One step preparation of a biocompatible, antimicrobial reduced graphene oxide–silver nanohybrid as a topical antimicrobial agent

Abstract

A reduced graphene oxide–silver nanohybrid (Ag–RGO) was prepared by simultaneous reduction of graphene oxide and silver ions, using the aqueous extract of the Colocasia esculenta leaf. The nanohybrid demonstrated better antimicrobial activity than the individual nanomaterials. Excellent cytocompatibility was observed for peripheral blood mononuclear cells (PBMCs) and mammalian red blood cells (RBCs). An acute dermal toxicity study on wistar rats confirmed no induction of direct or indirect toxicity to the host. Thus, this nanohybrid holds potential for applications as a non-toxic topical antimicrobial agent in dressings, bandages, ointments etc.

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Introduction

Graphene has recently been added to the class of nanomaterials. Being a nano-dimensional carbon based material, its compatibility with biological systems may be an interesting and relevant investigation. The outstanding attributes of graphene, including antimicrobial activity, are also present in reduced graphene oxide (RGO).¹ There are several reports on the preparation of RGO by direct reduction of graphene oxide (GO), using different reducing agents like sodium borohydride, hydrazine, hydroquinone etc.^2–4 Retention of trace amounts of reducing agents may be toxic to biological systems. In this context, attempts have been made to use benign materials, such as aqueous leaf extracts from the same laboratory.⁵ Recently, RGO has also been used to sequester some metal nanoparticles to obtain materials with multifaceted applications.⁶

Amongst different metal nanoparticles, silver has been widely used for various materials and biomedical applications.^7,8 The antimicrobial potency of these nanoparticles has attained copious attention from scientists.^7,9 Therefore, it was considered worthwhile to obtain a RGO and silver nanohybrid (Ag–RGO) to study whether there was any synergistic effect with respect to the antimicrobial potency of the material. In this respect, the simultaneous reduction of GO and silver ions was reported in literature.^10,11 Liu et al. recently reported the preparation of metal nanoparticles decorated with graphene by liquid phase exfoliation at 150 °C.⁶ Baby et al. also synthesized a similar material by using a mixture of NaBH₄ and NaOH as the reducing agents.¹⁰ Further, Xu et al. projected an idea for preparing a silver decorated graphene oxide nanocomposite at 60 °C.¹¹ On the contrary, the present method proposes the use of an aqueous plant extract as the reducing agent, which could simultaneously reduce GO and silver ions. Further, this study also puts forward a preparation protocol under ambient conditions.

Graphene-based materials with interesting properties have created new avenues for researchers in the domains of nanomedicine, sensors, catalysis, etc.^12–14 Therefore, the activity of nanomaterials at a bio-nano-interface is an area to be delved into thoroughly. Silver nanoparticles have been used recently in topical antimicrobial bandage applications.¹⁵ Ag–RGO may be a better option in such cases and would exhibit proficient activity in synergy. However, this demands a compatibility assessment of the material with blood cells. Peripheral blood mononuclear cells (PBMCs) are responsible for the contraction and healing of wounds.¹⁶ Therefore, the authors were interested in assessing the compatibility of the nanohybrid with PBMCs as well as with mammalian red blood cells (RBCs). Mere compatibility with blood cells is not a sufficient criterion of a material for application in the domain of biomedicine. A topical antimicrobial agent should also be devoid of toxic effects on the skin in particular and the host body in general. A recent report focused on the skin irritation study of a silver graphene hybrid on rat models.¹¹ However, they did not address the different parameters associated with dermal toxicity. A complete dermal toxicity study (in accordance with the Organization for Economic Cooperation and Development (OECD) guidelines) of Ag–RGO is reported here.

Thus, the overall work reports a facile green method for the preparation of antimicrobial Ag–RGO and its compatibility with mammalian RBCs and PBMCs. In addition, acute dermal toxicity was tested on wistar rat models.

Experimental

Materials

Graphite flakes (60 mesh, 99% purity, Loba Chemie, India) and AgNO₃ (Qualigens®, India) were used as received. 2 g of Colocasia esculenta leaves (Assam, India) were washed thoroughly with distilled water and ground using a domestic blender. Then the leaves were stirred for 20 min in 100 mL of water at 45 °C. The aqueous extract thus obtained was filtered using a muslin cloth.

Preparation of Ag–RGO

Graphene oxide (GO) was prepared by a modified Hummers method from graphite.⁵ 50 mg of GO was well dispersed in 100 mL of distilled water by ultrasonication for 20 min. Ten milliliters of the aqueous extract of the C. esculenta leaves was added into the solution with constant stirring by a magnetic stirrer. After 10 min, 0.01 M AgNO₃ solution was added to the reaction vessel and allowed to stir for another 8 h under the same reaction conditions. After the completion of reduction, the Ag–RGO settled down and it was washed several times with distilled water to remove undesired residual components. For comparison, AgNPs and RGO were separately prepared as per our previously reported procedures, using the same leaf extract as the reducing agent.^5,7 Briefly, AgNO₃ was reduced by the extract at pH7, under ambient conditions and GO was dispersed in water by ultrasonication and reduced by the same extract at room temperature.

Characterization of the nanohybrid

UV-visible spectra of the nanohybrid were taken using a Hitachi (U-2001, Tokyo, Japan) UV spectrophotometer. FTIR spectra of the samples were recorded on an Impact-410 FTIR spectrophotometer (Nicolet, USA) after mixing with a KBr pellet.¹⁷ X-ray diffractograms (Miniflex, Rigaku Corporation, Japan) were taken at room temperature (approx. 25 °C), at 5° min⁻¹, scanning over the range of 2θ = 30–80°. Transmission electron microscopy (TEM) images were taken on a JEOL 2100X electron microscope under an operating voltage of 200 kV. A double monochromator (SPEX 1403) was used to record the Raman spectra. The system was coupled to an air cooled argon ion laser (SPEX 1442) at a wavelength of 488 nm.

Antimicrobial assays

Antimicrobial activity of Ag–RGO was tested against the following microbial strains: Staphylococcus aureus (ATCC 11632), Escherichia coli (ATCC 10536) and Candida albicans (ATCC 10231). For comparative studies, the antimicrobial activity of silver nanoparticles (AgNPs) and RGO was also taken into account.

The minimum inhibitory concentration (MIC) of the nanohybrid against the mentioned strains was determined using a micro-dilution technique.¹⁸ 20 mg mL⁻¹ stock solutions were prepared for the samples. These were then diluted with 1% DMSO to obtain concentrations ranging from 1–40 μg mL⁻¹. Samples (100 μL) were added in the wells in various concentrations. 100 μL of the microbial inocula, corresponding to 10⁷ CFU per mL, was added to each well. DMSO (1%) was taken as the negative control and streptomycin and nystatin were taken as the positive controls. The plate was kept inside an incubator for 16 h at 37 °C. 40 μL of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) solution (0.2 mg mL⁻¹) was added to each well. This was further incubated for 30 min at 37 °C. Formation of a blue color signified bacterial growth, while the growth inhibition was indicated by no change in color.

The zone of inhibition for each sample at MIC was determined by the agar well diffusion assay. The bacteria and C. albicans were cultured on Mueller–Hinton and potato dextrose agar (Himedia, India) respectively.¹⁹ Samples (50 μL) at MIC were added to the wells (8 mm). Nystatin and streptomycin (8 μg mL⁻¹) were used as the positive controls for C. albicans and the bacteria respectively. DMSO (1%) was taken as the blank. The antimicrobial activity was examined after incubating the plates for 16 h at 37 °C by measuring the zone-diameter with a zone scale (Himedia, India). All the tests were carried out in triplicate.

Hemolytic assay

To scrutinize any lysis of the RBC membrane by the samples, a hemolytic assay was performed. Goat blood was received from an abattoir, collected in a heparinized tube (containing 4% sodium citrate) and centrifuged (MPW) for 20 min at 3000 rpm (503g). After centrifugation, the erythrocytes were washed with phosphate buffer saline (PBS). To obtain a 5% haematocrit, the packed erythrocytes were re-suspended in PBS. Sample concentrations of 0.25, 0.5, 0.75, 1.00, and 5.00 mg mL⁻¹ were prepared. In each microfuge tube (Eppendorf), 100 μL of the prepared samples was added along with 1900 μL of the haematocrit and incubated for 30 min at 37 °C. After incubation the cells were kept in an ice bath for 60 seconds, followed by centrifugation at 3000 rpm (503g) for 5 min. Lysis of RBC membranes was examined by determining the hemoglobin concentration with the help of UV absorbance at 540 nm.²⁰

In vitro cytotoxicity assessment with PBMCs

To assess the compatibility of the prepared samples with the mammalian PBMCs, goat blood was collected in a citrated container and diluted to a 1 : 1 ratio with PBS.¹⁹ The suspension was centrifuged at 2028 rpm, 400g for 15 min. Without disturbing the interface, plasma–PBS was carefully removed and diluted to 20 mL in a serum free RPMI-1640 medium. The PBMCs were cultured in RPMI-1640 in a 6 well plate (1 × 105 cells per well). Samples were added to the wells and the plate was incubated in a CO₂ incubator for 24 h at 37 °C. Cells were stained with trypan blue and counted on a hemocytometer. The cells devoid of the treatment of samples were taken as controls.

To evaluate the cell viability in the cultured PBMCs, an MTT assay was done.¹⁹ Twenty microlitres of a sterile MTT solution in PBS was added into each well. Formation of formazan crystals was observed after 4 h of incubation. These crystals were dissolved in DMSO (Merck, India) and absorbance was measured at a wavelength of 570 nm on an ELISA plate reader (Thermo Scientific). Relative cell viability (%) was calculated by the following formula:

Acute dermal toxicity study

Acute dermal toxicity was examined in wistar albino rats according to the OECD Guideline 402.²¹ Two groups of healthy rats were selected, weighing 200–250 g. One group was assigned as the nanohybrid-treated (T) group and the other was the control group (C). Each group contained 6 animals. A small ventral portion of the rats was clipped one day prior to the treatment. Ag–RGO powder was applied evenly to the skin of the rats on the following day. The primary irritation index (PII) was calculated for each animal at 24, 48 and 72 h, according to the Draize method.²² Skin sensitization reactions were observed by visual scoring according to the OECD Guideline 406.²³ Sensitization percentage, grade and classification were recorded. Possible mortality, food consumption and locomotion of the rats were observed regularly. After 15 days, the rats were sacrificed and the major organs (skin, kidney, liver, heart and brain) were carefully extracted and weighed. The toxicity (if any) of the material was examined by histopathological studies of the animals’ tissues.

The rats were subjected to overnight fasting before collecting the blood samples. Blood was extracted from orbital sinus veins by using a 75 mm heparinized capillary (Haematocrit capillary, Himedia, India) and collected in K3 EDTA collection tubes (Peerless Biotech, India). The hematological parameters were evaluated within 60 min of sample collection.

Serum biochemistry

Blood samples were collected as mentioned above in vacuum blood collection tubes (Peerless Biotech Pvt. Ltd, India). The tubes were allowed to stand at room temperature and serum was collected by centrifugation at 3000 rpm (503g) after the blood had clotted. Serum biochemistry was examined using a Coralyzer-100 (Tulip Diagnostics Pvt. Ltd, India) with the help of commercially available biochemical kits.²⁴

Results and discussion

Preparation of the Ag–RGO nanohybrid

In our previous work, we demonstrated the preparation of AgNPs and RGO by utilizing the aqueous extract of the C. esculenta leaf.^5,7 In this work thus, our endeavor was to prepare a Ag–RGO nanohybrid by a one step method using the same extract as the reducing agent. The extract is a natural source of many polyphenolic compounds.²⁵ These compounds have an affinity to form complexes with metal ions.¹⁹ Electrons and protons are released in such processes. The released electrons take part in the reduction process of Ag⁺ to Ag⁰. The mechanism of reduction of graphene oxide is well described in our previous work.⁵ The simultaneous reduction is schematically presented in Fig. 1. The present method depicts a greener and sustainable process, which is also a cost effective one for the preparation of such nanohybrids.

Fig. 1 A probable mechanism for the simultaneous reduction of GO and AgNO₃ by the C. esculenta leaf extract.

Characterization

The UV-visible spectroscopy demonstrated clear evidence of the reduction of AgNO₃ and GO. GO showed peaks at 228 nm and 302 nm, which are due to the π–π* and n–π* transitions of the aromatic carbon bonds and C=O bonds respectively (Fig. 2a). A red shift was observed for the first peak at 265 nm. This confirms the restoration of the electronic conjugation by the formation of RGO with a more graphitic structure. A peak at around 429 nm in the UV-visible spectrum of GO was observed, which indicates the formation of silver nanoparticles (Fig. 2a).

Fig. 2 (a) UV-visible spectra of (i) GO and (ii) Ag–RGO, (b) FTIR spectra of (i) GO and (ii) Ag–RGO, (c) Raman spectra of (i) GO and (ii) Ag–RGO and (d) XRD pattern of the Ag–RGO nanohybrid.

The final content of the Ag phase in the Ag–RGO nanohybrid was determined by an EDX study. It was observed that 15.53 weight% of Ag was loaded on the nanohybrid (ESI, Fig. S1 and Table S1†).

FTIR spectra of GO and Ag–RGO are shown in Fig. 2b. The presence of the bands at 1049 (C–O stretching), 1224 (C–O–C stretching) and 1720 cm⁻¹ (C=O stretching) with the broad band at around 3400 cm⁻¹ (–OH stretching) confirmed the presence of carbonyl, carboxylic, epoxy and hydroxyl moieties in GO.⁵ Removal of these oxygen-containing moieties of GO in Ag–RGO are clearly reflected by the absence of the aforementioned bands. The relative decrease in the intensity of the broad band at 3400 cm⁻¹ for the hydroxyl group is attributed to the successful reduction of GO.

Raman spectroscopy is a widely used tool to characterize disordered and defective structures of carbon-based materials. The Raman spectra of GO and Ag–RGO are shown in the Fig. 2c. In both the cases, two fundamental vibrations were observed at ∼1600 and ∼1350 cm⁻¹ that correspond to the G and D bands respectively.⁵ The G band represents the first-order scattering of the E_2g mode of sp² carbons while the D band represents the disorder-induced mode related to structural imperfections or defects. The degree of disorder in a graphitic structure can easily be measured from the ratio of the intensities of the D and G bands (I_D/I_G). This can be calculated from the relative integrated areas of the D and G Raman bands. In Ag–RGO, I_D/I_G was decreased over GO from 0.95 to 0.8 which suggests the reduction of the oxygenating functional groups on GO and the restoration of the sp² carbon network during the reduction process. However, the small difference in I_D/I_G is due to the fact that AgNPs are impregnated on the RGO sheets, which increases the I_D band.

The XRD patterns of the nanohybrid clearly indicated the presence of silver within it (Fig. 2d). Ag–RGO showed basal reflection peaks at 2θ = 38.4°, 44.2°, 64.4° and 74.3°, which correspond to (111), (200), (220) and (311) Bragg lattices of the face-centered cubic structure of silver (JCPDS 89-3722), respectively. This confirmed that silver nanoparticles with different average crystallite domain sizes were loaded onto the RGO.²⁶

HRTEM images of Ag–RGO are presented in Fig. 3. These indicate that silver nanoparticles with almost spherical morphology were impregnated within the RGO sheets. The histogram in Fig. 3a shows a narrow size distribution of silver nanoparticles; most of the particles ranging from 15–20 nm with an average particle size of 17.2 nm. A 0.270 nm lattice fringe spacing was observed for the particles, which represents the fcc lattice of AgNP (Fig. 3b, inset). It was observed that a nanoparticle was residing between two sheets of RGO with two different lattice fringe spacings of 0.345 and 0.364 nm.

Fig. 3 HRTEM images of Ag–RGO. Inset in (a): a histogram of the size distribution of silver nanoparticles. Inset in (b): a nanoparticle residing between three sheets of RGO.

Antimicrobial efficacy

Antimicrobial activity of the prepared nanohybrid was evaluated by calculating the MIC for each microbe (Table 1). From the table it can be assumed that the nanohybrid system has greater antimicrobial activity than both of the individual nanomaterials. This is reflected by the lower concentration of the nanohybrid which showed an inhibitory effect to microbial growth. This is due to the fact that Ag–RGO may have wrapped around the microbial cells, resulting in the intimate contact of silver with the microbial cell membranes.¹

Table 1 MIC of the nanohybrid against bacteria and fungi

Test organism	RGO (μg mL^−1)	AgNPs (μg mL^−1)	Ag–RGO (μg mL^−1)	Antibiotic (μg mL^−1)
Staphylococcus aureus	16	20	12.5	8
Escherichia coli	21	25	20	10
Candida albicans	19	20	16	6

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Zones of inhibition for each microorganism were determined at the MIC values of the samples (Table 2 and Fig. 4). Ag–RGO showed larger zones of inhibition than any of the individual nanomaterials. Thus the formation of the nanohybrid is advantageous compared to the individual nanomaterials from two perspectives; (1) this prevents the agglomeration of the nanomaterials and (2) this synergistically increases the antimicrobial potency. Further, the cytocompatibility assessment of the nanomaterials and the nanohybrid is an essential prerequisite.

Table 2 Antimicrobial activity (zone of inhibition, mm)

Test organism	RGO	AgNPs	Ag–RGO	Antibiotic
Staphylococcus aureus	17.42 ± 0.18	12.79 ± 0.3	23.82 ± 0.18	32.1 ± 0.2
Escherichia coli	17.06 ± 0.23	13.88 ± 0.06	18.84 ± 0.06	33.6 ± 0.1
Candida albicans	18.18 ± 0.14	12.48 ± 0.17	21.73 ± 0.13	35.55 ± 0.15

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Fig. 4 Representative antimicrobial activity of the nanomaterials against (a) Staphylococcus aureus, (b) Escherichia coli and (c) Candida albicans.

Hemolysis assay

RBC hemolysis protection assays were performed to ascertain the hemocompatibility of the materials. The materials showed excellent compatibility with the erythrocytes. RGO exhibited better compatibility than the silver nanoparticles (Fig. 5). Again, the nanohybrid had better compatibility than the individual nanomaterials. This suggests that the compatibility of the material was enhanced upon formation of the nanohybrid. On the other hand, Tween 20 demonstrated multifold rupture of the RBC membrane against the haematocrit, the negative control, which was clearly indicated by the values of hemoglobin absorbance. This assay confirmed the in vitro compatibility of the prepared material with mammalian RBCs. However, in vivo evaluation would be required for conclusive evidence before the Ag–RGO could be forwarded for consideration for biomedical applications.

Fig. 5 Anti-hemolytic activity assessment.

Compatibility with PBMCs

A material that is to be used in the biomedical field demands compatibility with biological systems. To get a better comparison with in vivo systems, cytotoxicity was evaluated with mammalian PBMCs. These cells are responsible for wound contraction and healing. No alteration of the cell morphology was witnessed when treated with the materials (Fig. 6). The MTT assay revealed that the cell survival rate of the treated groups was almost comparable with that of the control (ESI, Fig. S2†). This test provides evidence of the potential of the material for utilization in biomedical applications such as surgical dressings, antimicrobial ointments etc.

Fig. 6 Microscopic images of trypan blue stained PBMCs (control and treated).

Acute dermal toxicity

Clinical observations showed no mortality of the animals during the experiments. No abnormality was observed in the food consumption and locomotion of the treated rats. Terminal body weights were taken for both groups and no significant differences were observed from the weights on the first day. These parameters indicated that the nanohybrid had no visible toxicity in the rats. Dermal toxicity was tested by scoring the edema or erythema that occurred after application of the nanohybrid material on the skin of each wistar rat. A recent report indicated that there was no irritation in rats on application of graphene silver.¹¹ However, the dermal toxicity in the rats was not studied exhaustively. In the present study no skin irritation or sensitization was witnessed (Table 3 and ESI, Table S2†) for C and T. This confirmed that the material did not cause any allergic reaction to the host when applied transdermally.

Table 3 Dermal irritation study

Skin reaction	Erythema						Edema
Obs in (h)	24		48		72		24		48		72
Groups	C	T	C	T	C	T	C	T	C	T	C	T
Total score	0	0	0	0	0	0	0	0	0	0	0	0
Mean score	0	0	0	0	0	0	0	0	0	0	0	0
Total of mean score	0		0		0		0		0		0
Primary irritation index	0		0		0		0		0		0
Remark	Non-irritating						Non-irritating

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Hematological parameters for both C and T on the 15^th day are presented in Table 4. These showed no significant differences between the two groups. The parameters evaluated on the 0^th, 7^th and 14^th days ascertained that no immune response was generated after application of the nanohybrid. This suggests the utility of the prepared material as a potential topical antimicrobial agent.

Table 4 Hematological parameters of the control and the treated rats

Parameters	Control	Ag–RGO
Lymphocyte (%)	43.98 ± 0.54	42.23 ± 0.39
Monocyte (%)	9.8 ± 0.61	8.2 ± 0.89
Neutrophil (%)	9.6 ± 0.35	10.7 ± 0.94
Eosinophil (%)	3.5 ± 0.45	3.8 ± 0.53
Basophil (%)	0.3 ± 0.32	0.3 ± 0.78
RBC (m mm^−3)	8.23 ± 0.63	8.95 ± 0.45
Mean corpuscular volume (fl)	51.6 ± 0.22	50.3 ± 0.18
Hematocrit (%)	43.34 ± 0.63	44.4 ± 0.26
Mean corpuscular hemoglobin (pg)	15.1 ± 0.19	16.6 ± 0.16
Mean corpuscular hemoglobin concentration (g dL^−1)	28.2 ± 0.16	29.1 ± 0.56
Hemoglobin (g dL^−1)	10.2 ± 0.51	10.5 ± 0.78
Platelet (%)	0.73 ± 0.18	0.68 ± 0.25

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Biochemical parameters also advocated the non-toxic behavior of the material (Table 5). The table shows that the parameters are quite comparable for both of the groups. LDL, HDL, Chol and TGL levels were similar to that of the control, which signified that the lipid profiles of the treated rats were not perturbed by the application of the nanohybrid material. Similar observations were witnessed for the liver functions of the rats, which showed that the levels of ALT/SGPT and AST/SGOT were maintained. No nephrotoxic potential was observed after applying the materials, which was well evidenced by the Gluc, urea, UA and Cre levels of the treated rats. Toxicity of nanomaterials is becoming a global concern, which restricts their use in actual applications. In the present case it was ascertained that the prepared nanohybrid has not induced any direct or indirect toxicity to the host.

Table 5 Biochemical parameters (liver, kidney and lipid profiles)

Parameters	Control	Ag–RGO
Low density lipoprotein (mg dL^−1)	109.1 ± 0.61	109.5 ± 0.65
High density lipoprotein (mg dL^−1)	43.21 ± 0.35	44.68 ± 0.52
Cholesterol (mg dL^−1)	35.83 ± 2.18	33.01 ± 1.45
Triglycerides (mg dL^−1)	8.5 ± 0.23	7.2 ± 0.35
Glucose (mg dL^−1)	107.3 ± 1.52	110.2 ± 1.21
Urea (mg dL^−1)	67.66 ± 2.11	64.7 ± 1.44
Total protein (g dL^−1)	6.8 ± 0.39	6.9 ± 0.54
Uric acid (mg dL^−1)	1.34 ± 0.07	1.28 ± 0.05
Cholesterol (mg dL^−1)	0.63 ± 0.05	0.42 ± 0.22
Alanine aminotransferase (U L^−1)	75 ± 2.0	72.3 ± 1.8
Aspartate aminotransferase (U L^−1)	108.33 ± 1.52	104.1 ± 0.62

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The histopathological studies confirmed that the dermal contact of the nanohybrid did not cause any abnormalities to the vital organs of the host, such as the liver, kidneys, skin, brain and heart (Fig. 7). Fine skin sections exhibited well defined cellular organization with different epithelial layers. Similarly normal hepatocytes were visible in the liver sections. As evident from the brain section, no neurotoxicity was induced to the host tissue after treatment with the nanohybrid. Again, histological sections of the kidneys showed that no nephrotoxic effect occurred in the treated animals.

Fig. 7 Histopathological sections of the Ag–RGO treated wistar rats.

The overall study showed that Ag–RGO has immense potential for its utility as a topical antimicrobial agent in bandages, ointments etc.

Conclusion

A one-step green protocol was presented to synthesize a reduced graphene oxide–silver nanohybrid using Colocasia esculenta leaf extract. The characterization tools sufficed for the formation of the nanohybrid. The material possesses good antimicrobial activity. The cytocompatibility profile was found to be excellent for the mammalian PBMCs and RBCs. Applicability of the material was ascertained by an acute dermal toxicity study on wistar rats. The present investigation suggests that the nanohybrid material may be used in various domains of biomedical science especially as a topical antimicrobial agent in bandages, ointments etc. Thus, this calls for detailed in vivo toxicological studies of the material before any final recommendation for consideration for commercial applications can be made.

Acknowledgements

The authors express their gratitude to NRB for financial assistance through the grant no. DNRD/05/4003/NRB/251 dated 29.02.12, SAP (UGC), India through the grant no. F.3-30/2009(SAP-II) and FIST program-2009 (DST), India through the grant no. SR/FST/CSI-203/209/1 dated 06.05.2010. RSIC, NEHU, Shillong is acknowledged for the TEM imaging.

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c3ra46835f

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