In vitro antimicrobial activity of nanoconjugated vancomycin against drug resistant Staphylococcus aureus

International Journal of Pharmaceutics 436 (2012) 659–676

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International Journal of Pharmaceutics journal homepage: www.elsevier.com/locate/ijpharm

Pharmaceutical Nanotechnology

In vitro antimicrobial activity of nanoconjugated vancomycin against drug resistant Staphylococcus aureus Subhankari Prasad Chakraborty a , Sumanta Kumar Sahu b , Panchanan Pramanik b , Somenath Roy a,∗ a b

Immunology and Microbiology Laboratory, Department of Human Physiology with Community Health, Vidyasagar University, Midnapore 721102, West Bengal, India Nanomaterials Laboratory, Department of Chemistry, Indian Institute of Technology, Kharagpur 721302, West Bengal, India

a r t i c l e

i n f o

Article history: Received 8 June 2012 Received in revised form 17 July 2012 Accepted 19 July 2012 Available online 25 July 2012 Keywords: Vancomycin resistant Staphylococcus aureus Nanoconjugated vancomycin Minimum inhibitory concentration Cell wall thickness Cell viability Na+ , K+ -ATPase activity

a b s t r a c t The mounting problem of antibiotic resistance of Staphylococcus aureus has prompted renewed efforts toward the discovery of novel antimicrobial agents. The present study was aimed to evaluate the in vitro antimicrobial activity of nanoconjugated vancomycin against vancomycin sensitive and resistant S. aureus strains. Folic acid tagged chitosan nanoparticles are used as Trojan horse to deliver vancomycin into bacterial cells. In vitro antimicrobial activity of nanoconjugated vancomycin against VSSA and VRSA strains was determined by minimum inhibitory concentration, minimum bactericidal concentration, tolerance and disc agar diffusion test. Cell viability and biofilm formation was assessed as indicators of pathogenicity. To establish the possible antimicrobial mechanism of nanoconjugated vancomycin, the cell wall thickness was studied by TEM study. The result of the present study reveals that nano-sized vehicles enhance the transport of vancomycin across epithelial surfaces, and exhibits its efficient drug-action which has been understood from studies of MIC, MBC, DAD of chitosan derivative nanoparticle loaded with vancomycin. Tolerance values distinctly showed that vancomycin loaded into nano-conjugate is very effective and has strong bactericidal effect on VRSA. These findings strongly enhanced our understanding of the molecular mechanism of nanoconjugated vancomycin and provide additional rationale for application of antimicrobial therapeutic approaches for treatment of staphylococcal pathogenesis. © 2012 Elsevier B.V. All rights reserved.

1. Introduction

Abbreviations: AFM, atomic force microscopy; BHI, brain heart infusion; CFU, colony formation unit; CMC, carboxymethyl chitosan; CMC-EDBE-FA, carboxymethyl chitosan-2,2 -ethylenedioxy bis ethylamine-folate; CS, chitosan; DAD, disc agar diffusion; d-Ala-d-Ala, d-alanine-d-alanine; d-Ala-d-Lac, dalanine-d-lactate; DLS, dynamic light scattering; DMSO, dimethyl sulphoxide; EDBE, 2,2 -ethylenedioxy bis-ethylamine; EDC, 1-[3-dimethylamino) propyl]-3ethylcarbodiimide hydrochloride; FA, folic acid; FACS, fluorescence activated cell sorter; FITC, fluorescein isothiocyanate; FTIR, Fourier transform infrared spectroscopy; LB, luria broth; MBC, minimum bactericidal concentration; MHA, Mueller–Hinton agar; MHB, Mueller–Hinton broth; MIC, minimum inhibitory concentration; MRSA, methicillin resistant Staphylococcus aureus; MSSA, methicillin sensitive Staphylococcus aureus; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide; NA, nutrient agar; Nacl, sodium chloride; NAG, N-acetylglucosamine; NAM, N-acetylmuramic acid; NCCLS, National Committee for Clinical Laboratory Standards; NHS, N-hydroxysuccinimide; NV, nanoconjugated vancomycin; PBP, penicillin binding protein; PBS, phosphate buffer saline; PG, peptidoglycan; PRSA, penicillin resistant Staphylococcus aureus; Rh123, rhodamine 123; S. aureus, Staphylococcus aureus; SD, standard deviation; S.E.M., standard error of mean; TEM, transmission electron microscopy; Van, vancomycin; VISA, vancomycin intermediate Staphylococcus aureus; VRE, vancomycin resistant enterococci; VRSA, vancomycin resistant Staphylococcus aureus; VSSA, vancomycin sensitive Staphylococcus aureus. ∗ Corresponding author. Tel.: +91 3222 275329; fax: +91 3222 275329. E-mail address: [email protected] (S. Roy). 0378-5173/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ijpharm.2012.07.033

At present there is great concern about the emergence of multidrug resistant bacterial strains, and continuous research efforts are being devoted for the development of new and effective antimicrobial agents. Staphylococcus aureus produces many toxins and many extracellular products that may act as virulence factors (Archer and Climo, 2001). More than 90% of Staphylococcus strains are resistant to penicillin, methicillin, aminoglycosides, macrolides and lincosamide (Chambers, 2001; Levin et al., 2005; Schmitz et al., 2002). Resistance of S. aureus strains to penicillin is mediated by penicillinase (a form of ␤-lactamase) production, an enzyme which breaks down the ␤-lactam ring of the penicillin molecule. In 1961 S. aureus developed resistance to methicillin, invalidating almost all antibiotics including the most potent ␤-lactams (Jevons, 1961). The first report of a Japanese patient harboring MRSA intermediately resistant to vancomycin appeared in 1996 (Hiramatsu et al., 1997). Fully vancomycin-resistant S. aureus was first appeared in the USA in 2002 (Chang et al., 2003). Treatment of vancomycin-resistant S. aureus is a serious problem in globe. Vancomycin acts against Gram-positive bacteria by inhibiting the steps in murien (peptidoglycan) bio-synthesis and assembly of NAM-NAG-polypeptide into the growing peptidoglycan (PG) chain. It inhibits this process by

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reacting with d-Ala-d-Ala, which consequently blocks the release of terminal d-Ala and intra-chain bond formation (Saha et al., 2008a). Chitosan is a well-known biopolymer that possesses antibacterial activity against Gram-positive and Gram-negative bacteria, which has been exploited in a number of studies (Li et al., 2008; Rabea et al., 2003). Chitosan is a mucoadhesive polymer that is able to open tight junctions and allow the paracellular transport of molecules across mucosal delivery of vaccines (Van der Lubben et al., 2001). Some hypotheses indicate that polycationic chitosan could interact with anionic groups on the cell surface thereby causing an increase in membrane permeability and probably its disruption and subsequent leakage of cellular proteins (Qi et al., 2004). Another mechanism suggested involves the formation of chitosan chelates with trace elements or essential nutrients resulting in the inhibition of the activity of enzymes (Rabea et al., 2003). A generally accepted idea is that the antimicrobial activity of chitosan strongly depends on several factors including its molecular weight, degree of deacetylation, pH, etc., and that high molecular weight chitosan exhibits higher toxicity against Gram-positive bacteria compared to Gram-negative ones (Eaton et al., 2008). Due to its excellent biocompatibility, biodegradability and nontoxicity (Kean and Thanou, 2010), chitosan has been successfully used in nanomedicine for delivering therapeutic drugs (De Campos et al., 2004), proteins (Garcia-Fuentes et al., 2007) and genes (Roy et al., 1999). Chitosan nanoparticles had also been employed as a gene carrier to enhance gene transfer efficiency in cells (Mao et al., 2001; Kim et al., 2004). Chitosan microspheres have been used for gastric drug delivery and controlled release of active antimicrobial agents, such as amoxycillin and metronidazole in the gastric cavity (Portero et al., 2002). The unique character of nanoparticles for their small size and quantum size effect could make chitosan nanoparticles exhibit superior activities. Solubility in neutral and basic solutions can be achieved by further modification to the structure of chitosan, such as in carboxymethyl chitosan (CMC). This chitosan modification is synthesized by carboxylation of the hydroxyl and amine groups (Liu et al., 2001). The degree of carboxymethylation, which is controlled by reaction temperature and duration, strongly affects the solubility of CMC (Chen and Park, 2003). CMC is reported to have a higher sorption of metal ions than chitosan (Varma et al., 2004). It has been proposed that the higher sorption capacity is due to increased chain flexibility and higher concentrations of chelating groups (Guibal, 2004; Ngah and Liang, 1999). In our previous laboratory report, CMC-EDBE-FA nanoparticles was prepared based on CMC tagged with folic acid (FA) by covalently linkage through 2,2 -(ethylenedioxy) bis-(ethylamine) (EDBE). Physicochemical characteristics of this nanoparticles were examined by FTIR spectroscopy, DLS and TEM study; and it was also observed that the nanoparticles has no antimicrobial and toxic effect (Chakraborty et al., 2010, 2011a, 2012). Hence the present study was aimed to prepare nanoconjugated vancomycin by loading of vancomycin on CMC-EDBE-FA nanoparticles through physical adsorption and observes its antimicrobial activity against vancomycin sensitive and resistant S. aureus strains.

sulphate, NaOH, uranyl acetate, potassium chloride, ethanol, cell culture grade DMSO, magnesium sulphate, magnesium chloride, ouabain were procured from Merck Ltd., SRL Pvt. Ltd., Mumbai, India. Rhodamine 123 (Rh123), folic acid (FA), chitosan, dicyclohexyl carbodiimide (DCC), EDBE, di-tert-butyldicarbonate (BoC2 O), N-hydroxysuccinimide (NHS) and 1-[3-dimethylamino) propyl]3-ethylcarbodiimide hydrochloride (EDC), DNase, ATP, 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), ␣toxin antibody, fluorescein isothiocyanate (FITC) were purchased from Sigma Chemical Co., USA. All other the chemicals, reagents, were purchased from Himedia, India; SRL Pvt. Ltd., Mumbai, India and were of the highest grade available. 2.2. Bacterial strains Eight vancomycin sensitive S. aureus (VSSA) [MMC-1, MMC-2, MMC-3, MMC-5, MMC-6, MMC-7, MMC-8, MMC-15] and vancomycin resistant S. aureus (VRSA) [MMC-4, MMC-9, MMC-12, MMC-16, MMC-17, MMC-18, MMC-19, MMC-20] strains were selected for this study. All these strains were isolated from human post operative pus sample in our laboratory (Chakraborty et al., 2011b). 2.3. Preparation of nanoconjugated vancomycin 2.3.1. Loading of vancomycin on nanoparticle Vancomycin loading onto CMC-EDBE-FA nanoparticles was performed by the method of Cevher et al. (2006). Vancomycin loaded CMC-EDBE-FA nanoparticle (VNP1-VNP3) was prepared with polymer:drug ratios (w/w) of 1:1, 2:1 and 1:2 (Table 1). 10 mg, 20 mg and 10 mg CMC-EDBE-FA was dissolved in 1.0 ml PBS (pH 7.4) separately by sonication at 100 W for 1 min (10S working and 10S rest) in an ice water bath to which 10 mg, 10 mg and 20 mg vancomycin was dissolved respectively to obtain the nanoparticle: drug ratio of 1:1 (VNP1), 2:1 (VNP2) and 1:2 (VNP3). Each suspension was then emulsified by mechanical shaking at 200 ± 1 rpm for 36 h at 37 ± 0.1 ◦ C (Orbitek shaker incubator) to prevent aggregation. 10 mg, 20 mg and 10 mg CMC-EDBE-FA nanoparticle without vancomycin was parallely checked as negative control. 2.3.2. Actual drug content and encapsulation efficiency Actual drug content and encapsulation efficiency was measured by the method of Cevher et al. (2006). After 36 h of shaking, the mixtures were centrifuged at 3500 × g for 10 min to get vancomycin loaded CMC-EDBE-FA as pallet. Drug content was determined by analyzing the CMC-EDBE-FA solution and pallet (dissolve in 1.0 ml PBS pH 7.4) using Hitachi U2001 UV/vis spectrophotometer at a wavelength of 282 nm with PBS as reference. Drug content was determined by comparing with the standard curve of vancomycin which was achieved from vancomycin solution in PBS (pH 7.4) with concentration between 0.001 and 0.1 mg/ml. Actual drug content (AC) and encapsulation efficiency (EE) were calculated (Eqs. (1) and (2)). All analyses were carried out in triplicate. Results are expressed as the mean percentage (w/w) ± SD of three formulations:

2. Materials and methods 2.1. Culture media and chemicals Mueller–Hinton broth, nutrient agar, luria broth, tryptic soy broth, agar powder, vancomycin salt, vancomycin disc, crystal violet, alanine, lactate, lysostaphin, sucrose, RPMI 1640 were purchased from Himedia, India. Tris–HCl, Tris buffer, sodium chloride, sucrose, potassium dihydrogen phosphate (KH2 PO4 ), di potassium hydrogen phosphate (K2 HPO4 ), EDTA, sodium dodecyl

AC (%) =

Mact × 100 Mms

(1)

EE (%) =

Mact × 100 Mthe

(2)

where Mact is the actual vancomycin content in weighed quantity of CMC-EDBE-FA, Mms is the weighed quantity of powder of CMCEDBE-FA and Mthe is the theoretical amount of vancomycin in CMCEDBE-FA calculated from the quantity added in the process.

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Table 1 Actual drug content and encapsulation efficiency of vancomycin loaded CMC-EDBE-FA nanoparticle. Values are expressed as percentile ± SD, n = 6. Formulation

Polymer

Polymer:drug ratio

Actual drug content % ± SD

Theoretical drug content (%)

Encapsulation efficiency % ± SD

VNP1 VNP2 VNP3

CMC-EDBE-FA CMC-EDBE-FA CMC-EDBE-FA

1:1 2:1 1:2

32.47 ± 1.54 14.97 ± 1.92 23.55 ± 2.7

50.00 33.33 66.66

65.62 ± 3.87 44.9 ± 5.76 35.34 ± 4.05

2.3.3. DLS study of CMC-EDBE-FA nanoparticles and vancomycin loaded CMC-EDBE-FA nanoparticles The hydrodynamic (HD) size of the CMC-EDBE-FA nanoparticle and vancomycin loaded CMC-EDBE-FA nanoparticle were measured by laser light scattering by Zetasizer Nano ZS (Malvern Instruments) equipped with a 4 mW He–Ne laser at 633 nm at 25 ◦ C. For all measurements the concentration was maintained at 50 ␮g/ml.

2.3.4. TEM analysis of CMC-EDBE-FA nanoparticles and vancomycin loaded CMC-EDBE-FA nanoparticles Surface morphology of the CMC-EDBE-FA nanoparticle and vancomycin loaded CMC-EDBE-FA nanoparticle were observed by transmission electron microscope. TEM micrographs were obtained on a Phillips CM 200 with an acceleration voltage of 200 kV. The nanoparticles were thoroughly dispersed in water by ultra-sonication and placing a drop of solution on the 200 mesh carbon coated copper grid.

2.3.5. In vitro drug release study In vitro release profiles of vancomycin from vancomycin loaded CMC-EDBE-FA nanoparticle were examined in phosphate buffer (pH 7.4) according to Cevher et al. (2006). 1.0 ml of dissolution medium was put into eppendorf tube and 40 mg vancomycin loaded CMC-EDBE-FA nanoparticle was suspended in it. The tubes were put into orbital shaker at 37 ± 0.5 ◦ C at 120 ± 1 rpm. At scheduled time intervals (60 min interval), the tubes were taken and centrifuged at 2000 × g for 5 min; 30 ␮l samples were withdrawn and replaced with fresh medium to avoid saturation phenomena and maintain the sink condition. The samples were diluted with same buffer and analysed by Hitachi U2001 UV/Vis spectrophotometer at a wavelength of 282 nm. All analyses were carried out in triplicate. Results are expressed as the mean percentage of drug released as a function of time ± SD.

2.4. In vitro antimicrobial activity of nanoconjugated vancomycin 2.4.1. Drug preparation Several doses of vancomycin (0.5–1024 ␮g/ml) and nanoconjugated vancomycin (0.5–8 ␮g/ml) were prepared using sterile PBS (pH 7.4). In this study, all these doses were charged against VSSA and VRSA strains.

2.4.2. Determination of minimum inhibitory concentration (MIC) The minimum inhibitory concentration (MIC) of vancomycin and nanoconjugated vancomycin were determined against vancomycin sensitive and resistant S. aureus strains by broth dilution method using Mueller–Hinton broth (MHB), as recommended by the National Committee for Clinical Laboratory Standards (NCCLS, 2000). About 5 × 104 bacterial cells in MHB culture were treated with different concentrations of vancomycin and nanoconjugated vancomycin, and shaken for 16 h at 37 ◦ C. The minimum concentration at which there was no visible turbidity was taken as the MIC of that antibiotic.

2.4.3. Determination of minimum bactericidal concentration (MBC) The minimum bactericidal concentration (MBC) of vancomycin and nanoconjugated vancomycin were determined against vancomycin sensitive and resistant S. aureus strains according to Okore (2005). This is an extension of the MIC procedure. Vancomycin and nanoconjugated vancomycin treated bacterial culture showing growth or no growth in the MIC tests were used for this test. Bacterial culture used for the MIC test were inoculated onto the Mueller–Hinton agar (MHA) and incubated at 37 ◦ C for 24 h. Microbial growth or death were ascertained via no growth on Mueller–Hinton agar plate. The minimal concentration of the vancomycin and nanoconjugated vancomycin that produced total cell death is the MBC. 2.4.4. Determination of tolerance level Tolerance level of each strain against vancomycin and nanoconjugated vancomycin was determined according to the method of May et al. (2006) using the following formula: Tolerance = MBC/MIC. 2.4.5. Disc agar diffusion (DAD) test Susceptibility of vancomycin sensitive and resistant S. aureus strains to vancomycin and nanoconjugated vancomycin was determined by the disc agar diffusion (DAD) technique according to Acar and Lorian (1980) and Bauer et al. (1966). Bacterium taken from an overnight MHB culture was freshly grown for 4 h having approximately 106 CFU/ml. With this culture, a bacterial lawn was prepared on Mueller–Hinton agar. Filter paper discs of 6-mm size were used to observe susceptibility patterns against vancomycin and nanoconjugated vancomycin [amount of antibiotic per disc in microgram (␮g); vancomycin (30) and nanoconjugated vancomycin (30)]. Vancomycin disc was obtained commercially from Himedia and nanoconjugated vancomycin disc was prepared according to Bauer et al. (1966). The diameter of zone of bacterial growth inhibition surrounding the disc (including the disc) was measured and compared with the standard of vancomycin. 2.4.6. Fractional inhibiting concentration (FIC) The ability of nanoconjugated vancomycin against VSSA and VRSA strains was quantified using FIC values by the method of Eliopoulos and Moellering (1991), calculated using the formula: FIC =

[A] [B] + MICA MICB

where MICA is the MIC values of vancomycin, MICB is the MIC values of nanoconjugated vancomycin, [A] is the MIC values of vancomycin and nanoconjugated vancomycin in combination, and [B] is the concentration of nanoconjugated vancomycin. In this study, [B] was kept constant (0.5 ␮g/ml), and [A] for the bacterial strains was determined. Synergy is defined by FIC < 0.5 (Schmidt et al., 2001). 2.4.7. Killing kinetic studies Killing kinetic assay of VSSA and VRSA strains was studied against vancomycin and nanoconjugated vancomycin by the method of Vaudaux et al. (2005). Vancomycin and nanoconjugated vancomycin treated (at their respective MIC values) VSSA and VRSA

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cell suspension (107 CFU/ml) were grown in sterile glass tubes containing 1.0 ml of MHB, incubated at 37 ◦ C in a shaking water bath. The number of viable organisms was determined as total number of colonies by dilution plating method of broth on MHA from 0 h to 30 h of incubation having 3 h interval. The detection limit was 2 log 10 CFU/ml. 2.4.8. Cell viability assay Cell viability of VSSA and VRSA cells was performed after 12 h of treatment with vancomycin and nanoconjugated vancomycin by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method according to Mosmann (1983). Drug treated bacterial cultures were centrifuged at 1000 × g for 10 min at 4 ◦ C followed by repeated wash for two times with sterile PBS (pH 7.4). Thereafter, the medium was replaced with fresh RPMI (without phenol red and FBS) containing 0.5 mg/ml of MTT. After additional 3 h incubation at 37 ◦ C, HCl-isopropanolic solution was added and after 15 min of incubation at room temperature, absorbance of solubilized MTT formazan product was measured in Hitachi U2001 UV/Vis spectrophotometer at 570 nm. 2.4.9. Biofilm formation assay Virulence factor in terms of biofilm formation was measured by the method of Lauriano et al. (2004). Vancomycin and nanoconjugated vancomycin treated VSSA and VRSA cells were grown overnight in LB broth and then normalized to identical densities based on OD600 , and 5 ␮l was inoculated into 500 ␮l of LB broth in 10 ml borosilicate glass tubes. The tubes were then incubated statically at 30 ◦ C for 22 h. The tubes were rinsed with distilled water, incubated with 600 ␮l of 0.1% crystal violet for 30 min, and rinsed again with distilled water. 1.0 ml of dimethyl sulphoxide was then added, the tube was vortexed and allowed to stand for 10 min, and the optical density was measured in Hitachi U2001 UV/Vis spectrophotometer at a wavelength of 570 nm. 2.4.10. Measurement of membrane depolarization Membrane depolarization of VSSA and VRSA strains was carried out after the treatment of vancomycin and nanoconjugated vancomycin by the method of Saha et al. (2008b) with some modification. Vancomycin and nanoconjugated vancomycin treated VSSA and VRSA culture grown up to early exponential phase (OD620 ∼ 0.2–0.3) was harvested by centrifugation at 1000 × g for 10 min at 4 ◦ C, washed twice in 0.1 mol/l Tris buffer, pH 7.6 containing 0.25 mol/l sucrose and 0.005 mol/l MgSO4 , 7H2 O and suspended in the same buffer at an optical density of 0.09 at 620 nm. The Rh123 fluorescent dye was added giving 1.0 ␮ mol/l concentration and allowed to incorporate into the cells for 15 min. Excitation and emission wavelengths of 505 nm and 534 nm, respectively, were used to monitor depolarization. The fluorescence of Rh123 was measured at ambient temperature setting the fluorescence intensity of the cell suspension plus dye solution at zero. The sample was slowly stirred during the measurements that were taken every 30 s using a Hitachi F-7000 Spectrofluorimeter. 2.4.11. Fluorescence staining with rhodamine 123 Rh123 is a voltage sensitive cationic dye that is electrophoretically taken up into energized bacteria by virtue of the trans-membrane electrochemical potential (negative inside) of the plasma membrane. Vancomycin and nanoconjugated vancomycin was labeled with rhodamine 123 according to Mason et al. (1993). For this labeling, Rh123 dye (20 mg/ml in sterile water) was added to vancomycin and nanoconjugated vancomycin to give a concentration of 0.2 mg stain and it was kept at 37 ◦ C in darkness for 1 h. Growing VSSA and VRSA cultures were centrifuged at 1000 × g in 4 ◦ C for 10 min, washed twice with PBS (pH 7.4), replaced with

same buffer and charged Rh123 labeled vancomycin and nanoconjugated vancomycin as required concentration and placed it at 37 ◦ C in darkness for 12 h. Cells without Rh123 served as negative control. After 12 h of incubation, cells were washed and resuspended in PBS, and a drop of suspension was examined with a Olympus research phase contrast with fluorescence microscope (Model: CX41; Olympus Singapore Pvt. Ltd., Valley Point Office Tower, Singapore). 2.4.12. Confocal microscopy FITC–␣ toxin complex was prepared by adding FITC solution (20 ␮l, 1 mg/ml in ethanol) to ␣-toxin antibody of S. aureus to give a final concentration of 20 ␮g/ml and allowed to react in a shaking incubator at 50 ◦ C for 2 h. Rh123 labeled vancomycin and nanoconjugated vancomycin were prepared by the procedure as described in Section 2.4.11. Growing VSSA and VRSA cultures were centrifuged at 1000 × g for 10 min at 4 ◦ C, washed twice with PBS (pH 7.4), replaced with same buffer and charged FITC–␣-toxin complex to give a final concentration of 0.5 ␮g/ml and kept in incubator at 37 ◦ C for 2 h in darkness. Rh123 labeled vancomycin and nanoconjugated vancomycin as required concentration (respective MIC values) is added to it and placed at 37 ◦ C in darkness for 12 h. After 12 h of incubation, cells were washed and resuspended in PBS, and a drop of suspension was examined with Olympus FV1000 confocal microscope. Cells without Rh123 and FITC–␣-toxin complex served as negative control. 2.4.13. Cell viability count by fluorescence activated cell sorter Rh123 is used as a viability stain that stains the mitochondria of living cell (Mason et al., 1993). Bacteria with membrane potential exclude the dye, but non-viable bacteria with depolarized membranes allow it to enter the cell. VSSA and VRSA cultures were centrifuged at 1000 × g for 10 min at 4 ◦ C, washed twice with PBS (pH 7.4), replaced with same buffer and charged Rh123 labeled vancomycin and nanoconjugated vancomycin as required concentration (respective MIC values) and placed it at 37 ◦ C in darkness for 12 h. Cells without Rh123 served as negative control. After 12 h of incubation, cells were washed twice in PBS (pH 7.4), and analysed by flow cytometry (Model: FACS calibur flow cytometer, Becton Dickinson) for fluorescence at an excitation and emission wavelengths of 505 nm and 534 nm. 2.5. In vitro antimicrobial mechanism of nanoconjugated vancomycin against vancomycin sensitive and resistant S. aureus strains 2.5.1. Bonding of nanoconjugated vancomycin with alanine and lactate The binding of nanoconjugated vancomycin with alanine and lactate was analysed by FTIR spectroscopy with a model Thermo Nicolet Nexux model 870. The FTIR values were taken within 400–4000 wave numbers (cm−1 ). In brief, 1.0 mg sticky mass with 100 ␮l KBr medium and a thin film was prepared on the NaCl plate by drop casting method and under atmosphere separately. The FTIR value was taken within 400–4000 wave numbers (cm−1 ). 2.5.2. Transmission electron microscopy of bacterial cell wall The cell wall of vancomycin and nanoconjugated vancomycin treated VSSA and VRSA cells was studied by transmission electron microscopy according to Cui et al. (2000). Suspensions of VSSA and VRSA cells (107 CFU/ml) were treated with vancomycin and nanoconjugated vancomycin at their respective MIC concentrations for 8 h at 37 ◦ C. Control experiments (without vancomycin and nanoconjugated vancomycin) were performed in parallel. Organisms were harvested by centrifugation at 6500 × g for 8 min at 4 ◦ C, and the pellet was resuspended in 1 ml of phosphate buffer 0.1 mol/l (pH 7.4). After centrifugation (10,000 × g for 10 min, 4 ◦ C),

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the pellets were fixed with 4% glutaraldehyde in 0.1 mol/l phosphate buffer for 2 h at 4 ◦ C. Microbial pellets were embedded in 2% low fusion-point agarose (Serva Electrophoresis GmbH, Germany) and maintained at 4 ◦ C for 40 min. The cells were then post fixed in 1% buffered osmium tetroxide for 10 h, stained with 1% uranyl acetate, dehydrated in a graded alcohol series, washed twice in propylene oxide and embedded in durcupan resin (Aname Instrumentacion Cientıfica, Madrid, Spain). Ultra-thin sections were examined with a Jeol JEM 1010 transmission electron microscope at an accelerated voltage of 100 kV. Morphometric evaluation of cell wall thickness was performed by using photographic images at a final magnification of 30,000×, and the cell wall thickness was measured (Cui et al., 2000). Thirty cells of each strain with nearly equatorial cut surfaces were measured for the evaluation of cell wall thickness, and results were expressed as means ± SD. 2.5.3. Atomic force microscopy study The antibacterial process of nanoconjugated vancomycin against vancomycin sensitive and resistant S. aureus was elucidated by AFM observation. Vancomycin and nanoconjugated vancomycin were treated to bacterial cultures grown to the late exponential phase (1× MIC values). Samples were removed onto the surface of a piece of mica plate for AFM observation. AFM worked under the same conditions as described above. AFM imaging was performed using Si3 N4 probes with a spring constant of 0.06 N/m. 2.5.4. Preparation of bacterial cell membrane Crude bacterial cell membrane was prepared by the method of Cui et al. (2000). Overnight growth VSSA and VRSA cultures were centrifuged at 15,000 × g for 10 min at 4 ◦ C, washed twice with phosphate buffer (pH 7.4), resuspended in same buffer to reach a cell density of 107 CFU/ml (OD550 ∼ 0.3) and treated with vancomycin and nanoconjugated vancomycin for 8 h. After treatment period, the cells were harvested and washed twice with cold 20 mM TE (Tris–EDTA) buffer (pH 7.6). The crude extracts were obtained by digestion with 0.1% lysostaphin in 0.2 ml of TE buffer (10 mM Tris–HCl, 1 mM EDTA; pH 8.0) at 37 ◦ C for 10 min, followed by the addition of 0.1 ml of 0.2% DNase for overnight. Crude cell membrane was resuspended in 25 mM Tris, pH 7.4. Protein was estimated in the samples by the method of Lowry et al. (1951). 2.5.5. Assay of membrane Na+ , K+ -ATPase activity Na+ , K+ -ATPase activity of crude cell membrane of vancomycin and nanoconjugated vancomycin treated VSSA and VRSA cells was measured according to Mallick et al. (2000). Briefly, an aliquot of the membrane suspension (100 ␮l containing 100–250 ␮g protein) was added to a reaction mixture containing 100 mM NaCl, 10 mM KCl, 6 mM MgCl2 and 3 mM ATP in 25 mM Tris, pH 7.4 in the presence or absence of 2 mM ouabain and incubated for 15 min at 37 ◦ C. The inorganic phosphate (Pi) liberated was measured spectrophotometrically at 660 nm and the enzyme expressed as ␮mol of Pi liberated/mg protein/h. 2.5.6. Release of intracellular potassium Intracellular potassium released was measured according to Leon and Moujir (2008) and Herbert et al. (1971). Overnight growth VSSA and VRSA cultures were centrifuged at 15,000 × g for 10 min at 4 ◦ C, washed twice with saline buffer, resuspended in same buffer to reach a cell density of 107 CFU/ml (OD550 ∼ 0.3) and treated with vancomycin and nanoconjugated vancomycin for 8 h. The samples were membrane-filtered and K+ release was measured in Hitachi U2001 UV/vis spectrophotometer at a wavelength of 766 nm. 2.5.7. Leakage of cellular constituents absorbing at 260 nm To observe the leakage of cellular constituents we adopted the procedure of Chou and Pogell (1981). Overnight growth VSSA and

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VRSA cultures were centrifuged at 15,000 × g for 10 min at 4 ◦ C, washed twice with 0.05 mol/l potassium phosphate buffer (pH 7.3) containing 0.05 mol/l sucrose, and resuspended in the same buffer to reach a cell density of 107 CFU/ml (OD550 ∼ 0.3) and treated with vancomycin and nanoconjugated vancomycin for 8 h. The liberation of the cellular constituents was determined by measuring the optical density of the suspensions and supernatant (after removing cells by centrifugation at 8000 × g for 10 min, 4 ◦ C) in Hitachi U2001 UV/Vis spectrophotometer at a wavelength of 550 nm and 260 nm, respectively. 2.5.8. Protein estimation Protein concentration was determined according to Lowry et al. (1951) using bovine serum albumin as standard. Protein reacts with Folin-Ciocalteau reagent to give a colored complex, which was measured in Hitachi U2001 UV/Vis spectrophotometer at a wavelength of 660 nm. 2.6. Statistical analysis The experiments were performed three times and the data are presented as mean ± S.E.M., n = 8. Comparisons of the means of control, and experimental groups were made by two-way ANOVA test (using a statistical package, Origin 6.1, Northampton, MA 01060, USA) with multiple comparison t-tests, P < 0.05 as a limit of significance. 3. Results 3.1. Vancomycin loading and characterization of nanoconjugated vancomycin 3.1.1. Actual drug content and encapsulation efficiency Vancomycin loaded CMC-EDBE-FA nanoparticles (VNP1-VNP3) were successfully prepared in different polymer:drug ratios. Actual drug contents were approximately 32%, 15%, 24% for the vancomycin loaded CMC-EDBE-FA nanoparticle with polymer:drug ratios 1:1, 2:1, 1:2, respectively. High drug encapsulation efficiency (over 65%) was achieved with polymer:drug ratio 1:1 formulation (Table 1). 3.1.2. DLS study of CMC-EDBE-FA nanoparticles and nanoconjugated vancomycin The sizes of CMC-EDBE-FA self-assembled nanoparticles and vancomycin loaded CMC-EDBE-FA nanoparticles in aqueous medium were measured by dynamic laser light scattering ranged from 210 ± 40 nm and 260 ± 35 nm, respectively (Fig. 1). 3.1.3. TEM analysis of CMC-EDBE-FA nanoparticles and nanoconjugated vancomycin TEM images were taken to investigate the morphology of CMCEDBE-FA self-aggregated nanoparticles and vancomycin loaded CMC-EDBE-FA nanoparticles. The nanoaggregate shows a spherical geometry having a uniform size. TEM images of CMC-EDBE-FA nanoparticles and vancomycin loaded nanoparticles in aqueous solution were presented in Fig. 2. At lower magnification nanoparticles having an average size of about 50 nm were observed. On the other hand, after vancomycin loading the size of the nanoparticles increased with an irregular shape. 3.1.4. In vitro release profile of vancomycin from nanoconjugated vancomycin The release profile of vancomycin from vancomycin loaded CMC-EDBE-FA nanoparticle was displayed in Fig. 3. The highest dissolution rate (95.24%) was achieved at 8 h.

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(a)

100

100

80

Number (%)

Number (%)

80

60

40

20

0

(b)

60

40

20

0

100

200

300

400

500

Mean Diameter (nm)

0 0

100

200

300

400

500

Mean Diamteter (nm)

Fig. 1. The size determination of (a) CMC-EDBE-FA nanoparticles and (b) vancomycin loaded CMC-EDBE-FA nanoparticles by dynamic light scattering.

Fig. 2. TEM images of (a) CMC-EDBE-FA nanoparticles and (b) vancomycin loaded CMC-EDBE-FA nanoparticles.

3.2. In vitro antimicrobial activity of nanoconjugated vancomycin 3.2.1. Minimum inhibitory concentration (MIC) Particular drug concentration was noted where no visible growth appears in broth culture, both in case of vancomycin and nanoconjugated vancomycin treatment. In case of vancomycin

3.2.2. Minimum bactericidal concentration (MBC) Drug concentration was noted where no visible growth appears on agar plate, both in case of vancomycin and nanoconjugated vancomycin treatment. The MBC value was decreased significantly (P < 0.05) by 50% and 99.75% in vancomycin sensitive and resistant strain when charged with nanoconjugated vancomycin (Fig. 5).

100

% Released Vancomycin

sensitive strain, the MIC value was decreased significantly (P < 0.05) by 46.15% when charged with nanoconjugated vancomycin where as in case of vancomycin resistant strain, the MIC value was decreased significantly (P < 0.05) by 97.52% when charged with nanoconjugated vancomycin (Fig. 4).

80

3.2.3. Tolerance level Tolerance level of each strain against vancomycin and nanoconjugated vancomycin was calculated from the respective MIC and MBC value. In case of vancomycin sensitive strain, the tolerance level was decreased significantly (P < 0.05) by 28.35% when charged with nanoconjugated vancomycin where as in case of vancomycin resistant strain, the tolerance level was decreased significantly (P < 0.05) by 90.63% when charged with nanoconjugated vancomycin (Fig. 6).

60

40

20

0 0

60

120 180 240 300 360 420 480 540

Time (minute) Fig. 3. In vitro release profiles of vancomycin from vancomycin loaded CMC-EDBEFA nanoparticle. Experimental results are presented as mean ± SD, n = 3 of data obtained from three independent experiments that yielded similar results.

3.2.4. Disc agar diffusion The diameter of zone of inhibition was increased significantly (P < 0.05) by 30.77% in case of vancomycin sensitive strain when charged with nanoconjugated vancomycin, where as in case of vancomycin resistant strain, it was increased significantly (P < 0.05) by 136.90% (Fig. 7).

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665

Fig. 4. Minimum inhibitory concentration of VSSA and VRSA strains against vancomycin and nanoconjugated vancomycin. Here A = 0.5 ␮g/ml, B = 1 ␮g/ml, C = 2 ␮g/ml, D = 4 ␮g/ml, E = 8 ␮g/ml, F = 16 ␮g/ml, G = 32 ␮g/ml, H = 64 ␮g/ml, I = 128 ␮g/ml, J = 256 ␮g/ml, K = 512 ␮g/ml, and L = 1024 ␮g/ml. All the experimental results are presented as mean ± S.E.M., n = 8. Data obtained from three independent experiments that yielded similar results. *Statistically significant (P < 0.05) difference as compared to control group.

Table 2 Fractional inhibitory concentration values of nanoconjugated vancomycin against VSSA and VRSA strains. Bacterial strains

MIC values of vancomycin and NV in combination [A]

MIC values of vancomycin [MICA ]

Concentration of NV [B]

MIC values of NV [MICB ]

FIC index

MMC-1 MMC-2 MMC-3 MMC-5 MMC-6 MMCc-7 MMCc-8 MMC-15 MMC-4 MMC-9 MMC-12 MMC-16 MMC-17 MMC-18 MMC-19 MMC-20

1.0 1.0 1.0 1.0 1.5 1.0 1.5 1.0 8.0 5.0 19.0 7.0 7.5 5.0 11.0 15.5

2.0 1.0 1.0 2.0 2.0 1.0 2.0 2.0 64 32.0 128.0 64.0 32.0 32.0 64.0 128.0

0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5

1.5 0.5 0.5 1.0 1.5 0.5 1.0 0.5 1.5 2.0 1.5 1.5 2.0 1.5 2.0 1.5

0.83 2.0 2.0 1.0 1.08 2.0 1.25 1.5 0.46 0.41 0.48 0.44 0.48 0.49 0.42 0.45

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Fig. 5. Minimum bactericidal concentration of VSSA and VRSA strains against vancomycin and nanoconjugated vancomycin. Here A = 0.5 ␮g/ml, B = 1 ␮g/ml, C = 2 ␮g/ml, D = 4 ␮g/ml, E = 8 ␮g/ml, F = 16 ␮g/ml, G = 32 ␮g/ml, H = 64 ␮g/ml, I = 128 ␮g/ml, J = 256 ␮g/ml, K = 512 ␮g/ml, and L = 1024 ␮g/ml. All the experimental results are presented as mean ± S.E.M., n = 8. Data obtained from three independent experiments that yielded similar results. *Statistically significant (P < 0.05) difference as compared to control group.

3.2.5. Fractional inhibitory concentration (FIC) In this experiment, the concentration at which nanoconjugated vancomycin inhibited bacterial growth was pre-determined and the value was 0.5 ␮g/ml. It was calculated from the formula described earlier (Eliopoulos and Moellering, 1991), that for vancomycin sensitive and resistant S. aureus species tested and for different values of MICA , MICB and [A]. FIC values were less than 0.5 in case of resistant strains. The exception was sensitive strains, which exhibited FIC values more than 0.5. Thus, the condition for synergistic effect (Schmidt et al., 2001) of nanoconjugated vancomycin and vancomycin combination was satisfied by all multiple antibiotic resistant S. aureus (Table 2).

3.2.6. Killing kinetic assay (KKA) The time-kill curve of vancomycin and nanoconjugated vancomycin against VSSA and VRSA strains is presented in Fig. 8. Bactericidal activity (>59.9% reduction) was observed after 12 h exposure of the isolates to vancomycin and nanoconjugated vancomycin at their respective MIC concentrations. Bactericidal activity of nanoconjugated vancomycin is higher after 12 h exposure to VSSA strains as compared to VRSA. Nanoconjugated vancomycin showed a time-dependent and rapid bactericidal activity against the test VSSA and VRSA, leads bacteria to early stationary phase, as shown in time-kill curves (Fig. 8).

S.P. Chakraborty et al. / International Journal of Pharmaceutics 436 (2012) 659–676 18

Vancomycin Treated

NV TReated

9

16

VSSA+Van VRSA+Van

VSSA+NV VRSA+NV

8

14

7

12 -1

log10(c.f.u.ml )

Tolerance Level

VSSA VRSA

667

10 8 6

6 5 4 3

4

*

2

*

2 1

0

VSSA

VRSA

Fig. 6. Tolerance level of VSSA and VRSA strains against vancomycin and nanoconjugated vancomycin. All the experimental results are presented as mean ± S.E.M., n = 8. Data obtained from three independent experiments that yielded similar results. *Statistically significant (P < 0.05) difference as compared to control group.

3.2.7. Biofilm formation Vancomycin significantly decreased (P < 0.05) the biofilm formation of VSSA by 25.19%; and 1.30% of VRSA which is not significant; where as nanoconjugated vancomycin significantly decreased (P < 0.05) the biofilm formation of VSSA and VRSA by 53.11% and 42.86%, respectively (Fig. 9).

0 0 Hr 3 Hr 6 Hr 9 Hr 12 Hr 15 Hr 18 Hr 21 Hr 24 Hr 27 Hr 30 Hr

Time (hr) Fig. 8. Killing kinetic studies of VSSA and VRSA strains against vancomycin and nanoconjugated vancomycin. All the experimental results are presented as mean ± S.E.M., n = 8. Data obtained from three independent experiments that yielded similar results. *Statistically significant (P < 0.05) difference as compared to control group.

3.2.8. Cell viability by MTT assay Vancomycin significantly decreased (P < 0.05) the cell viability of VSSA by 43.88%; and 4.27% of VRSA which is not significant; where as nanoconjugated vancomycin significantly decreased (P < 0.05)

Fig. 7. Diameter of inhibition zone of VSSA and VRSA strains against vancomycin and nanoconjugated vancomycin. All the experimental results are presented as mean ± S.E.M., n = 8. Data obtained from three independent experiments that yielded similar results. *Statistically significant (P < 0.05) difference as compared to control group.

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Control

Vanco Treated

110

NV Treated

Control

Vanco Treated

NV Treated

100 0.25

80

0.20

0.15

0.10

* *

70 60

* *

50 40

*

30

*

0.05

Cell Viability (%)

Biofilm Formation (OD570)

90

20 10

0.00

VSSA

VRSA

0

VSSA Fig. 9. Biofilm formation of VSSA and VRSA strains against vancomycin and nanoconjugated vancomycin. All the experimental results are presented as mean ± S.E.M., n = 8. Data obtained from three independent experiments that yielded similar results. *Statistically significant (P < 0.05) difference as compared to control group.

the cell viability of VSSA and VRSA by 73.58% and 64.89%, respectively (Fig. 10).

VRSA

Fig. 10. Cell viability of VSSA and VRSA strains against vancomycin and nanoconjugated vancomycin. All the experimental results are presented as mean ± S.E.M., n = 8. Data obtained from three independent experiments that yielded similar results. *Statistically significant (P < 0.05) difference as compared to control group.

3.2.9. Cell viability count by FACS Flow cytometric susceptibility test showed that vancomycin slaughtered VSSA and VRSA cells by 55.30% and 11%, respectively; where as, nanoconjugated vancomycin slaughtered VSSA and VRSA cells by 75.40% and 61.10%, respectively (Fig. 11).

conditions. Rapid increase of fluorescence of the dye was observed in nanoconjugated vancomycin treated VSSA and VRSA cell suspension (Fig. 12). For assessment the nanoconjugated vancomycin uptake by VSSA and VRSA, fluorescence images showed that vancomycin treated VSSA cell has 20% rhodamine emission and VRSA cell has no such emission; where as nanoconjugated vancomycin treated VSSA and VRSA cells have 50% and 55% rhodamine emission (Figs. 12 and 13).

3.2.10. Membrane depolarization and Rh123 uptake The fluorescent probe (Rh123) was scanned to verify its excitation (505 nm) and emission (534 nm) maxima at experimental

3.2.11. Confocal microscopy Results of confocal microscopy suggest that vancomycin penetrate into VSSA cell by 33.33% but not in VRSA cell; where as

Fig. 11. Flow cytometric antibiotic susceptibility test of VSSA and VRSA strains against vancomycin and nanoconjugated vancomycin.

S.P. Chakraborty et al. / International Journal of Pharmaceutics 436 (2012) 659–676

a.u. of Rhodamine 123 fluorescence intencity

Control

Vanco Treated

NV Treated

*

2.6

669

nanoconjugated vancomycin penetrate into VSSA and VRSA cell by 67.31% and 56.48%, respectively (Fig. 14).

*

2.4

3.3. In vitro mechanism of action of nanoconjugated vancomycin

2.2 2.0

*

1.8 1.6 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0

VSSA

VRSA

Fig. 12. Membrane depolarization of VSSA and VRSA cells against vancomycin and nanoconjugated vancomycin. All the experimental results are presented as mean ± S.E.M., n = 8. Data obtained from three independent experiments that yielded similar results. *Statistically significant (P < 0.05) difference as compared to control group.

3.3.1. FTIR spectroscopy FTIR is an appropriate technique to study the chemical adsorption or chemical interaction. The FTIR spectra of CMC-EDBE-FA nanoparticles and the conjugation with vancomycin, lactate and alanine are presented in Fig. 15. The peak assignment of CMC-EDBEFA nanoparticles was as follows: 3455 (O H stretch overlapped with N H stretch), 2922 and 2879 (C H stretch), 1650–1550 (C O stretch of carboxyl methyl group overlapped with amide N H bend), 1409 (C H bend), 1327 (C N stretch), 1155 (bridge O stretch), and 1077 (C O stretch). After conjugation with vancomycin, alanine and lactate molecules with CMC-EDBE-FA nanoparticles through physical cross linking the variation in the band was observed. 3.3.2. TEM analysis study of VSSA and VRSA cell wall After treatment with vancomycin and nanoconjugated vancomycin, VSSA and VRSA cells were subjected to morphometric study using transmission electron microscopy. It was evident in transmission electron micrographs, VRSA strains had significantly thicker cell walls than VSSA strains (Figs. 16 and 17). The results of cell wall thickness measurement for control and treated VSSA and VRSA strains are presented in Fig. 16. The mean (SD) cell wall thicknesses of VSSA and VRSA strains were 37.2 (4.11) and 59.74 (7.37) nm, respectively. Vancomycin significantly decreased the

Fig. 13. Fluorescence (Rh123) uptake in VSSA and VRSA cells against vancomycin and nanoconjugated vancomycin. Vancomycin and nanoconjugated vancomycin were labeled with rhodamine 123, charged against VSSA and VRSA and examined by fluorescence microscope.

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Fig. 14. Membrane permeabilization of nanoconjugated vancomycin in VSSA and VRSA cell. VSSA and VRSA cells were first stained with FITC. Vancomycin and nanoconjugated vancomycin were labeled with rhodamine 123, charged against VSSA and VRSA and examined by confocal microscope.

(P < 0.05) cell wall thickness of VSSA by 31.34%; and 8.07% of VRSA which is not significant; where as nanoconjugated vancomycin significantly decreased (P < 0.05) the cell wall thickness of VSSA and VRSA by 46.88% and 59.26% (Fig. 17). 3.3.3. AFM imaging of the antimicrobial effects of nanoconjugated vancomycin on VSSA and VRSA In this study we used AFM to investigate the morphological changes and structural damage induced in VSSA and VRSA

cells after exposure to nanoconjugated vancomycin. The AC mode was used to acquire the topographical AFM images during the scan (Fig. 18). Nanoconjugated vancomycin as shown in Fig. 18A by AFM observation exhibits an irregular assemblage shape like snowflakes. The topographical images show the typical nearspherical shaped form of a single S. aureus cell before antibiotic exposure (Fig. 18B and C). The VSSA cells were degraded from a spherical shape to irregularly condensed masses when treated with vancomycin as shown in Fig. 18D. When the VRSA were treated with

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85 80

a=CMC-EDBE-FA b=CMC-EDBE-FA + Vancomycin c=CMC-EDBE-FA + Vancomycin + Lactate d=CMC-EDBE-FA + Vancomycin + Alanine

70

Cell wall thickness (nm)

% of Transmitance

70

d

60 55 50 45

Vanco Treated

NV Treated

60

75

65

Control

671

a

50

40

* #

30

#

20

b

40

10

c

35 0

30

VSSA

25 4000

3500

3000

2500

2000

Wave number/cm

1500

1000

500

-1

VRSA

Fig. 17. Cell wall thickness of VSSA and VRSA strains against vancomycin and nanoconjugated vancomycin. All the experimental results are presented as mean ± S.E.M., n = 8. Data obtained from three independent experiments that yielded similar results. *Statistically significant (P < 0.05) difference as compared to control group.

Fig. 15. The FT-IR spectra of CMC-EDBE-FA alone and in combination with vancomycin, alanine and lactate.

vancomycin, the cells were surrounded by vancomycin as shown in Fig. 18F. When VSSA and VRSA cells were treated with nanoconjugated vancomycin, cells were disrupted to a considerable degree with the leakage of cytosolic components, membrane sloughing, breaching, bledding and began to fragment (Fig. 18E and G).

3.3.4. Membrane Na+ , K+ -ATPase activity Vancomycin significantly increased (P < 0.05) the membrane Na+ , K+ -ATPase activity of VSSA by 76.71%; and 5.54% of VRSA which is not significant; where as nanoconjugated vancomycin significantly increased (P < 0.05) the membrane Na+ , K+ -ATPase activity of VSSA and VRSA by 189.51% and 162.49% (Fig. 19A).

Fig. 16. The TEM images of VSSA and VRSA cell wall after vancomycin and nanoconjugated vancomycin treatment.

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Fig. 18. Atomic force micrographs of VSSA and VRSA cells after treatment with vancomycin and nanoconjugated vancomycin. Nanoconjugated vancomycin (A), non treated VSSA cells (B), non treated VRSA cells (C); vancomycin treated VSSA cells (D), nanoconjugated vancomycin treated VSSA cells (E), vancomycin treated VRSA cells (F), nanoconjugated vancomycin treated VRSA cells (G).

4.5

Vanco Treated

NV Treated

*

5

*

A

Control

Vanco Treated

NV Treated

* 4

Potassium leakage (µg/ml)

3.5 3.0

* 2.5 2.0 1.5

+

+

Na , K -ATPase activity (µ moles of Pi liberated/mg protein/h)

4.0

Control

1.0

B

*

3

*

2

1

0.5

0

0.0

VSSA

VRSA

VSSA

Control

Vanco Treated

260nm absorbing material (%) leakage of metabolic pool

7.5 7.0 6.5

VRSA

NV Treated

*

C

*

6.0 5.5 5.0 4.5

*

4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0

VSSA

VRSA

Fig. 19. Membrane Na+ , K+ -ATPase activity (A), intracellular potassium release (B) and leakage of cellular constituents absorbing at 260 nm (C) of VSSA and VRSA strains against vancomycin and nanoconjugated vancomycin. All the experimental results are presented as mean ± S.E.M., n = 8. *Statistically significant (P < 0.05) difference as compared to control group.

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3.3.5. Intracellular potassium release Vancomycin significantly increased (P < 0.05) the intracellular potassium release of VSSA by 100.77%; and 1.44% of VRSA which is not significant; where as nanoconjugated vancomycin significantly increased (P < 0.05) the intracellular potassium release of VSSA and VRSA by 247.69% and 200% (Fig. 19B). 3.3.6. Leakage of cellular constituents Vancomycin significantly increased (P < 0.05) the leakage of cellular constituents of VSSA by 114.44%; and 1.56% of VRSA which is not significant; where as nanoconjugated vancomycin significantly increased (P < 0.05) the leakage of cellular constituents of VSSA and VRSA by 273.26% and 218.23% (Fig. 19C). 4. Discussion S. aureus resistant to antibiotics appears within a few years after the onset of the antibiotic epoch (Kirby, 1944) and currently spans all known classes of antibiotics (D’Costa et al., 2006). Increasing resistance of S. aureus to vancomycin in developed and developing countries is alarming. Vancomycin acts against Gram-positive bacteria only by inhibiting the incorporation of NAM-NAG-polypeptide into the growing peptidoglycan chain and exerts its bactericidal action. Resistance of vancomycin develops due to uncontrolled use of vancomycin as an antibiotic that causes the thickening of cell membrane; results of which, the cell membrane become impermeable and further use of vancomycin cannot permeable through cell membrane and remains inactive state. Vancomycin resistance in S. aureus highlights the need for development of new and novel anti-VRSA antibiotics (Bax et al., 2001; Livermore, 2004). In this study, to develop the anti-VRSA antibiotics, folic acid modified carboxymethyl chitosan nanoparticles was used to which vancomycin was loaded and charged against sensitive and resistant strains of S. aureus. In the present study, polymer:drug ratio of 1:1 formulations achieved high drug encapsulation efficiency (over 65%) and dissolution rate (95.24%) at 8 h (Table 1 and Fig. 3). Based on this result, VNP1 was selected for this study. CMC-EDBE-FA conjugates formed monodisperse self-aggregated nanoparticles by sonicating their dispersions in water at a concentration of 0.2 mg/ml. On probe sonication, the size of nanoparticles was found to decrease with increasing sonication time, reaching a limiting value after a maximum of 3 min. After vancomycin loading, the size of nanoparticles became slightly larger (Fig. 1), suggesting that a few bits of crosslinking reaction take place between the free carboxyl groups of the nanoparticles and the amine groups present in vancomycin molecules. TEM images shows that after vancomycin loading the size of the nanoparticles increased with an irregular shape (Fig. 2). TEM described the size in the dried state of the sample, whereas DLS measured the size in the hydrated state, so that the size measured by DLS was a hydrodynamic diameter and had a larger value. Result of this study shows significant decrease of minimum inhibitory concentration (Fig. 4), minimum bactericidal concentration (Fig. 5), onset of stationary phase (Fig. 8), virulence factor (Fig. 9), cell viability (Fig. 10) and flow cytometric susceptibility test (Fig. 11) of VSSA and VRSA strains when charged with nanoconjugated vancomycin. It was also observed from our study, diameter of inhibition zone of VSSA and VRSA strains was increased significantly when charged with nanoconjugated vancomycin (Fig. 7). FIC index interpretations of the activities of nanoconjugated vancomycin against VSSA and VRSA strains predominantly showed difference; the drug combinations were each synergistic for vancomycin resistant S. aureus (FIC index range for synergism, <0.5). The interpretations of the activity of nanoconjugated vancomycin combined with vancomycin produced a determination of antagonism for vancomycin sensitive S. aureus (FIC index, >0.5) (Table 2).It

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may be due to the penetration of nanoconjugated vancomycin into the bacterial cell that inhibits the bacterial growth and acts as a bactericidal agent followed by bacteriostatic activity. It was also observed from our study that the tolerance level of VSSA and VRSA strains was decreased significantly when charged with nanoconjugated vancomycin (Fig. 6). It may be due to decrease MIC and MBC value of nanoconjugated vancomycin treated VSSA and VRSA strains as compared with vancomycin treatment. The fluorescence response of the Rh123 changes with the cell membrane potential. In this experiment, the rapid increase of fluorescence intensity of nanoconjugated vancomycin treated VSSA and VRSA cell suspension demonstrates the membrane depolarizing activity of this compound. Hence, it can be stated that nanoconjugated vancomycin permeabilizes the outer membrane of the VSSA and VRSA strains by depolarizing their outer membrane (Figs. 12–14). The surface properties of the nanoparticles are important to enable design and tailoring of drug delivery systems. The nanoparticles are synthesized using carboxylic group ( COOH) of the folic acid and COOH group of functionalized carboxymethyl chitosan connected through the end-amino groups hydrophilic spacer, 2,2 (ethylenedioxy)-bis ethylamine. Considering that there is a large surface-to volume ratio and a number of functional groups on the surface of the nanoparticles. These functional groups on the surface of nanoparticles tend to adsorb ions or molecules from the solution. Thus, to analyse the surface properties of the nanoparticles, FTIR studies of the CMC-EDBE-FA nanoparticles in combination with vancomycin, alanine and lactate were carried out. After conjugation with vancomycin, alanine and lactate molecules with CMC-EDBEFA nanoparticles through physical cross linking the variation in the band was observed. The appearance of these variant peaks confirms the successful conjugation of vancomycin, alanine and lactate molecules on the nanoparticles (Fig. 15). It seems likely that the thickening of the cell wall is closely associated with the mechanism of vancomycin resistance in the VRSA strains. As other researchers proposed previously, trapping of vancomycin molecules in the cell wall peptidoglycan would be the essential contributor (Hiramatsu, 1998; Sieradzki et al., 1999). The thicker the cell wall, the more vancomycin molecules would be trapped within the cell wall, thus allowing a decreased number of vancomycin molecules to reach the cytoplasmic membrane where the real functional targets of vancomycin are present (Breukink et al., 1999; Hiramatsu, 1998). Vancomycin binds to the stem peptide of the membrane-anchored murein monomer (lipid II) at its Lys-d-Ala-d-Ala residue and thus prevents the murein monomer from being incorporated into the nascent peptidoglycan chain (Reynolds and Somner, 1990; Walsh, 1999). Thickened cell wall not only traps a greater number of vancomycin molecules but also significantly reduces the time that vancomycin completely inhibits peptidoglycan synthesis. In view of their low-level vancomycin resistance and any alteration in the terminal d-alanyl-d-alanine residues of peptidoglycan, it would be reasonable to consider the cell wall thickening as the major contributor to the vancomycin resistance of S. aureus clinical strains (Cui et al., 2000; Hiramatsu, 1998, 2001). The morphology of nanoconjugated vancomycin treated VSSA and VRSA cells was examined by atomic force microscopy. Several mechanisms for the antimicrobial action of chitosan have been postulated (Fig. 18). There are as follows: (a) chitosan could chelated with trace elements or essential nutrients so as to inhibit the growth of bacteria (Roller and Covill, 1999); (b) chitosan could interact with anionic groups on the cell surface and form polyelectrolyte complexes with bacterial surface compounds, thereby forming an impermeable layer around the cell, which prevents the transport of essential solutes into the cell (Muzzarelli et al., 1990; Choi et al., 2001). A similar AFM result was recently reported by Rai et al. (2010) and Potara et al. (2011). Rai et al. found that cefaclor reduced gold nanoparticles have the ability to directly interact with

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Fig. 20. Possible in vitro antimicrobial mechanism of action of nanoconjugated vancomycin (NV).

S. aureus cell walls and to penetrate through the rigid peptidoglycan layer. Potara et al. found that dramatic changes in morphology of S. aureus cells due to disruption of bacterial cell wall integrity after incubation with chitosan–silver nanoparticles was observed as revealed by atomic force microscopy. The possible cause of the membrane disruption may be due to the huge differences in pressure between the intracellular and extracellular environments when the regulation of internal pressure through the pores in the peptidoglycan layer is strongly perturbed by the interaction with nanoconjugated vancomycin. An important component of the S. aureus cell wall is the peptidoglycan layer which confers strength and rigidity to the cell, allowing its growth and division, keeping the cell shape and protecting against osmotic lysis (Cabeen and Jacobs-Wagner, 2005). As the outer membrane is absent in S. aureus, the multilayered peptidoglycan represents the site for exchange and interactions between this strain of bacteria and the external environment. The peptidoglycan layer consists of linear polysaccharide chains cross linked by short peptides. This architecture provides small number of anchoring sites, which makes the interaction with antibacterial agents. Peptidoglycan, thus, represents an important challenge for the development of antimicrobial agents. The ability of nanoconjugated vancomycin to penetrate the peptidoglycan layer demonstrates once again their superior efficiency as antimicrobial agents. Chitosan nanoparticles exhibit higher antibacterial activity than chitosan on account of the special character of the nanoparticles.

The negatively charged surface of the bacterial cell is the target site of the polycation (Avadi et al., 2004). Therefore, the polycationic chitosan nanoparticles with higher surface charge density interact with the bacteria to a greater degree than chitosan itself. Chitosan nanoparticles provide higher affinity with bacteria cells for a quantum-size effect. Because of the larger surface area of the chitosan nanoparticles, nanoparticles could be tightly adsorbed onto the surface of the bacteria cells so as to decrease cell wall thickness (Figs. 16 and 17); disrupt the membrane, which would lead to increase Na+ , K+ -ATPase activity, intracellular potassium release and leakage of intracellular components (Fig. 19), thus killing the bacteria cells. From this study it was evident that chitosan tagged with folic acid acts as a drug delivery vehicle which overcome the barrier of cell membrane and deliver drug into the bacterial cell. The enticement by folic acid which is an essential nutrient required for nucleotide synthesis for the bacteria, helps to transport the nanoconjugated vancomycin through endocytosis across the plasma membrane into the cytoplasm (Russell-Jones, 1996; Tamai and Tsuji, 1996). 5. Conclusion In conclusion, nanoconjugated vancomycin shows an effective drug delivery system for drug resistance S. aureus. Nanoconjugated vancomycin may binds with lactate, which was replaced in place

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