (i.e. Swiss mice), of clean grade, weighing from 28to 32 g, were obtained from Qinglongshan animal farm of Nanjing and weremaintained in high efficiency particulate air-filtered barrier units kept inside bio-logical safety cabinets for the duration of the experiments. Mice were given freeaccess to tap water and pelleted rodent food and were kept at 25 �C with alternating12-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, bacteriawere grown in TSB, centrifuged at 5000 rpm for 5min, washedand suspended in sterile PBS, and maintained at 37 �C. Fc2SBCp1 suspension wasprepared in accordance with the method mentioned in Section 2.4. Animals wereclearly moribund or on the verge of death were killed with an overdose of pento-barbital sodium (150 mg/kg).

2.9. Bacteremia model and blood bacterial counts

Mice were lightly anesthetized with isoflurane and inoculated by the tail veininjection 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 immediatelyadministered 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 insterile saline and determined by plating serial dilutions onto TSA plates for viablecount (cfu/ml blood).

2.10. Analysis of neutrophil alkaline phosphatase (NAP) activity

Using the relevant venous blood samples of the above animal model, thecytochemistry analysis of NAP activity was evaluated according to the methoddescribed by Kaplow [19] and performed in accordance with the kit protocol. Toinvestigate whether Fc2SBCp1 can interfere with the test results, a group of normalmice inject Fc2SBCp1 at the same dosage was supplemented. The blood smears weremade of blood samples and fixed with formaldehyde/methanol (v/v) ¼ 1:9 after the

Fig. 1. Molecular structure of Fc2SBCp1. The single crystals were grown in petroleum etheatoms are omitted for clarity. Selected bond lengths (Å) and angles (�): C(1)eC(2) 1.767(4), C1.543(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) 1.481.541(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(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)e107.2(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).

incubation of blood smears with naphthol ASeBI phosphate at 37 �C for 15 min, thesamples were stained with 1% hematoxylin for 3 min. The degree of the enzymeactivity in each cell, as detected by red spots, was rated from0 to V on the basis of thenumber of precipitated red granules in the cytoplasm. The sum of the rating of 100cells was considered the NAP score in a given sample. The entire analysis of NAPactivity 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 stringby their front teeth and allowed to aspirate the inoculum for 10 min, before beingreturned to cages to recover. After administering 60 mg Fc2SBCp1/kg body weight/day for different time, the mice were killed with pentobarbital sodium and removedlung tissue. The bacterial density in the lung tissue samples were homogenized, andplated onto TSA plates for bacterial colony counts. Results were expressed as thenumber of CFU per lung.

2.12. Histopathology

The method was the same as the above section except the difference of handlinglung. The experimental method of histopathology was performed according to theexisting research [21] with appropriate modifications. Briefly, the fresh removedlungs were fixed in 10% formalin overnight before being embedded in paraffin. Five-micrometer sections of tissue were stained with hematoxylin/eosin before beingexamined.

2.13. Statistical methods

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

r/CH2Cl2 (v/v) ¼ 1:1. Thermal ellipsoids are depicted at 30% probability; all hydrogen(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)6(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)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)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)

Table 1In vitro antibacterial activity and antibiofilm effect of Fc2SBCp1 against two clinicalisolates of S. aureus and P. aeruginosa.

Organism MICa MBCb MBIC50c MBIC90d

S. aureus 36 72 4 14P. aeruginosa 36 72 6 16

a Minimum inhibitory concentration.b Minimum bactericidal concentration.c Minimum biofilm inhibition concentration of Fc2SBCp1 that showed 50% inhi-

bition on the biofilm formation.d Minimum biofilm inhibition concentration of Fc2SBCp1 that showed 90% inhi-

bition on the biofilm formation.

S. Li et al. / Biomaterials 34 (2013) 902e911 905

3. Results and discussion

3.1. Characterization of Fc2SBCp1

Single crystals for X-ray diffraction of Fc2SBCp1 were grown inpetroleum ether/CH2Cl2 (v/v)¼ 1:1 and the solid structure is shownin Fig. 1. The synthetic compound Fc2SBCp1 is a reddish-brownsolid, mp 240 �C (dec.), 1H NMR (CDCl3) (Fig. S1): d 7.38 (d, 1H,J¼ 10Hz, C(3)H), 6.75 (d,1H, J¼ 10Hz, C(4)H), 6.55 (d,1H, J¼ 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, J¼ 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, J¼ 9.0 Hz, C(15)H),1.68 (d,1H, J¼ 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 forC33H36B10O2S2Fe2: C, 52.95; H, 4.85. Found: C, 52.79; H, 4.95%. The1H NMR and 13C NMR spectra and the molecular structure wereclearly identified by the two-dimensional NMR spectroscopy of1H-1H correlation spectroscopy (COSY) (Fig. S3) and 1H-13C-connectivity (HMQC, HMBC) (Fig. S4). IR (KBr) (Fig. S5): n (cm�1):1660.5 and 1625.2 (C]O), 2591.8 (BeH). In UVeVisible absorptionspectrum (in DMSO/PBS (v/v) ¼ 1/50 (pH ¼ 7.2), the absorptionpeak of compound Fc2SBCp1 appears at 484 � 1 nm and this signalwas stable for at least 56 days (Fig. S6).

Fig. 2. Time-kill curves of Fc2SBCp1 against S. aureus (A) and P. a

3.2. MIC, MBC, MBIC50 and MBIC90 determination

The MIC, MBC, MBIC50 and MBIC90 distributions of Fc2SBCp1against S. aureus and P. aeruginosa were shown in Table 1. It isobserved that Fc2SBCp1 demonstrated nearly similar antimicrobialand antibiofilm activity against the two strains with the same MIC/MBC values and similar MBIC50/MBIC90. For both strains, itexhibited bacteriostasis activity at 36 mg/mL (MIC value) andbactericidal activity at 72 mg/mL (MBC value). The MBIC50 values ofFc2SBCp1 were observed for S. aureus (4 mg/ml) and P. aeruginosa(6 mg/ml). Meanwhile, our results demonstrated that Fc2SBCp1induced prevention of 90% of biofilm formation of S. aureus andP. aeruginosa when used at 14 and 16 mg/ml. All of the MBIC50 andMBIC90 values were lower than their MIC values, suggesting itsstrong biofilm inhibition efficiency.

3.3. Time-kill assay

Time-kill assay is capable of detecting differences in the rate andextentofantibacterialagentactivityover timeandarebetter suited forassessing changes in the antibacterial agent activity [22,23]. A bacte-ricidal effect is defined as a�3 log10 decrease in theCFU/mLor a 99.9%kill over a specified time [24,25]. Aspresented in Fig. 2, Fc2SBCp1 showa concentration- and time-dependent manner against S. aureus(Fig. 2A) and P. aeruginosa (Fig. 2B), whilst its antibiotic effects grad-ually change the effect from a bacteriostatic to a bactericidal actionwith the increasing of the Fc2SBCp1 concentration. The bactericidalactivity of Fc2SBCp1was fast-acting against S. aureus and P. aeruginosaat concentrationof 1�MBC(72mg/mL) and2�MBC (144mg/mL); thereduction in the CFU/mL was >3 log10 CFU/mL (99.99%). The bacte-ricidal endpointsofFc2SBCp1against S. aureuswere2hat1�MBCand4hat2�MBC,meanwhile its endpoint againstP. aeruginosawas6hat2 � MBC. Thus, it is evident that Fc2SBCp1 has effective antibacterialactivity against both S. aureus and P. aeruginosa.

3.4. CLSM visualization of biofilm formation

Acridine orange is usually used as a fluorescent biofilm biomassindicator as this compound stains all cells in a biofilm, alive or dead[26,27]. In this study, the Leica confocalmicroscope andsoftwarewasused for analysis of biofilm images, which allowed for collectionof relevant three-dimensional (3D) reconstruction. Images wereacquired fromrandompositions of biofilms formedon theupper side

eruginosa (B) at different concentrations with different time.

S. aureus only H=5.5µµm

P. aeruginosa only H=5.8µm

+ 0.25 × MIC H=4.3µm + 0.5 × MIC H=2.0µm

+ 0.5 × MIC H=2.8µm+ 0.25 × MIC H=3.9µm

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 thereconstructed 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.)

S. Li et al. / Biomaterials 34 (2013) 902e911906

of the cover slips. Fig. 3 shows spatial biomass distributions ofS. aureus and P. aeruginosa in 48-h-old biofilms in the absence orpresence of Fc2SBCp1, as they appear by visual inspection of CSLMimages characterizing the 3D structures. As shown in Fig. 3, CLSM z-section analyses can provide surface coverage of the biofilms as well

Fig. 4. SEM micrographs of S. aureus and P. aeruginosa treated w

as the entire thickness of bacterial biofilms, where S. aureus andP. aeruginosa formed thick biofilms with compact architecture char-acterized by large clumps that were separated by water channelswhen grown in the absence of Fc2SBCp1. In contrast, the biofilm ofS. aureus formed smaller aggregate of microorganisms and looser

ith different concentration of Fc2SBCp1. Scale bars ¼ 1 mm.

S. aureus only

S. aureus only

P. aeruginosa only+ 1 × MBC

+ 1 × MBC

+ 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 thenucleic acids leakage, while the bottom four fluorescence images show the proteins leakage.

S. Li et al. / Biomaterials 34 (2013) 902e911 907

structure than that of P. aeruginosa. Meanwhile, in the presence ofFc2SBCp1, at a concentration of 0.25 � MIC, the biofilms of the bothstrains were much thinner than those of the respective strainsincubated without Fc2SBCp1, with the biofilm thickness decreasingfrom5.5 mmto 4.3 mmand5.8 mmto 3.9 mm, respectively. Besides, thesurface coverage and the density of bacteria of biofilms was signifi-cantly decreased. At concentrations of 0.5 � MIC, the biofilmformation was almost completely inhibited by Fc2SBCp1 and thusonly few biofilms were observed, indicating that it can effectivelyprevent biofilm formation on solid surfaces at very lowconcentration.

3.5. Characterization of bacterial cell damage

Moreover, SEMwas used to examine the ultrastructural changesin bacteria induced by Fc2SBCp1. It is noted that the untreatedS. aureus and P. aeruginosa cells displayed a smooth and intact

Fig. 6. Blood bacterial counts of bacteremia mice infected with S. aureus (A) or P. aeruginrespectively; T-1 and T-3: treated with Fc2SBCp1 mice for 1 and 3 days respectively.

surface (Fig. 4A1 and B1). After incubation with 1 � MBC ofFc2SBCp1 for 1 h, some of S. aureus cells had single blisters anddents in their cell wall (Fig. 4A2). After treatment of S. aureus with2 �MBC of Fc2SBCp1, some bacteria had burst with deep destroy intheir 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 rele-vant bacterial cell wall and cause membrane damage.

To further investigate whether the permeability of cellmembranes dramatically changed in the presence of Fc2SBCp1, therelevant samples were treatedwith propidium iodide (PI) and FITC-I. PI can bind DNA or RNA specifically to acquire enhanced fluo-rescence, but it cannot cross the membrane and is excluded fromviable cells [28]. Thus, another general indication of drug-inducedbacterial cell injury is the leakage of nucleic acid and proteinfrom cells, where intracellular staining of PI can identify dead cells.Fluorescein Isothiocyanate (FITC) is widely used in biology and

osa (B). N: normal mice; I-1 and I-3: infected but untreated mice for 1 and 3 days

Fig. 8. Lung bacterial counts of mice suffered from S. aureus pneumonia. N: normalmice; 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.

S. Li et al. / Biomaterials 34 (2013) 902e911908

medicine as a fluorescent marker for labeling various proteins.After treating suspensions of S. aureus or P. aeruginosa withFc2SBCp1 at 37 �C for 4 h and staining them with PI and FITC-I,fluorescence images show that the permeability of treatedbacteria increases (Fig. 5A and B). The diffused fluorescence clustersoccurring beyond cells imply that some nucleic acids have leakedout of the cells.

Nucleic acids arewell known to absorbUV light at awavelength of260 nm. The amount of nucleic acids released into the cell suspensionof the twobacterial strainswasanalyzedbymeasuring theabsorbanceat 260 nm (Fig. S7A). It is observed that the amount of leaked nucleicacid from the cells increased with the increasing of the Fc2SBCp1concentration of the cell suspension, and the leakage of nucleic acidfrom S. aureuswas higher than that from P. aeruginosa, implying thatS. aureusmay suffer greater membrane damage than P. aeruginosa. Inagreement with the released nucleic acids, the amount of proteinreleased into the cell suspension analyzed by Bradford assay in bothstrains also increased with the increasing of the Fc2SBCp1 concentra-tion of the cell suspension (Fig. S7B), indicating that Fc2SBCp1 candamage to the cell membrane to effectively kill the relevant bacteria.

3.6. Preliminary biocompatibility evaluation of Fc2SBCp1

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 buttreated with Fc2SBCp1 mice for 3 days.

S. Li et al. / Biomaterials 34 (2013) 902e911 909

this study. As shown in Fig. S8, the cytotoxicity assay was used tocharacterize the probability of Fc2SBCp1 induced cell death andthe release of hemoglobin was used to quantify the membrane-damaging properties [29]. It is observed that the CC50 values ofFc2SBCp1 to these cell lines are much higher than its MBCs againstS. aureus and P. aeruginosa (Fig. S8). Moreover, the viability ofL-929 cells and MRC-5 cells were 88.7% and 96.5% respectively at

Fig. 9. Histology of lungs after intranasal infection with S. aureus. Lungs were inflated andparaffin section of lungs were observed by low-(A1-D1, 100�) or high-powered (A2-D2, 40infected and treated mice for 3 days; D, infected and treated mice for 5 days.

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

fixed with 10% formalin, and 5-mm sections were stained with hematoxylin/eosin. The0�) optical microscope. A, healthy mice; B, infected but untreated mice for 5 days; C,

S. Li et al. / Biomaterials 34 (2013) 902e911910

3.7. In vivo antibacterial effect

Based on the above observations, the in vivo therapeutic effect ofFc2SBCp1 against bacteremia caused by S. aureus and P. aeruginosawas further investigated and evaluated by blood bacterial countsand NAP activity analysis. Fig. 6 shows the number of viable cells inthe blood of the testing samples. The results indicate that thenumber of bacteria in the blood of mice was significantly decreasedafter 1-day treatment of Fc2SBCp1 for S. aureus bacteremia, andbacterial counts reach normal levels after 3-day treatment (Fig. 6A).The identical results were found for Fc2SBCp1 against P. aeruginosabacteremia (Fig. 6B). Moreover, the activity of NAP was evaluated asanother diagnosis marker of infection diseases. The NAP activity isusually significantly increased in acute bacterial infection, so theNAP positive rate and score generally reflect the severity of infec-tion. The NAP positive rate and score of mice blood samples wereexamined by following routine medical pathology blood tests. Asshown in Fig. 7, the NAP positive rate (Fig. 7A and C) and NAP score(Fig. 7B and D) was significantly increased after infected withS. aureus or P. aeruginosa for 1 and 3 days and they were reducedremarkably after treatment with Fc2SBCp1. After treatment for 3days, the NAP positive rate and score returned back to normallevels, implying that the S. aureus bacteremia or P. aeruginosabacteremia had been cured.

Furthermore, the therapeutic effect of Fc2SBCp1 againstS. aureus pneumonia was examined by lung bacterial counts andlung histopathology. By compared with untreated group, thenumber of bacteria in the lung tissue was significantly decreasedwith the increase of treatment time (Fig. 8). After 5 days ofFc2SBCp1 treatment, The number of bacteria in the lung tissue wasalmost reduced to normal levels. The results of lung histopa-thology also confirmed the therapeutic effect of Fc2SBCp1 againstS. aureus pneumonia (Fig. 9). The infected mice (Fig. 9B1 and B2)show the symptoms of interstitial pneumonia compared withnormal control group (Fig. 9A1 and A2). Interstitial pneumoniasare a confusing and frustrating set of diseases both for the treatingphysician and for the diagnostic pathologist. The pathologicalfeatures of infected 5 days mice (Fig. 9B1 and B2) showed alveolarinterval widened, a larger number of lymphocytes infiltrated,pulmonary capillary hyperemia, etc. After 3 days treatment,inflammatory cells (mainly lymphocytes) were obviously reducedbut the situation of alveolar interval and hyperemia has notimproved (Fig. 9C1 and C2). After 5 days treatment (Fig. 9D1 andD2), inflammatory cells and alveolar interval has been almostcompletely returned to its normal state, but the hyperemia has notbeen improved.

4. Conclusion

In summary, a ferrocene-substituted carborane derivativeFc2SBCp1 has been synthesized in this study and utilized asa promising antibacterial therapeutic agent against MDR bacterialinfections. The results demonstrate the significant antibacterialeffect of Fc2SBCp1 against two clinical common MDR pathogens(i.e., Gram-positive S. aureus and Gram-negative P. aeruginosa), bothin vitro and in vivo, with no/or little toxicity to normal cells andtissues. It is evident that this ferrocene-substituted carboranederivative could act on bacteria via damaging the cell walls,destabilizing cell membranes and inducing the leakage of cellularcontents including nucleic acids and proteins. Meanwhile, Fc2SBCp1can effectively prevent biofilm formation at sub-MIC and quicklykill bacteria at MBC. This raises the possibility to utilize thisferrocene-substituted carborane derivative as a promising andeffective antibacterial agent against MDR infections in future clin-ical practice.

Acknowledgments

This work is supported by National Key Basic ResearchProgram (2010CB732404), National Nature Science Foundation ofChina (21175020, 20925104 and 21021062), Key Project of Scienceand Technology of SuZhou (ZXY2012028), Doctoral Fund ofMinistry of Education of China (20090092110028), National HighTechnology Research and Development Program (2007AA022007), Graduate Research and Innovation Program of JiangsuProvince (CXLX_0145), and State Key Laboratory of Electroana-lytical Chemistry, Changchun Institute of Applied ChemistryChinese Academy of Sciences.

Appendix A. Supplementary data

Supplementary data related to this article can be found online athttp://dx.doi.org/10.1016/j.biomaterials.2012.10.069.

References

[1] Spellberg B, Powers JH, Brass EP, Miller LG, Edwards Jr JE. Trends in antimi-crobial drug development: implications for the future. Clin Infect Dis 2004;38:1279e86.

[2] Huang CM, Chen CH, Pornpattananangkul D, Zhang L, Chan M, Hsieh MF, et al.Eradication of drug resistant Staphylococcus aureus by liposomal oleic acids.Biomaterials 2011;32:214e21.

[3] Høiby N, Bjarnsholt T, Givskov M, Molin S, Ciofu O. Antibiotic resistance ofbacterial biofilms. Int J Antimicrob Agents 2010;35:322e32.

[4] Lewis K. Riddle of biofilm resistance. Antimicrob Agents Chemother 2001;45:999e1007.

[5] Dombrowski KE, Baldwin W, Sheats JE. Metallocenes in biochemistry,microbiology & medicine. J Organomet Chem 1986;302:281e306.

[6] van Staveren DR, Metzler-Nolte N. Bioorganometallic chemistry of ferrocene.Chem Rev 2004;104:5931e86.

[7] Fouda MFR, Abd-Elzaher MM, Abdelsamaia RA, Labib AA. On the medicinalchemistry of ferrocene. Appl Organometal Chem 2007;21:613e25.

[8] Fish RH, Jaouen G. Bioorganometallic chemistry: structural diversity oforganometallic complexes with bioligands and molecular recognitionstudies of several supramolecular hosts with biomolecules, alkali-metalions, and organometallic pharmaceuticals. Organometallics 2003;22:2166e77.

[9] Caldwell1 G, Meirim1 MG, Neuse1 EW, van Rensburg CEJ. Antineoplasticactivity of polyaspartamide-ferrocene conjugates. Appl Organometal Chem1998;12:793e9.

[10] Wu X, Tiekink ERT, Kostetski I, Kocherginsky N, Tan ALC, Khoo SB, et al.Antiplasmodial activity of ferrocenyl chalcones: investigations into the role offerrocene. Eur J Pharm Sci 2006;27:175e87.

[11] Biot C, Daher W, Ndiaye CM, Melnyk P, Pradines B, Chavain N, et al. Probingthe role of the covalent linkage of ferrocene into a chloroquine template.J Med Chem 2006;49:4707e14.

[12] Biot C, François N, Maciejewski L, Brocard J, Poulain D. Synthesis and anti-fungal activity of a ferrocene-fluconazole analogue. Bioorg Med Chem Lett2000;10:839e41.

[13] Chantson JT, Verga Falzacappa MV, Crovella S, Metzler-Nolte N. Solid-phasesynthesis, characterization, and antibacterial activities of metalloceneepeptide bioconjugates. ChemMedChem 2006;1:1268e74.

[14] Armstrong AF, Valliant JF. The bioinorganic and medicinal chemistry of car-boranes: from new drug discovery to molecular imaging and therapy. DaltonTrans 2007;38:4240e51.

[15] Kim DH, Ko J, Park K, Cho S, Kang SO. Addition reactions of the novelmononuclear dithio-o-carbora-nylcobalt(III) complex (h5-C5H5)Co(h2-S2C2B10H10). Organometallics 1999;18:2738e40.

[16] Barriga S, Marcos CF, Riant O, Torroba T. Synthesis of poly-ferroceneheterocycles by cycloaddition of mono- or bis(ferrocenecarbonyl)acety-lenes and bis[1,2]dithiolo[1,4]thiazinethiones. Tetrahedron 2002;58:9785e92.

[17] Bruker. SMART, version 5.0; SAINT, version 6; SHELXTL, version 6.1; SADABS,version 2.03. Madison, WI: Bruker AXS Inc.; 2000.

[18] Clinical and Laboratory Standards Institute. Methods for dilution antimicro-bial susceptibility tests for bacteria that grow aerobically: approved standard.Document M7eA8. 8th ed. Wayne, PA: CLSI; 2008.

[19] Kaplow LS. Leukocyte alkaline phosphatase cytochemistry: applications andmethods. Ann NY Acad Sci 1968;155:911e47.

[20] Lee MH, Arrecubieta C, Martin FJ, Prince A, Borczuk AC, Lowy FD.A postinfluenza model of Staphylococcus aureus pneumonia. J Infect Dis 2010;201:508e15.

[21] Lathem WW, Crosby SD, Miller VL, Goldman WE. Progression of primarypneumonic plague: a mouse model of infection, pathology, and bacterialtranscriptional activity. P Natl Acad Sci U S A 2005;102:17786e91.

S. Li et al. / Biomaterials 34 (2013) 902e911 911

[22] Keele DJ, DeLallo VC, Lewis RE, Ernst EJ, Klepser ME. Evaluation of ampho-tericin B and flucytosine in combination against Candida albicans and Cryp-tococcus neoformans using time-kill methodology. Diagn Micr Infec Dis 2001;41:121e6.

[23] Klepser ME, Malone D, Lewis RE, Ernst EJ, Pfaller MA. Evaluation of vor-iconazole pharmacodynamics using time-kill methodology. AntimicrobAgents Chemother 2000;44:1917e20.

[24] Pendland SL, Neuhauser MM, Prause JL. In vitro bactericidal activity and post-antibiotic effect of ABT-773 versus co-amoxiclav against anaerobes.J Antimicrob Chemother 2002;49:879e82.

[25] National Committee for Clinical Laboratory Standards. Methods for deter-mining bactericidal activity antimicrobial agents; Tentative guideline; NCCLSdocument M26-T. Villanova, Pa: National Committee for Clinical LaboratoryStandards; 1992.

[26] Wang XQ, Yao X, Zhu ZA, Tang TT, Dai KR, Sadovskaya I, et al. Effect ofberberine on Staphylococcus epidermidis biofilm formation. Int J AntimicrobAgents 2009;34:60e6.

[27] Branda SS, Vik S, Friedman L, Kolter R. Biofilms: the matrix revisited. TrendsMicrobiol 2005;13:20e6.

[28] Zhao YY, Tian Y, Cui Y, Liu WW, Ma WS, Jiang XY. Small molecule-capped goldnanoparticles as potent antibacterial agents that target Gram-negativebacteria. J Am Chem Soc 2010;132:12349e56.

[29] Fischer D, Li YX, Ahlemeyer B, Krieglstein J, Kissel T. In vitro cytotoxicitytesting of polycations: influence of polymer structure on cell viability and-hemolysis. Biomaterials 2003;24:1121e31.

[30] International Standardization Organization. Biological evaluation of medicaldevices. Sample preparation and reference materials. British Standard ISO10993e12; 2009.