Evaluation of physicochemical properties, and ...

Evaluation of physicochemical properties, and antimicrobial efficacy of monoclinic sulfur-nanocolloid Samrat Roy Choudhury, Amrita Mandal, Dipankar Chakravorty, Madhuban Gopal & Arunava Goswami Journal of Nanoparticle Research An Interdisciplinary Forum for Nanoscale Science and Technology ISSN 1388-0764 Volume 15 Number 4 J Nanopart Res (2013) 15:1-11 DOI 10.1007/s11051-013-1491-y

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Author's personal copy J Nanopart Res (2013) 15:1491 DOI 10.1007/s11051-013-1491-y

RESEARCH PAPER

Evaluation of physicochemical properties, and antimicrobial efficacy of monoclinic sulfur-nanocolloid Samrat Roy Choudhury • Amrita Mandal • Dipankar Chakravorty • Madhuban Gopal • Arunava Goswami

Received: 6 July 2012 / Accepted: 7 February 2013 Ó Springer Science+Business Media Dordrecht 2013

Abstract Stable nanocolloids of monoclinic sulfur (b-SNPs) were prepared through ‘water-in-oil microemulsion technique’ at room temperature after suitable modifications of the surface. The morphology (rod shaped; *50 nm in diameter) and allotropic nature (monoclinic) of the SNPs were investigated with Transmission Electron Microscopy and X-ray Diffraction technique. The surface modification, colloidal stability, and surface topology of b-SNPs were evaluated with Fourier Transform Infrared Spectroscopy, zeta potential analysis, and Atomic Force Microscopy. Thermal decomposition pattern of these nanosized particles was determined by Thermo Gravimetric Analysis (TGA). b-SNPs-colloids expressed excellent antimicrobial activities against a series of

Electronic supplementary material The online version of this article (doi:10.1007/s11051-013-1491-y) contains supplementary material, which is available to authorized users. S. Roy Choudhury (&) A. Goswami Biological Sciences Division, Indian Statistical Institute, 203 B. T. Road, Kolkata 700108, West Bengal, India e-mail: [email protected]; [email protected] A. Mandal D. Chakravorty Indian Association for the Cultivation of Science, Kolkata 7000032, West Bengal, India M. Gopal Divisions of Agricultural Chemicals, Indian Agricultural Research Institute, New Delhi 110012, India

fungal and bacterial isolates with prominent deformities at their surface. In contrast, insignificant cytotoxicity was achieved against the human derived hepatoma (HepG2) cell line upon treatment with b-SNPs. A simultaneous study was performed to determine the stock concentration of b-SNP-colloids using a novel high phase liquid chromatographic method. Cumulative results of this study hence, elucidate the stabilization of nanosized monoclinic sulfur at room temperature and their potential antimicrobial efficacy over micron-sized sulfur. Keywords Monoclinic Sulfur nanoparticles Nanocolloids Antimicrobial Cytotoxicity Biocompatibility

Introduction Several allotropes of elemental sulfur (ES) are known to exist in nature with their respective physicochemical and biological properties (Meyer 1976; Mitchell 1996). Among them, the orthorhombic (a-sulfur) and monoclinic (b-sulfur) allotropes are the most commonly occurring forms in nature. Both these allotropes of ES and their compounds (H2S, pentathionic acid, or SO2) have long been known for their microbicidal efficacy (Williams and Young 1929; McCallan 1949). ES-allotropes are believed to exert the antimicrobial effects through contact action and respiratory inhibition (McCallan

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1949; Beffa et al. 1987; Baldwin 1950). Furthermore, it is worth mentioning that ES is the only naturally accumulating inorganic phytoalexin in plants to combat the microbial invasions (Williams and Cooper 2003, 2004). Sulfur-conjugated antimicrobial drugs (sulfonamides) are also used in biomedical sectors for treating severe bacterial infections. However, non-specific contact action, insolubility in water, microbial resistance, and commonly reported allergenicity to the sulfur drugs reinforce the necessity of developing alternative synthesis methods for the sulfurous antimicrobial agents to sustain the use of sulfur (Neafsey et al. 2010; Sun et al. 2004). Incompatibility of insoluble drugs (including sulfurous) with water-based physiological environments such as blood, cytoplasm, and other body fluids presents a major challenge in their delivery, biodistribution, and bioavailability (Wang et al. 2005). Stable nanocolloids of such insoluble drugs have the potential to facilitate enhanced cellular uptake and ensure targeted delivery into the cytoplasm (Leo et al. 2004; Roy Choudhury et al. 2012e). Orthorhombic nano-forms (a-SNPs) of insoluble sulfur particles with remarkable in vitro microbicidal efficacy have already been reported by our consortium (Roy Choudhury et al. 2011, a, b). It has been successfully demonstrated that nanoencapsulations of a-SNPs significantly enhanced their delivery into microbial cytosol and minimize toxicity against the tested human-derived lung fibroblast cell line. (Roy Choudhury et al. 2013). However, the aforementioned allotrope works best up to the temperature of *95 °C, over which it is naturally converted into the monoclinic allotrope. This natural inter-conversion between a- and b- sulfur implies an internal tensional state, which is followed by disintegration and reduction in the volume of sulfur (Blight et al. 1978). Hence, the antimicrobial treatment of thermophilic microorganisms with a-sulfur would be impossible to be achieved at high temperatures. Several approaches have already been attempted to lessen the sulfur crystallization at differential temperature, especially with the use of organic polymers as the additives or modifiers (Blight et al. 1978; Bordoloi and Pearce 1978). Here we introduce the concept of stabilization of b- sulfur of nanodimension encapsulated within polymeric surfactants, and essentially at room temperature. The present study also elucidates the microbicidal efficiency of

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monoclinic sulfur nanoparticles (b-SNPs) against both the fungal and bacterial isolates of clinical and agricultural importance. A series of common pathogenic fungi and bacteria were chosen to demonstrate the microbicidal efficacy of the b-SNPs. In the present study, b-SNPs were stabilized at room temperature using ‘water in oil micro-emulsion’ technique with suitable modifications of their surfaces. Size (*50 nm), shape (rod-shaped), and allotropic nature (monoclinic) of SNPs were characterized with Transmission Electron Microscopy (TEM) and X-ray diffraction (XRD) technique, respectively. Surface topological features and surface encapsulations of bSNPs were determined with Atomic Force Microscopy (AFM) and Fourier Transform-Infra red (FT-IR) spectroscopy, respectively. Purity and elemental composition of the b-SNPs were determined with Energy Dispersive X-ray (EDX) spectroscopy. Colloidal stability and thermal decomposition pattern of the prepared nanocolloids were determined with zeta potential values and Thermo-gravimetric analysis (TGA), respectively. Evaluation of ES as an active ingredient in the prepared nanocolloids was carried out, prior to determining its dosage and stock concentration. A simultaneous study was carried out to assess the biocompatible profile of b-SNPs against the Human derived hepatoma (HepG2) cell line using WST-1 assay.

Materials and methods Materials Aspergillus niger strains (MTCC-282, 2196) were purchased from the Microbial Type Culture Collection (MTCC), IMTECH, Council of Scientific and Industrial Research (Chandigarh, India). The wild strains of A. niger (MTCC-10180) and A. fumigatus were procured from the environment, characterized, and deposited in MTCC. Fusarium oxysporum strains (NCL-1072, NCL-1008, and NCL-1043) were obtained from the National Collection of Industrial Microorganisms, National Chemical Laboratory (Pune, India). Gram-negative [Escherichia coli (n = 4), Klebsiella pneumoniae (n = 4), Acinetobacter baumannii (n = 3), and Pseudomonas aeruginosa (n = 2)] and gram-positive [Staphylococcus aureus (n = 2),, Staphylococcus kloosii (n = 1), and

Author's personal copy J Nanopart Res (2013) 15:1491

Stenotrophomonas maltophilia (n = 1)] bacterial isolates were collected from both environmental and nosocomial sources. Wild strains were identified using ID-32E or ID-32GN and ID-32STAPH kit (bioMe0 rieux, Marcy l’Etoile, France). Standard bacterial strains of E. coli (ATCC-25922) and K. pneumoniae (ATCC700603) were obtained from the American Type Culture Collection Centre (ATCC) (VA, USA). All the chemicals used for the present study, unless specified, were purchased from Sigma-aldrich (MO, USA). Deionized water (18 MX, arium 61316 reverse osmosis system, Sartorius Stedium Biotech, Aubagne, France) was used for all the experiments. Preparation of the monoclinic sulfur nanocolloids b-SNP-colloids were synthesized after suitable modifications to the method originally proposed by Guo et al. 2006 (Guo et al. 2006). Briefly, two reverse microemulsion systems were prepared. Each of the microemulsion (I & II) systems comprised identical components of oil phase (cyclohexane or oleylamine), surfactants (a mixture of Span and Tween-80 in 8:1 ratio or TritonX-100), and co-surfactants (1-butanol or n-hexanol), but differed in their aqueous phase (sodium/ammonium/calcium polysulfide for the microemulsion-I and hydrochloric acid for the microemulsion-II). For transparent solution of the microemulsion systems, oil phase, surfactant, cosurfactant, and aqueous phases were mixed in the ratio of 90:12:2:5. Two clear microemulsions, thus prepared, were mixed with each other using an emulsifier. However, depending upon the colloidal stability (Roy Choudhury et al. 2012c) and bioefficacy, the batch of b-SNPs prepared from the combination of sodium polysulfide as inorganic sulfur reactant, conjugate of Span-80 and Tween-80 as surfactant, 1-butanol as co-surfactant, and cyclohexane as oil phase was selected for all the assays included in the present study. Combined microemulsion, thus formed, was centrifuged (8,0009 rpm for 15 min), the precipitate was washed with acetone to eliminate excess surfactant and co-surfactants from SNP’s surface, and subjected to rotary vacuum evaporation for the removal of residual acetone. The resultant nanocolloid was then refrigerated (4 °C) for long time storage and future use. At 4 °C, the average particle size (*50 nm) of b-SNPs remains almost unaltered (with a variation of 5 nm) for around 3 months.

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Physicochemical characterization of the prepared nanocolloids Preparation of samples for physicochemical characterizations was carried out as described in previous studies (Roy Choudhury et al. 2011, c). The distributions of hydrodynamic diameters of SNPs were analyzed using Dynamic Light Scattering (DLS) [Zetasizer nano series: Nano-S, Malvern, Worcestershire, UK] at three different temperatures (45, 55, and 65 °C). The size, morphology, and allotropic nature of the synthesized SNPs were determined by TEM [JEOL Model JEM 2010, Japan] operated at 200 kV after mounting the samples on a carbon coated copper grid. The allotropic nature was further confirmed with the XRD analysis [X’Pert PRO-MRD, PANalytical B. V, Netherlands]. The XRD system was equipped with graphite monochromatized Cu Ka radiation (k = 1.54 A°) with a scan rate of 5°/min in the 2h range from 10° to 80°. Purity and composition of the prepared SNPs were determined by Energy Dispersive X-ray (EDX) [FEI Quanta-200 MK-2, Oregon, USA] spectroscopy. Topological features and modification of the surface of b-SNPs were evaluated using AFM [di-CP-II with Proscan-19 software, Veeco, New York, USA] and FT-IR [Shimazdu Corp., Kyoto, Japan] spectroscopy, respectively. The surface charge distribution of the electrostatically stabilized b-SNP-colloids was assayed by measuring the zeta potential [Zeta-sizer nano series; Malvern, Worcestershire, UK] using Smoluchowski model. Thermal decomposition pattern of the b-SNPs was studied with TGA [SII with Pyris manager software, Perkin Elmer, MA, USA] analysis in the temperature range 50–500 °C. Estimation of active ingredient and stock concentration Extraction and estimation of ES from the PEGylated a-SNPs formulation were carried out earlier by our group (Kumar et al. 2011). Principle of the reported method has been applied to the present study for effective extraction and estimation of ES in the prepared nanocolloids. Briefly, ES was extracted from the b-SNP-colloids with 50 mL of cyclohexane. The cyclohexane extract (bottom layer in the separator funnel) was then filtered through anhydrous sodium sulfate. The filtrate was subjected to a rotary vacuum evaporator and the concentrated extract, thus obtained,

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was adjusted to 50 mL in volume with additional cyclohexane. One milliliter of this cyclohexane extract was then further diluted with 25 mL of cyclohexane and subjected to the High Phase Liquid Chromatography (HPLC) [MERCK, HITACHI] for determining the concentration of ES under the wavelength (k) of 290 nm, flow rate of 0.5 mL/min, 100 % methanol as the mobile phase, and RP-18 as the chromatographic column. Technical grade sulfur powder (MERCK, Germany) was used as the standard for obtaining the concentration of ES in the b-SNPcolloids. The antimicrobial assay with b-SNPs The fungal strains were cultured (104 spores/mL) in potato dextrose agar (PDA), while the bacterial strains were incubated (104 CFU/spot) in Muller Hinton agar (MHA) [BD, NJ, USA] prior to the performed assays. The antimicrobial susceptibility of the target pathogens to b-SNPs was determined in terms of minimum inhibitory concentration (MIC) via agar dilution method (ADM) (Roy Choudhury et al. 2012b; Wiegand et al. 2008). MICs were determined as the lowest concentration at which there was no detected growth in the test media (agar). All the microbial (fungi and bacteria) strains were cultured in their respective medium, supplemented with differentially diluted concentrations of b-SNPs starting from their stock concentration. Interaction and effects of b-SNPs on the microbial surface were visualized in comparison to the untreated replica using Field Emission Scanning Electron Microscopy (FE-SEM) at 5.0 kV. Such nano-bio interaction studies are important to understand the contact effect of these novel nanomaterials on the microbial membrane. One representative isolate of A. niger (MTCC-10180) and E. coli (ATCC-25922) have been included for the FE-SEM analysis in the present study. Furthermore, distribution of b-SNPs within the microbial bodies was determined with TEM coupled with EDX analysis. Assessment of biocompatibility of the b-SNPcolloids The WST-1 assay was performed to evaluate the effect of b-SNPs on the viability and decrease in the metabolically active cells of the Human derived

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Hepatoma cell line (HepG2) (Kikkawa et al. 2005). HepG2 cell line (ATCC-HB8065) was obtained from the American Type Culture Collection. Cells were incubated in Dulbecco’s modified Eagles medium (DMEM) supplemented with 10 % heat-inactivated fetal bovine serum (FBS), 19 glutamine (0.584 mg/ L), 19 sodium pyruvate (110 mg/L), and 19 non essential amino acids under 5 % CO2 at 37 °C. 104 cells/well were seeded in a 96-well plate in 200 lL medium. The pure medium was then replaced with 200 lL of b-SNPs supplemented media at five different concentrations (144, 288, 576, 1,152, 2,304, and 4,608 lg/mL) and incubated in triplicate for 72 h. Ten microliters of WST-1 reagent was then added to each well and further incubated for 4 h before measuring the absorbance at 450 nm.

Results Physicochemical properties of the b-SNPs The x-ray diffraction pattern of SNPs (Fig. 1a) revealed that the nanoparticles consisted of monoclinic phase. The peak positions were matched with the JCPDS (file number: 77- 0227) data for standard monoclinic sulfur phase, and it was found that the peaks corresponded to (223), (402), (606), (135), and (356) planes, respectively. The XRD pattern did not show any broad peaks. Agglomeration of the SNPs to form the rod like structure could be a possible reason for the absence of line broadening. The TEM micrographs, as shown in Fig. 1b, b inset, c revealed that Span-80-Tween-80 encapsulated b-SNPs were rod like in shape with an average diameter of *50 nm and length of *550 nm. The formation of b-SNPs was also confirmed from selected area electron diffraction (SAED) pattern of rod like structures. The interplanar spacing (dhkl) values estimated from SAED patterns were 0.16, 0.18, 0.22, and 0.37 nm, which are in good agreement with the standard dhkl values for (135), (351), (243), and (313) planes, respectively. The dynamic light scattering (DLS) measurements revealed size distributions (Fig. 1e) in hydrodynamic diameters in the range of 30–70 nm for b-SNPs, which is consistent with the diameter obtained from TEM studies. A non-contact mode atomic force microscopy was performed to analyze the surface topological features


Fig. 1 a X-ray diffractogram of the monoclinic sulfur nanoparticles (b-SNPs) prepared via water-in-oil microemulsion technique, b Transmission Electron Micrograph (TEM) of rodshaped b-SNPs. (Inset) Low resolution TEM image showing multiple rod-shaped structures of b-SNPs. c TEM image of a

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single rod-shaped structure having diameter *50 nm. d SAED pattern of rod-shaped b-SNPs e Size distribution curves obtained from three consecutive rounds of DLS measurements at 25 °C

Fig. 2 a AFM image of arbitrarily selected individual b-SNP, b 3-dimensional view of b-SNPs

of b-SNPs. Four individual b-SNPs (Fig. 2) were arbitrarily selected, and their gross surface features have been determined at 20 % bearing ratio (Table 1).

AFM micrographs reveal an average size of *50 nm with the mean surface roughness of *0.5130 nm of bSNPs, which also indicates their homogeneity in the

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Table 1 Topological features of arbitrarily selected b-SNPs as obtained from the atomic force microscopy (AFM) images Selected b-SNPs

Rp-v (nm)

Rms roughness (nm)

Average roughness (nm)

Mean height (nm)

Median height (nm)

Bearing ratio @20.0 % (nm)

Peak (nm)

Valley (nm)

Volume (lm3)

Surface area (lm2)

Projected area (lm2)

1

5.811

0.7999

0.5524

2.72

2.712

3.23

3.091

-2.72

0.0003916

0.1458

0.1439

2

5.811

0.7774

0.5299

2.695

2.712

3.1

3.116

-2.695

0.0003605

0.1355

0.1338

3

5.424

0.7273

0.5018

2.638

2.712

3.1

2.785

-2.638

0.0003314

0.1277

0.1256

4

4.261

0.6633

0.4682

2.559

2.712

3.1

1.702

-2.559

0.0002953

0.1168

0.1154

Rp-v: valley roughness Rms: mean surface roughness

Fig. 3 FT-IR spectra of a pure Span-80–Tween-80 solumer and b Span-80–Tween-80 conjugate encapsulated b-SNPs

synthesized nanocolloid. The surface encapsulation of b-SNPs with the conjugate of Span-80 and Tween-80 was analyzed by FT-IR spectra (Fig. 3). The major peaks have been indexed in the figure. The broad band centered at 3,454 cm-1 is assigned to O–H stretching (Coates 2000; Ludvigsson et al. 2000). After incorporation of sulfur, this band was red shifted to 3,434 cm-1 probably due to attachment of sulfur ions with H atoms of hydroxyl groups by weak electrostatic force of attraction. At the same time, the intensity (at 3,434 cm-1) increases as electronegative sulfur ions tend to attach the carbon of C=O groups, and in acidic medium, it converts the C=O to C–OH. As a result, the intensity of C=O stretching band at 1,742 cm -1 decreases (Dluhy et al. 1983). The absorption at 2,928 and 2,854 cm-1 are assigned to C–H stretching of the methylene group (Coates 2000). The intensity of these peaks decreases because sulfur ions have a probability

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to attach with sp2 carbon center and convert it to sp3 center. Due to the same reason, the peak intensity at 1,465 cm-1 (C–H bending of methylene group) (Coates 2000) was also decreased . The peaks at 1,087 and 725 cm-1 are assigned to C–O–C stretching (Innocenzi et al. 2010) and CH2 rocking modes (Rabe et al. 1986), respectively, which remain almost unchanged. According to the spot-EDX analysis of the arbitrary b-SNPs, an average of 38.88 wt% of sulfur was detected in the sample (Fig. 4a). Other major peaks, corresponding to carbon (15.42 wt%) and oxygen (45.7 wt%), might come from the surfactant (Span80–Tween-80 conjugate) layer. A negligible amount of N (1.85 %) was also traced, which was within the standard error value. The average of zeta potential values of Span-80– Tween-80 conjugate-coated b-SNPs-colloid was found to be around -5.33 mV at a pH of 4.19. Surface charge distribution (Fig. 4b) of the aforementioned b-SNPs suggests that the nanoparticles of our interest exhibit a tendency of flocculation. Colloidal stability and coagulation kinetics of this batch of bSNPs were determined earlier along the gradient of temperature, time drive, and dilution (Roy Choudhury et al. 2012c). Zeta potential of the b-SNPs-colloids prepared other than the Span-80–Tween-80 conjugate encapsulation, were found to be less negative and represent their enhanced tendency of agglomeration (Table S1). Thermal decomposition of b-SNPs took place following a non-isothermal pattern. The TGA graph (Fig. 4c) revealed almost half (43.46 %) of the weight loss at 122.39 °C, which can be correlated to the liberation of water molecules from the SNPcolloids. Furthermore, it was found that the b-SNPs underwent complete degradation at 413.13 °C with a


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Fig. 4 a EDX spectrum of b-SNP-colloids, b zeta potential distribution of b-SNPs, c TGA graph of b-SNPs

total mass loss of *92.31 % due to the loss of sulfur. There was a small amount of inert residue retained (0.15 %) at the highest applied temperature (500 °C). Thermal decomposition pattern of b-SNPs also suggest their suitability as antimicrobial agents at higher ranges of temperature.

Stock concentration of the b-SNP-colloids Estimation of ES in the b-SNPs-colloids was carried out successfully using the HPLC method. The limit of detection in HPLC method was 0.1 ppm (lg/mL). Calibration parameters were obtained from a least

Table 2 Antimicrobial susceptibility of the fungal and bacterial isolates expressed in terms of Minimum Inhibitory Concentrations (MIC) against the monoclinic sulfur nanoparticles and elemental sulfur Name of the organism with strain designation

MICs of monoclinic SNPsa (lg/mL)

MICs of ESb molar

Aspergillus niger (MTCC-282, 2196, 10180)

2,304

Resistant B 1 Mc

Aspergillus fumigatus (AF-2,3)

1,296

Resistant B 1 M

Fusarium oxysporum (NCL-1008, 1045, 1072)

1,843.2

1M

Escherichia coli (SG, NJ, S205, GNB-27, ATCC-25922)

2,304

Resistant B 1 M

Klebsiella pneumoniae (37-BL, 128-BL, 104-BL, ATCC-700603)

2,304

Resistant B 1 M

Acinetobacter baumannii (93, 211, 252)

1,152

Resistant B 1 M

Pseudomonas aeruginosa (247, 236)

1,152

Resistant B 1 M

Staphylococcus auerus (184, 198)

576

1M

Staphylococcus kloosii (46, 244)

576

1M

Stenotrophomonas maltophilia (1253)

576

1M

a

SNPs: sulfur nanoparticles

b

ES: elemental sulfur

c

Molar concentration

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square regression in a concentration range of 1.032–51.4 ppm. Its limit of quantification was 1.032 ppm. The concentration of ES in the stock solution of the b-SNP-colloid was estimated to be 18 mg/L. The antimicrobial properties of b-SNPs The MIC values of respective microbial isolates against micron-sized sulfur (ESM) and b-SNPs were evaluated and were summarized in Table 2. The ADM studies revealed that b-SNPs inhibited bacterial growth at a concentration as low as 576 lg/mL for the Staphylococcus aureus isolates. In contrast, the Klebsiella pneumoniae and Escherichia coli isolates expressed least susceptibility toward b-SNPs, and were inhibited at a concentration of 2,304 lg/mL (Table 2, Fig. S1). MICs of the fungal strains were obtained at 1,296, 1843.2, and 2,304 lg/mL for the A. fumigatus, F. oxysporum, and A. niger, respectively (Table 1, Fig. S2). These organism-specific nano-bio interactions are of immense interest and are yet to be understood (Nel et al. 2009). ESM, in contrast, was found to be ineffective against most of the tested pathogens. However, ESM expressed their bactericidal property against the isolates of S. aureus, S. kloosii and S. maltophilia only at 1 molar concentration. FE-SEM micrographs revealed that the surface of treated fungi (Fig. 5f) was unevenly crumpled and significantly deformed after treatment with b-SNPs. In contrast, the treated bacterial cells were found to shrink heavily at many sites of their body (Fig. 5b). The physiological facts behind these surface deformities and their patterns are yet to be understood. Furthermore, TEM coupled with EDX analysis was used to determine the internalization and distribution of b-SNPs within the microbial bodies. As evident from the TEM micrographs, b-SNPs were found to form precipitates (indicated by arrows), nonspecifically, at many sites of the bacterial cells and fungal hyphae (Fig. 5c, g). Suspected precipitates were analyzed further using EDX spectroscopy. Specific spots were selected and analyzed for their elemental composition (Fig. 5d, h). From EDX spectra of the internalized SNPs, around 29.52 and 18.80 wt% of elemental sulfur was detected. Result of the biocompatibility (Cytotoxicity) assay The WST-1(4-[3-(4-iodophenyl)-2-(4-nitrophenyl)2H-5-tetrazolio]-1,3-benzene disulfonate)-based assay

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revealed no significant cytotoxicity against the HepG2 cells upon treatment with b-SNPs, up to the concentration of 2,304 lg/mL. However, the cell viability gradually decreased from 100.04 to 41.7 % upon serial exposure to b-SNPs, and LD50 was achieved at a concentration of 4,608 lg/mL. The results were plotted onto a fitted curve and expressed as treatment over control values for cell survival after 72 h of incubation (Fig. 6).

Discussion In the present study, b-SNPs were prepared via ‘waterin-oil microemulsion technique’ which is considered as one of the best methods for retaining the size of the nanoparticles in a non-aggregated configuration under stable thermodynamic condition (Ethayaraja et al. 2007). Choice of polysulfide agents, surfactants, cosurfactants, and oil phases are some of the important factors for tuning the size of b-SNPs. Encapsulation with Span 80–Tween-80 conjugate not only stabilizes the monoclinic form of the ES at room temperature but also increases their biocompatibility against the mammalian cells. Span-80–Tween-80 conjugate also contributes to the prolonged stability of b-SNP-colloid by lowering the chance of allotropic phase transition of sulfur. Noteworthily, Span-80 is a neutral lipid, while ES is lipophilic in nature. The selected surfactant agent, hence, is expected to facilitate the sustained release of b-SNPs in the colloidal media and ensure their prolonged activity as an antimicrobial agent. Sodium polysulfide as the inorganic sulfur reactant has already been reported for its efficacy to generate lower sized SNPs in comparison to the other polysulfides (Guo et al. 2006). Moreover, the choice of cyclohexane as the oil phase instead of theolin, forms b-SNPs substantially different from that of the earlier study (Guo et al. 2006) in terms of their size, shape, and chemical properties. Determination of ES as an active ingredient in the prepared nanocolloids was another crucial step prior to fixing their stock concentration and effective dosage against the microbes. The HPLC method has turned out to be an ideal technique for the analysis of ES in the prepared nanocolloids due to its high sensitivity and selectivity. The results of ADM with ESM and b-SNPs validated that microbial growth inhibition was directly


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Fig. 5 ESEM micrographs for the untreated and b-SNPs treated isolate of E. coli (a, b) and A. niger (e, f). TEM coupled with EDX analysis after the incorporation of b-SNPs inside the body of E. coli (c, d) and A. niger (g, h)

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Fig. 6 The percentage of viability of HepG2 cells, treated with twofold diluted concentrations of b-SNPs (from 144 to 4,608 lg/mL) at 72 h of incubation

proportional to the increase in concentration and inversely proportional to the increase in particle size. Earlier, we have determined that nanoallotropes of ESM have marked lipogenic effect (Roy Choudhury et al. 2012d). With increasing concentration gradient, b-SNPs significantly depleted the lipid content with subsequent down-regulation of the desaturases in fungi. Lipophilicity can thus be considered as one of the reasons for the SNPs-mediated deformities at the fungal surface. It is yet to be confirmed whether the same lipogenic effect is also responsible for the bacterial surface deformities, or some additional factors are involved in it. The cytotoxic profile of b-SNPs can be attributed to the use of Span-80–Tween-80 conjugate as the surface stabilizer. Application of Span-80 and Tween-80 is already popular for their use as the biocompatible surfactants of numerous drugs (Murthy et al. 2003). Both the polymers are also approved for human administration via drugs and foodstuffs. Insignificant cytotoxicity against the HepG2 cell line within their effective antimicrobial dosage also supports the view that b-SNPs are not potentially toxic against the robust mammalian cells in the presence of the chosen surfactants and can be disseminated in nature without any severe health hazards. Orthorhombic allotrope is, undoubtedly, the most favorable form of ES to be used as an antimicrobial agent. The aforementioned allotrope works best up to the temperature of *95 °C, over which it might be converted into the monoclinic allotrope. Hence, the microbicidal treatment against pathogenic

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thermophilic bacteria (e.g., selected sp. of Campylobacter) and fungi (e.g., selected sp. of Humicola) (Rabkin et al. 1985; Bergey 1919; Hughes and Crosier 1973) with a-sulfur would be impossible to be achieved at high range of temperature. Earlier reports are available only on the synthesis and characterization of b-SNPs, but not on their biological applications (Guo et al. 2006). The present study, hence, is novel in itself, which utilizes the intelligent applications of nanotechnology to stabilize b-sulfur in nanosized configuration at room temperature and demonstrate their microbicidal and cytotoxic activities. The antimicrobial efficacy of b-SNPs now demands their microbicidal profiling against the pathogenic thermophiles. Acknowledgments Samrat Roy Choudhury is a recipient of Senior Research Fellowship [09/93 (0,143)/12 EMR-I] from the Council of Scientific and Industrial Research (CSIR), India (2012-2013). Authors would like to thank Mr. Kishore K. Nair (Senior Research Fellow in the division of Agricultural Chemicals at Indian Agricultural Research Institute, New Delhi, India) for his extended technical support. The authors are also thankful to Dr. Sulagna Basu (Scientist C in the Division of Bacteriology at National Institute of Cholera and Enteric Diseases) for contributing the bacterial strains and Mrs. Arpita Roy Choudhury for improving the image qualities and Ms. Moni Baskey and Mrs. Prativa Mazumder for helping in the analysis of FTIR spectra. The research was partially funded by NAIPICAR-World Bank (Comp-4/C3004/2008-09), ICAR-National Fund (NFBSFARA/GB-2019/2011-12), Department of Biotechnology (DBT), Govt. of India (BT/PR8931/NNT/28/ 07/2007) and ISI plan project for 2008-2012.

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Supplementary material

Evaluation of physicochemical properties, and antimicrobial efficacy of monoclinic sulfur-nanocolloid

Samrat Roy Choudhurya*, Amrita Mandalb, Dipankar Chakravortyb, Madhuban Gopalc, Arunava Goswamia a

Biological Sciences Division, Indian Statistical Institute, 203 B. T. Road, Kolkata, West Bengal,

India, bIndian Association for the Cultivation of Science, Kolkata-7000032, West Bengal, India, c

Divisions of Agricultural Chemicals, Indian Agricultural Research Institute, New Delhi-110012, India

*Corresponding author: Samrat Roy Choudhury, Biological Sciences Division, Indian statistical Institute, 203 B. T. Road, Kolkata-700108, India, Phone: +91-9433337755, Fax (office): +91-332577-3049, E-mail ID: [email protected]

Table S1: Highest aggregate stability (in terms of zeta potential) of monoclinic sulfur nanoparticles, as obtained at a specific pH Alkaline polysulfides Sodium polysulfide Sodium polysulfide Sodium polysulfide Sodium polysulfide Ammonium polysulfide Ammonium polysulfide Calcium polysulfide Calcium polysulfide

Surfactant

Co-surfactant

Oil phase

Span 80-Tween 80 (8:1 ratio) Triton X-100

1-butanol

Span 80-Tween 80 (8:1 ratio) Span 80-Tween 80 (8:1 ratio) Span 80-Tween 80 (8:1 ratio) Triton X-100 Span 80-Tween 80 (8:1 ratio) Triton X-100

Cyclohexane

Zeta potential (mV) -5.33

pH 4.19

1-butanol

Cyclohexane

-4.9

2.90

1-butanol

Oleylamine

-3.3

5.00

n-hexanol

Oleylamine

-4.66

3.20

1-butanol

Cyclohexane

-0.62

4.64

1-butanol

Cyclohexane

-2.19

4.17

1-butanol

Cyclohexane

-3.52

5.26

1-butanol

Cyclohexane

-1.37

4.20

Fig.S1. The antibacterial effect of ß selected bacterial isolates (Table 2. in the main document) at different concentration represents untreated bacterial cultures. Bacterial growth was also observed for the concentration of 576 µg/ml (S1b), 1152 µg/ml plates.

Fig. S2. The antifungal effect of ß-SNPs was assayed via agar dilution method against the representative isolates of Aspergillus Fumigatus (s2a, s2b, and s2c) and Fusarium oxysporum (s2d, s2e, and s2f)