- Email: [email protected]
Fabrication of pure and moxifloxacin functionalized silver oxide nanoparticles for photocatalytic and antimicrobial activity
Accepted Manuscript Fabrication of pure and moxifloxacin functionalized silver oxide nanoparticles for photocatalytic and antimicrobial activity
Sirajul Haq, Wajid Rehman, Muhammad Waseem, Vera Meynen, Saif Ullah Awan, Shaukat Saeed, Naseem Iqbal PII: DOI: Reference:
S1011-1344(18)30564-5 doi:10.1016/j.jphotobiol.2018.07.011 JPB 11297
To appear in:
Journal of Photochemistry & Photobiology, B: Biology
Received date: Revised date: Accepted date:
23 May 2018 3 July 2018 11 July 2018
Please cite this article as: Sirajul Haq, Wajid Rehman, Muhammad Waseem, Vera Meynen, Saif Ullah Awan, Shaukat Saeed, Naseem Iqbal , Fabrication of pure and moxifloxacin functionalized silver oxide nanoparticles for photocatalytic and antimicrobial activity. Jpb (2018), doi:10.1016/j.jphotobiol.2018.07.011
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Fabrication of pure and moxifloxacin functionalized silver oxide nanoparticles for Photocatalytic and antimicrobial activity Sirajul Haq1,3, Wajid Rehman1, Muhammad Waseem*2, Vera Meynen3, Saif Ullah Awan4,
Department of Chemistry, Hazara University, Mansehra Pakistan
IP
1
T
Shaukat Saeed5 and Naseem Iqbal6
Department of Chemistry, COMSATS University Islamabad (CUI), Islamabad Pakistan
3
Laboratory of Adsorption and Catalysis, University of Antwerp, Universiteitsplein 1, B-
CR
2
Department of Electrical Engineering, NUST College of Electrical and Mechanical
AN
4
US
2610 Wilrijk, Belgium
Department of Metallurgy and Materials Engineering, Pakistan Institute of Engineering and
PT
US-Pakistan Centre for Advanced Studies in Energy, NUST Islamabad, Pakistan Email: [email protected] (Muhammad Waseem)
CE
6
ED
Applied Sciences, Islamabad, Pakistan
AC
5
M
Engineering, Islamabad, Pakistan
ACCEPTED MANUSCRIPT ABSTRACT This paper reports the synthesis of silver oxide (Ag2O) and moxifloxacin functionalized silver oxide (M-Ag2O) nanoparticles for photocatalytic and antimicrobial activity. The Ag2O nanoparticles were synthesized by using 2 dimethyl amino ethanol as reducing agent. The BET
IP
T
surface area measured from N2 adsorption method was found to be 16.89 m2/g. The mix (cubic and hexagonal) phase of silver oxide (Ag2O) nanoparticles was confirmed by X-rays diffraction
CR
(XRD). The extra diffracted peaks were observed after moxifloxacin fictionalization. The
US
scanning electron micrographs display spherical shaped particles of different sizes. The elemental composition and weight percent of both samples were studied by energy dispersive X-
AN
ray (EDX). The decrease in the weight percent of silver with the subsequent increase in the
M
weight percent of carbon and oxygen revealed the successful loading of moxifloxacin onto Ag2O NPs. The two stages of weight loss due to the removal of physisorbed and chemisorbed water
ED
was examined during thermogravimetric analysis (TGA). The optical band gap derived from the
PT
diffuse reflectance spectrum (DRS) was 1.83 eV, which corresponds to the transmittance edge of 676 nm. The Fourier transform infrared (FTIR) band at 668.56 cm-1 confirms the successful
CE
synthesis of moxifloxacin functionalized silver oxide (Ag2O) nanoparticles. The pure Ag2O
AC
nanoparticles were used for the degradation of rhodamine 6G and 98.56 % dye was degraded in 330 minutes. The bacterial species selected for the present study were Bacillus subtilis, Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Candida albicans and Aspergillus Niger. Both pure and functionalized Ag2O NPs were screened against selected bacterial and fungal species and they showed improved activity with the volume of samples taken in wells. However, the activity of Ag2O NPs against fungi was found less effective than bacteria which may be due to the difference in the composition of the cell wall. Further gram-
ACCEPTED MANUSCRIPT positive bacteria showed more resistance toward both samples as compared to the gram-negative bacteria. It was concluded that Ag2O NPs upon conjugation with moxifloxacin displayed promising antimicrobial activity.
IP
T
Keywords: Antimicrobial; Characterization; Moxifloxacin; Nanoparticles; Photocatalytic; Silver
AC
CE
PT
ED
M
AN
US
CR
Nanoparticles.
ACCEPTED MANUSCRIPT
1.
INTRODUCTION
T
Noble metals have attracted much attention due to their technological and medical
IP
applications (Khodashenas, Bahareh 2005; Kohl et al. 2011). Among these metals, silver is one
CR
of the most famous therapeutic agent, used since last century. It shows promising antimicrobial activity due to high thermal stability and low toxicity to the cell (Poon and Burd 2004; Chen and
US
Schluesener 2008; Rai et al. 2012). Silver Nanoparticles (AgNPs) and other silver containing
AN
compounds are widely used as an antimicrobial agent in cosmetic and hygienic products (Edwards-Jones 2009; Wilkinson et al. 2016) as well as for the elimination of microorganisms
M
(Kim et al. 2007; Hebeish et al. 2014). Silver oxide nanoparticles (Ag2O NPs) are not limited to
ED
bacteria and fungi but also have significant antiviral and larvicidal effects (Zodrow et al. 2009; Salunkhe et al. 2011). The silver oxide nanoparticles are also applicable to drug delivery, nano-
PT
electronics, data storage, catalytic and antimicrobial activities (Soltani et al. 2016).
CE
In literature, AgNPs were assimilated with some organic compounds to develop new
AC
therapeutic agents (Li et al. 2005; Kong and Jang 2008; Wang et al. 2016). Recently, Ag and Ag2O NPs were incorporated with chitosan-based matrix (Alananbeh et al. 2017a), doxycycline (Lalit et al. 2015), chloramphenicol (Haghighi Pak et al. 2016), carboxymethyl chitosan (Pei et al. 2015), curcumin (Ravindra et al. 2012), doxorubicin and alendronate (Benyettou et al. 2015). These conjugates possess broad-spectrum antimicrobial activity and thus offering an opportunity to develop new generation antibiotics (Morones and Elechiguerra 2005; Yoksan and Chirachanchai 2009; Rai et al. 2012; Mohamed and Sabaa 2014; Youssef et al. 2014;
ACCEPTED MANUSCRIPT Alananbeh et al. 2017a). Moxifloxacin is one of the fourth-generation broad-spectrum fluoroquinolone antibiotics, usually used for the treatment of ocular infection (Speciale et al. 2002). The moxifloxacin has been reported to have excellent antimicrobial activity than ciprofloxacin and ofloxacin. It was found more effective against gram-negative bacteria due to
T
better penetration as compared to ciprofloxacin, levofloxacin and ofloxacin (Dajcs et al. 2004;
IP
Yağci et al. 2007). In some earlier studies, moxifloxacin loaded chitosan-dextran and PLG
CR
nanoparticles were developed for antimicrobial activity against Staphylococcus aureus, Pseudomonas aeruginosa and Mycobacterium tuberculosis respectively (Ahmad et al. 2013;
US
Kaskoos 2014). Therefore, moxifloxacin encapsulated nano silver oxide could be a better choice
AN
for a bactericidal activity.
M
The selection of microorganisms in the present study were purely based on their abundance in nature and numerous toxic effects toward human health. The microorganisms
ED
usually contaminate food and water. The Bacillus subtilis is rod shaped Gram-positive bacteria,
PT
usually found in soil, vegetation and contaminate food-stuff (Stein et al. 2005). Staphylococcus aureus is round shaped Gram-positive bacteria found in human body and cause skin infection
CE
including abscesses and food poisoning (Mack et al.; Dajcs et al. 2004). The Escherichia coli and
AC
Pseudomonas aeruginosa are rod shaped Gram-negative bacteria; the former one cause serious food poisoning while the later one associated with serious diseases i.e. pneumonia and various sepsis. The Candida albicans is an opportunistic pathogenic yeast and a cause of infection in human known as candidiasis while Aspergillus niger is fungus and cause black mould that causes mental impairment, breathing problems as well as damage to internal organs (Romani 2000; Dijck 2002).
ACCEPTED MANUSCRIPT The present study is focused on the synthesis of silver oxide nanoparticles (Ag2O NPs) by modified chemical precipitation method. The silver oxide and moxifloxacin were selected on the basis of their excellent antimicrobial efficacy. Individually, both systems were found effective against many bacterial strains. However, based on literature review, to the best of our
T
knowledge, the combined effects of Ag2O and moxifloxacin functionalized Ag2O has never been
IP
reported. The samples were characterized by N2 adsorption method, X-rays diffraction (XRD),
CR
scanning electron microscopy (SEM), energy dispersive X-ray (EDX), thermogravimetric analysis (TGA), diffuse reflectance spectroscopy (DRS) and Fourier transform infrared (FTIR)
US
spectroscopy. The pure Ag2O NPs were used the degradation of rhodamine 6G in the presence of
AN
solar light source. Both pure and moxifloxacin functionalized samples (M-Ag2O) were tested against selected bacteria and fungi species. The selection of microorganisms was because of their
EXPERIMENTAL
2.1.
Materials
PT
2.
ED
M
abundance in nature and numerous toxic effects toward human health.
CE
Silver nitrate (99.99%), ammonium hydroxide (33.33%), 2-dimethyl amino ethanol (99.5%) and rhodamine 6G (95%) were purchased from Merck and used as received. The
AC
Moxifloxacin was obtained from Santa Cruz Biotechnology and tryptic soya agar was obtained from Sigma-Aldrich. All the solutions were prepared in deionized water. 2.2.
Synthesis of silver oxide nanoparticles Stock solution of 3 mM silver nitrate was prepared and 85 mL of which was mixed
slowly with 15 mL of 2-dimethyl amino ethanol. The dropwise mixing of these contents was performed on hot plate at a stirring speed of 200 rpm under constant heating of 55 °C. After 15
ACCEPTED MANUSCRIPT minutes, it was observed that the colorless solution turned completely brown. At this stage, the contents were agitated further for 45 minutes and the heating was switched off. Afterward, the suspension was aged for 10 h and the particles thus obtained were separated from the solvent by centrifuging at 4000 rpm for 20 minutes. The Ag2O NPs were then washed three times each with
polymeric bottles for further investigations. Moxifloxacin loading on silver oxide nanoparticles
CR
2.3.
IP
T
ethanol and water. The final product was dried at 50 °C in an oven for 10 h and then stored in
US
For 25 % drug loading (250 mg per gram), 0.25 g moxifloxacin was first dissolved in ethanol and then transferred into burette. On the other hand, 1g Ag2O NPs were dispersed in 20
AN
mL ethanol which has already been taken in conical flask. The particles were mixed at room
M
temperature with the stirring speed of 200 rpm for 10 minutes. To this, moxifloxacin solution
ED
was added in dropwise manner at 55 °C under stirring speed of 200 rpm for 20 minutes. The reaction mixture was then irradiated ultrasonically (Model W-225) at room temperature (20 °C)
PT
for more 15 minutes and then aged for 1 h. Finally, the filtrate was dried at 50 °C in drying oven
Characterization
AC
2.4.
CE
and stored in polyethylene bottle for further use.
The surface area and pore size of Ag2O NPs were measured by surface area analyzer Micromeritics model Gemini VII 2390i. The X-rays diffraction (XRD) pattern was acquired by X-ray diffraction model Panalytical X-Pert Pro equipped with Cu-K?? radiation (?? = 1.54056 Å) at 40 kV voltage and the current was 30 mA. The field emission scanning electron microscope (SEM) model JED-2300F JEOL was used to study the morphology and composition at 20 kV with an EDX detector. The thermal stability of particles was tracked by TG/DTA analyzer model
ACCEPTED MANUSCRIPT Perkin Elmer model 6300. The sample was heated till 1000 °C with heating rate 10 °C/min. The optical property of Ag2O NPs was studied by diffuse reflectance spectroscopy (DRS) model UVVIS/ NIR spectrometer lambda 950 with integrating sphere of 200-2500 nm. The FTIR spectra were recorded by Nicolet 6700 (USA) in the wavenumber range 4000-500 cm-1.
T
Photocatalytic activity
IP
2.5.
CR
The photocatalytic study of Ag2O NPs was conducted to degrade rhodamine 6G in aqueous solution. The photocatalytic activity was carried out in a Pyrex beaker in the presence of
US
Ag2O NPs and solar light source (US-800 (250 W)). 50 ml rhodamine 6G solution (15 ppm) was transferred into a beaker containing 20 mg of the Ag2O NPs (0.4 g/L), covered with aluminum
AN
foil to avoid the interaction of solar light. The solution mixture was stirred for 30 minutes in dark
M
to established adsorption/desorption equilibrium and was then exposed to simulated solar light
ED
irradiation. After a specific interval, (i.e. 5, 15, 30, 50, 75, 105, 140, 180, 225, 275 and 330 minutes) 3 mL of the sample was centrifuged at 4000 rpm for 4 minutes to remove the catalyst. centrifuged
sample
was
spectrophotometrically
examined
using
double
beam
PT
The
CE
spectrophotometer (Thermo Spectronic UV 500) and the absorbance maxima at 526 nm were noted in function of time. Bioactivity assay
AC
2.6.
The antimicrobial activity of pure and drug loaded Ag2O NPs was evaluated by using agar well diffusion methods (Ahmad et al. 2013). The microorganisms selected for antimicrobial screening were Bacillus subtilis and Staphylococcus aureus as Gram-positive bacteria, Escherichia coli and Pseudomonas aeruginosa as Gram-negative bacteria and Candida albicans and Aspergillus Niger as fungal species. The stock solution of pure and drug loaded Ag2O NPs
ACCEPTED MANUSCRIPT were prepared by dissolving 1mg of each sample in 1mL of Milli Q water. The effect of concentration on the antimicrobial activity, each well was loaded independently with different volume of prepared solutions i.e. 40 µL and 60 µL. The bacterial and fungal inoculum were prepared by culturing onto tryptic soya agar and then incubated at 37 °C for 24 h and at 25 °C for
3.
RESULTS AND DISCUSSION
3.1.
Surface area and pore size distribution
CR
IP
T
4 days respectively. The zone of inhibition was measured and shown in the unit of millimeter.
US
The N2 adsorption study of the silver oxide nanoparticles was carried out to measure the
AN
surface area by applying single and multipoint BET, Langmuir, t-plot external surface area and BJH adsorption cumulative surface area. The BET surface area (SBET) of the Ag2O NPs was
M
found to be 16.89 m2/g. The surface area measured by different methods are summarized in
ED
Table I. Both BET and BJH cumulative methods were used to measure pore volume and pore size which were found in the range 0.000639-0.00879 cm3/g and 12.97-15.19 Å respectively.
PT
The variation occurred in the surface area of Ag2O NPs was due to the difference in geometry
CE
and crystallites size. The results further confirmed that the particles were mesoporous in nature with high surface area. This anomaly in the surface area and porosity are correlated with
AC
different morphological nature of the silver oxide crystals as given by the equation 1(Alananbeh et al. 2017a).
(1)
Where Ksv is the shape factor, Sm is the surface area, p is the density and Dsv is the diameter of the particle. The equation shows that the surface area varies directly with the morphology of the particles, as the difference in geometrical shapes was confirmed by XRD analysis. The average
ACCEPTED MANUSCRIPT particles size was estimated from BET surface area of the particles by utilizing equation 2 (Viswanathan and Raj 2009). (2)
T
Where D is the particle size in nm, SBET is the surface area (m2/g) and p is the density of
IP
particles (silver oxide =7.14 g/ml). The particle size derived from the surface area was 49.76 nm,
CR
which is almost similar with average crystallite size calculated by using Debye-Scherer’s equation (Haq et al. 2018). Therefore, the particle size calculated from the surface area was
X-rays diffraction (XRD) analysis
AN
3.2.
US
found in good agreement with the average crystallite size derived from XRD data.
M
Fig. 1 represents the XRD patterns of Ag2O and (M-Ag2O) NPs. The XRD pattern displays diffraction peaks at 2θ values with corresponding hkl planes 32.84o (111), 38.07o (200),
ED
55.07o (220), 65.59o (311) and 68.82o (222). These planes were matched with reference card 00-
PT
041-1104, corresponding to the cubic geometry. Whereas the diffraction peak at 56.06 o (003) was associated with the hexagonal geometry (Reference card no 00-042-0874 and 00-019-1155).
CE
Debye-Scherer’s equation was used to measure the crystallite size calculated from the full width
AC
at half maxima (FWHM). The crystallite sizes were found to be 51.26 nm for cubic and 49.75 nm for hexagonal phase respectively. The XDR data therefore, confirms the presence of mixed phase in the sample. After drug loading, the new reflection was detected at 2θ values 21.62o, 24.82o and 29.61o which can be associated to the moxifloxacin. Mudgil and Pawar found reflections at 2θ values 8.0o, 8.4o, 10.0o, and 17.3o while studying the diffraction pattern of moxifloxacin (Mudgil and Pawar 2013). In this manuscript, the diffractions were observed at relatively higher intensities which may be due to the presence of Ag2O in the core material. The
ACCEPTED MANUSCRIPT crystallite size after functionalization has been increased to 53.26 nm for cubic and 50.87 nm for hexagonal phase respectively. This increase further supports the surface coverage of Ag2O NPs with moxifloxacin. Scanning electron microscopy (SEM) Energy dispersive X-rays (EDX) and mapping
T
3.3.
IP
The SEM micrograph, EDX and mapping studies are shown in Figs. 2 and 3 respectively.
CR
The micrograph shows that the particles before drug loading (Fig. 2 a) were irregularly distributed with little agglomeration. Most of the particles have distinct boundaries with various
US
morphological shapes and sizes. However, after drug loading, the distinct boundaries were diminished, resulting in the particles covered with moxifloxacin (Fig. 2 b). The EDX analysis
AN
shows that the weight percent of silver and oxygen in Ag2O NPs was 89.87 % and 9.18 %
M
respectively. A little amount of sodium (0.95 %) has also been detected as an impurity (Fig. 2 c
ED
and d). The EDX mapping micrographs for silver, oxygen and sodium were shown in the form of respective green, red and blue color in the Fig. 3 (a-c). In the drug loaded sample, the signals for
PT
the C atoms are prominent in both EDX (Fig 2 d) and mapping micrograph (Fig 3 d). For a drug
CE
loaded sample, the respective percent weight of silver, oxygen, carbon and sodium was found to be 83.11%, 12.55%, 3.11% and 1.22 % respectively. The decrease in the weight percent of silver
AC
and the subsequent increase in the weight percent of carbon and oxygen points towards the successful loading of moxifloxacin onto Ag2O NPs. 3.4.
Thermal analysis and diffuse reflectance (DRS) studies The TGA/DTG curves (Fig. 4 A) of Ag2O NPs shows two stages of weight loss. The first
weight loss was associated with the removal of solvent molecules while the second was linked to the loss of chemisorbed water. The DTG curve has a sharp endothermic peak 70 °C and small
ACCEPTED MANUSCRIPT one at 110 °C which is due to the loss of ethanol traces and moisture absorbed by the sample while a board endothermic peak in the range 380-530 °C corresponds to slow weight loss of chemisorbed water. The heat treatment shows that Ag2O NPs was stable after 530 °C as it displays no significant weight loss after the release of absorbed water.
IP
T
The band gap energy of a semiconductor is the minimum amount of energy to excite an outer most electron from the valance shell to the conduction band. The diffuse reflectance
CR
spectrum is the most important tool to calculate the band gap energy (Eg) of semiconductor
US
(Ullah et al. 2016). The Fig. 4 B represents the reflectance spectrum from which the transmittance edge and band gap energy of Ag2O NPs were calculated. The transmittance edge
AN
was calculated by extrapolating the sharp raising edge of the spectrum with a horizontal portion
M
and was found to be 676 nm. Based on transmittance edge, the calculated band gap was 1.83 eV
3.5.
ED
(Kuzhalosai et al. 2013; Vaida et al. 2016).
Fourier transform infrared (FTIR) spectroscopy
PT
The FTIR spectra of Ag2O NPs are given in Fig. 5. A broad band around 3319-3080 cm-1
CE
and 1588 cm-1 are due to the water molecules (Sripriya et al. 2013). The band at 1038 cm-1 is attributed to O-Ag-O (Haghighi Pak et al. 2016) while a couple of bands at 878 and 708 cm-1 are
AC
due to stretching vibrations of Ag-O-Ag bond (Vanaja et al. 2014), whereas two small bands positioned at 678 and 618 cm-1 are linked to Ag-O (Kumar and Rani 2013; Wankhede et al. 2013). The bands appeared in the spectrum of the drug loaded sample at 3538 and 3468 cm-1 are attributed to starching vibrations of N-H and O-H respectively (Lalit et al. 2015). The bands at 1733 and 1618.89 cm-1 are assigned to vC=O in COOH and C=C vibration respectively. The other bands appeared at 1698.38, 1643.04 and 1516.75 cm-1 are due to N-H and C-N stretching vibration of amine. The band centered at 1456.84 cm-1 is due to the stretching vibration C-O-C
ACCEPTED MANUSCRIPT while the band at 1323.28 cm-1 is assigned to symmetric vibration of -C-O functionality of alcohol, ether or carboxylic acid. The transmittance bands at 1043.15 and 944.89 cm-1 are due to C=C and C-F stretching respectively. The disappearance of O-H band and the appearance of a new band at 668.56 cm-1 is associated with Ag-O bond which confirms the incorporation of
T
silver into moxifloxacin. The FTIR study revealed that Ag2O NPs and drug had formed a
Photocatalytic activity
CR
3.6.
IP
bridging complex at phenolic oxygen of the moxifloxacin.
US
The photocatalytic activity of Ag2O NPs was examined against rhodamine 6G in the presence of the solar light source. A clear decrease in absorbance maxima was observed after
AN
solar light exposure and directly related to time. The % degradation and reaction rate constant
M
were calculated by the equations 3 and 4 respectively. The rate constant calculated is
ED
0.0128/min, proposed that 98.56 % rhodamine 6G was degraded in 330 minutes. The Fig. 6 (b and d) also demonstrated that % degradation increases while C/Co decreases with solar light
)
(3)
(4)
AC
(
CE
PT
exposure time.
The rhodamine 6 g was degraded in the presence of Ag2O NPs and solar light source, as the Ag2O NPs ability to oxidize the organic dye depends on the intensity of light. When light beam strike on the surface of Ag2O NPs crystal, the electron from an inner (4d) shell excited into outer shell (5sp) having high energy. The hole (h+) generated in the inner d shell have ability to capture an electron from the adsorbed dye (rhodamine 6G) and allow the dye to degrade (Chen et al. 2010).
ACCEPTED MANUSCRIPT 3.7.
Antimicrobial activity Antimicrobial activity of pure and drug loaded Ag2O NPs was carried out against
selected gram positive, gram negative bacterial and fungal species, utilizing agar well diffusion method as shown in Fig. 7. The standard drugs used were Levofloxacin for bacteria and
IP
T
Fluconazole for fungi. The clear inhibition zones were measured as the activity of pure and loaded silver oxide nanoparticles against microorganisms and compiled in Table II. The
CR
experiment shows that the activity of both pure and loaded Ag2O NPs increases with increasing
US
volume of prepared solutions (from 40µL to 60µL). Both samples have effectively inhibited the growth of microorganisms and the results are in agreement with those reported earlier
AN
(González-Sánchez et al. 2015; Haq et al. 2018). Similarly silver and silver oxide nanoparticles
M
were utilized for antibacterial and antifungal activities (Alananbeh et al. 2016, 2017b). The silver nanoparticles dispersed in chitosan solution showed larger inhibition zone against Gram-negative
ED
bacteria as compared to Gram-positive bacteria (Yoksan and Chirachanchai 2009). Silver
PT
nanoparticles and its composite with chitosan were also screened for antifungal activity, which suggest that the incorporation of silver into chitosan matrix enhances its antifungal activity
CE
(Alananbeh et al. 2017a).
AC
The surface of Ag2O NPs can release silver cation (Ag+), so even simple colloidal solution contains three forms of silver, i.e. solid silver, free silver ion (Ag+) or silver complexes and surface- adsorbed silver cation (Ag+) (González-Sánchez et al. 2015). The activity was high against gram-negative bacteria as compared to gram-positive bacteria. This is due to the difference in the composition of the cell wall of gram-positive and gram-negative bacteria. The cell wall of gram-positive bacteria is composed of teichoic acid which is negatively charged due to phosphate in their structure while outer membrane of gram-negative bacteria has
ACCEPTED MANUSCRIPT phospholipids and lipopolysaccharide which impart a strong negative charge to the bacterial surface. The less resistivity of gram-negative bacteria toward Ag2O NPs are due to the interaction of its strong surface negative charge with Ag+ ions. The strong growth inhibiting effects of both samples toward gram-negative bacteria than gram-positive bacteria are possibly
T
due to the difference in peptidoglycan layer of the cell wall and presence of porine (Prameela
IP
Devi et al. 2013). Gram-positive bacteria contain a thick and rigid peptidoglycan layer than a
CR
gram-negative bacteria, which provide extra strength to the cell wall and hence shows more resistance. However, both pure and Moxifloxacin loaded Ag2O NPs show less activity against
US
fungi as compared to the bacteria. This is due to the difference in electrostatic charges and
AN
cellular structure of bacteria and fungi. The fungus cell wall is more complex followed by cell membrane which provides more strength and shows more resistance to upcoming penetrating
M
agents (therapeutic agents). The Ag2O NPs have the ability to generate free radical which
ED
interact with negatively charges outer membrane and disturbed its functions. Bacterial cell wall possesses strong negative charge on their surface which can easily interact with Ag+ ions. The
PT
Ag+ have not only the ability to attach with the outer surface but also can penetrate inside the
CE
cell, interacting the phosphorous containing compounds like DNA, and hence can interrupt the replication process (Prabhu and Poulose 2012). On the other hand, the fungus cell wall due to the
AC
weak electrostatic force of attraction and provides a weak binding site for Ag+ ions. In the present study, the antimicrobial activity of M-Ag2O NPs has been found more active than the pure Ag2O NPs. This is due to the coordination of Ag2O NPs with moxifloxacin, which enhances the lipophilic character of the bridging complex. The enhanced activity of functionalized samples was attributed to the additive effect of both Ag+ and moxifloxacin. The
ACCEPTED MANUSCRIPT possible chemical reaction for the functionalization of Ag2O NPs with moxifloxacin is given in
US
CR
IP
T
the form of the following reaction.
The Ag2O NPs after hydration result in the formation of AgOH, which in turn, will react with
AN
moxifloxacin to form a bridging complex at phenolic oxygen along with the removal of H2O. This has also been confirmed from the FTIR analysis, where the absence of a band at 3468 cm-1
M
and the appearance of 668.56 cm-1 were associated with O-H and O-Ag moieties respectively.
ED
After the incorporation of Ag2O into moxifloxacin structure, it is expected to be present in the
PT
form of silver ion in the moxifloxacin structure. Therefore, the enhanced activity was associated with the additive effect of both silver ion and moxifloxacin.
CE
The increase in the activity M-Ag2O NPs may be explained by overtone’s concept and
AC
Tweede’s chelation theory (Johnson and Sivasankaran 2013; Emam et al. 2017). According to overtone’s concept of cell permeability, the lipid membrane that surrounds the cell favors the passage of only the lipid-soluble materials due to liposolubility which is an important factor to enhance the activity of the sample. According to Tweede’s theory, chelation considerably reduced the polarity of the metal ion because of the partial sharing of its positive charge with the donor groups and the electron delocalization over the whole chelate ring. Such chelation could enhance the lipophilic character of the Ag2O NPs, which subsequently favors its
ACCEPTED MANUSCRIPT permeation through the lipid layer of the cell membrane. The order of antimicrobial activity was noted as gram-negative bacteria > gram-positive bacteria > fungi. 4.
Conclusion
T
Finally, it can be concluded that we have developed mixed phase Ag2O NPs i.e. cubic
IP
and hexagonal phases by using 2-dimethyl amino ethanol as reducing and precipitating agents.
CR
The phase confirmation and crystallite size of Ag2O NPs was confirmed by XRD analysis. The functionalization of Ag2O NPs with moxifloxacin was confirmed by FTIR spectroscopy. The
US
photocatalytic experiment shows that 98.56 % rhodamine 6G was degraded in 330 min with the degradation rate of 1.28 × 102 min. Both pure and loaded samples were found to have significant
AN
activity against examined microorganisms, although they were found less active than standard
M
drugs (Levofloxacin for bacteria and Fluconazole for fungi).
ED
Conflict of interest
AC
CE
PT
The authors declare that they have no conflict of interest.
ACCEPTED MANUSCRIPT REFERENCES Ahmad T, Wani IA, Manzoor N, et al (2013) Biosynthesis, structural characterization and antimicrobial activity of gold and silver nanoparticles. Colloids Surfaces B Biointerfaces
T
107:227–234. doi: 10.1016/j.colsurfb.2013.02.004
IP
Alananbeh KM, Al-Qudah Z, El-Adly A, Al Refaee WJ (2016) Impact of Silver Nanoparticles
CR
on Bacteria Isolated From Raw and Treated Wastewater in Madinah, KSA. Journal, Arab Sci F O R Arab Saudi. doi: 10.1007/s13369-016-2133-3
US
Alananbeh KM, Al-Refaee WJ, Al-Qodah Z (2017a) Antifungal Effect of Silver Nanoparticles
AN
on Selected Fungi Isolated from Raw and Waste Water. Indian J Pharm Sci 79:559–567. doi: 10.4172/pharmaceutical-sciences.1000263
M
Alananbeh KM, Al-Refaee WJ, Al-Qodah Z (2017b) Antifungal Effect of Silver Nanoparticles
ED
on Selected Fungi Isolated from Raw and Waste Water. Indian J Pharm Sci 79:559–567.
PT
doi: 10.4172/pharmaceutical-sciences.1000263 Benyettou F, Rezgui R, Ravaux F, et al (2015) Synthesis of silver nanoparticles for the dual
CE
delivery of doxorubicin and alendronate to cancer cells. J Mater Chem B 3:7237–7245. doi:
AC
10.1039/C5TB00994D
Chen X, Schluesener HJ (2008) Nanosilver: A nanoproduct in medical application. Toxicol Lett 176:1–12. doi: 10.1016/j.toxlet.2007.10.004 Chen X, Zheng Z, Ke X, et al (2010) Supported silver nanoparticles as photocatalysts under ultraviolet and visible light irradiation. Green Chem 12:414–419. doi: 10.1039/b921696k Dajcs JJ, Thibodeaux BA, Marquart ME, et al (2004) Effectiveness of ciprofloxacin,
ACCEPTED MANUSCRIPT levofloxacin, or moxifloxacin for treatment of experimental Staphylococcus aureus keratitis. Antimicrob Agents Chemother 48:1948–1952. doi: 10.1128/AAC.48.6.19481952.2004 Dijck PWM Van (2002) On the safety of Aspergillus niger – a review. 426–435. doi:
IP
T
10.1007/s00253-002-1032-6
CR
Edwards-Jones V (2009) The benefits of silver in hygiene, personal care and healthcare. Lett Appl Microbiol 49:147–152. doi: 10.1111/j.1472-765X.2009.02648.x
US
Emam SM, El-Tabl AS, Ahmed HM, Emad EA (2017) Synthesis, structural characterization,
AN
electrochemical and biological studies on divalent metal chelates of a new ligand derived from pharmaceutical preservative, dehydroacetic acid, with 1,4-diaminobenzene. Arab J
M
Chem 10:S3816–S3825. doi: 10.1016/j.arabjc.2014.05.019
ED
González-Sánchez MI, Perni S, Tommasi G, et al (2015) Silver nanoparticle based antibacterial
PT
methacrylate hydrogels potential for bone graft applications. Mater Sci Eng C 50:332–340. doi: 10.1016/j.msec.2015.02.002
CE
Haghighi Pak Z, Abbaspour H, Karimi N, Fattahi A (2016) Eco-Friendly Synthesis and
AC
Antimicrobial Activity of Silver Nanoparticles Using Dracocephalum moldavica Seed Extract. Appl Sci 6:69. doi: 10.3390/app6030069 Haq S, Rehman W, Waseem M, et al (2018) Effect of heating on the structural and optical properties of TiO2 nanoparticles: antibacterial activity. Appl Nanosci 8:11–18. doi: 10.1007/s13204-018-0647-6 Hebeish A, El-Rafie MH, EL-Sheikh MA, et al (2014) Antimicrobial wound dressing and anti-
ACCEPTED MANUSCRIPT inflammatory efficacy of silver nanoparticles. Int J Biol Macromol 65:509–515. doi: 10.1016/j.ijbiomac.2014.01.071 Johnson A, Sivasankaran N (2013) Synthesis, characterization and biological studies on some metal complexes with Schiff base ligand containing pyrazolone moiety. J Saudi Chem Soc
IP
T
20:1–8. doi: 10.1016/j.jscs.2013.04.007
CR
Kaskoos RA (2014) Investigation of moxifloxacin loaded chitosan-dextran nanoparticles for topical instillation into eye: In-vitro and ex-vivo evaluation. Int J Pharm Investig 4:164–73.
US
doi: 10.4103/2230-973X.143114
AN
Khodashenas, Bahareh G (2005) Synthesis of silver nanoparticles with different shapes. Mater Lett 59:1760–1763. doi: 10.1016/j.matlet.2005.01.061
M
Kim JS, Kuk E, Yu KN, et al (2007) Antimicrobial effects of silver nanoparticles. Nanomedicine
ED
Nanotechnology, Biol Med 3:95–101. doi: 10.1016/j.nano.2006.12.001
PT
Kohl Y, Kaiser C, Bost W, et al (2011) Preparation and biological evaluation of multifunctional PLGA-nanoparticles designed for photoacoustic imaging. Nanomedicine Nanotechnology,
CE
Biol Med 7:228–237. doi: 10.1016/j.nano.2010.07.006
AC
Kong H, Jang J (2008) Antibacterial properties of novel poly(methyl methacrylate) nanofiber containing silver nanoparticles. Langmuir 24:2051–2056. doi: 10.1021/la703085e Kumar H, Rani R (2013) Structural Characterization of Silver Nanoparticles Synthesized by Micro emulsion Route. Int J Eng Innov Technol 3:344–348. Kuzhalosai V, Subash B, Senthilraja A, et al (2013) Synthesis, characterization and photocatalytic properties of SnO 2-ZnO composite under UV-A light. Spectrochim Acta -
ACCEPTED MANUSCRIPT Part A Mol Biomol Spectrosc 115:876–882. doi: 10.1016/j.saa.2013.06.106 Lalit M, Chanan R, Singh N (2015) Drug Loaded Cyanobacterial Nano-formulation : Preparation , Characterization and Bioactivity Evaluation. 5:423–430.
T
Li P, Li J, Wu C, et al (2005) Synergistic antibacterial effects of β-lactam antibiotic combined
IP
with silver nanoparticles. Nanotechnology 16:1912–1917. doi: 10.1088/0957-4484/16/9/082
CR
Mack D, Davies AP, Harris LG, et al Staphylococcus epidermidis in Biomaterial-Associated
US
Infections.
Mohamed RR, Sabaa MW (2014) Synthesis and characterization of antimicrobial crosslinked
M
doi: 10.1016/j.ijbiomac.2014.05.025
AN
carboxymethyl chitosan nanoparticles loaded with silver. Int J Biol Macromol 69:35–99.
Morones JR, Elechiguerra JL (2005) The bactericidal effect of silver nanoparticles.
ED
Nanotechnology 16:2346–53. doi: 10.1088/0957-4484/16/10/059
PT
Mudgil M, Pawar PK (2013) Preparation and in Vitro/Ex Vivo evaluation of moxifloxacin-
CE
loaded PLGA nanosuspensions for ophthalmic application. Sci Pharm 81:591–606. doi: 10.3797/scipharm.1204-16
AC
Pei Z, Sun Q, Sun X, et al (2015) Preparation and characterization of silver nanoparticles on silk fibroin/carboxymethylchitosan composite sponge as anti-bacterial wound dressing. Biomed Mater Eng 26:S111–S118. doi: 10.3233/BME-151296 Poon VKM, Burd A (2004) In vitro cytotoxity of silver: Implication for clinical wound care. Burns 30:140–147. doi: 10.1016/j.burns.2003.09.030 Prabhu S, Poulose EK (2012) Silver nanoparticles: mechanism of antimicrobial action, synthesis,
ACCEPTED MANUSCRIPT medical applications, and toxicity effects. Int Nano Lett 2:32. doi: 10.1186/2228-5326-2-32 Prameela Devi T, Kulanthaivel S, Kamil D, et al (2013) Biosynthesis of silver nanoparticles from Trichoderma species. Indian J Exp Biol 51:543–547. doi: 10.1007/s13205-013-0138-0
T
Rai MK, Deshmukh SD, Ingle AP, Gade AK (2012) Silver nanoparticles: The powerful
IP
nanoweapon against multidrug-resistant bacteria. J Appl Microbiol 112:841–852. doi:
CR
10.1111/j.1365-2672.2012.05253.x
Ravindra S, Mulaba-Bafubiandi AF, Rajinikanth V, et al (2012) Development and
US
Characterization of Curcumin Loaded Silver Nanoparticle Hydrogels for Antibacterial and
AN
Drug Delivery Applications. J Inorg Organomet Polym Mater 22:1254–1262. doi: 10.1007/s10904-012-9734-4
M
Romani L (2000) Innate and adaptive immunity in Candida albicans infections and
ED
saprophytism. 68:175–179.
PT
Salunkhe RB, Patil S V., Patil CD, Salunke BK (2011) Larvicidal potential of silver nanoparticles synthesized using fungus Cochliobolus lunatus against Aedes aegypti
CE
(Linnaeus, 1762) and Anopheles stephensi Liston (Diptera; Culicidae). Parasitol Res
AC
109:823–831. doi: 10.1007/s00436-011-2328-1 Soltani M, Jamali-sheini F, Yousefi R (2016) Effect of growth condition on structure and optical properties
of
hybrid
Ag-CuO
nanomaterials.
Adv
Powder
Technol.
doi:
10.1016/j.apt.2016.08.004 Speciale A, Musumeci R, Blandino G, et al (2002) Minimal inhibitory concentrations and timekill determination of moxifloxacin against aerobic and anaerobic isolates. Int J Antimicrob
ACCEPTED MANUSCRIPT Agents 19:111–118. doi: 10.1016/S0924-8579(01)00486-1 Sripriya J, Anandhakumar S, Achiraman S, et al (2013) Laser receptive polyelectrolyte thin films doped with biosynthesized silver nanoparticles for antibacterial coatings and drug delivery applications. Int J Pharm 457:206–213. doi: 10.1016/j.ijpharm.2013.09.036
IP
T
Stein T, Mikrobiologie I, Goethe- JW (2005) MicroReview Bacillus subtilis antibiotics :
CR
structures , syntheses and. 56:845–857. doi: 10.1111/j.1365-2958.2005.04587.x Ullah S, Hasanain SK, Aftab M (2016) In fl uence of Li 1 þ co-doping defects on luminescence
US
and bandgap narrowing of ZnO : Co 2 þ nanoparticles due to band tailing effects. J Lumin
AN
172:231–242. doi: 10.1016/j.jlumin.2015.12.022
M
Vaida M, Duteanu N, Grozescu I (2016) The effects of doping on the structural, optical and
ED
electric properties of Zn4Sb3 material（吸收谱，电子带隙）. J Serbian Chem Soc 81:4– 4. doi: 10.2298/JSC150918004V
PT
Vanaja M, Paulkumar K, Baburaja M, et al (2014) Degradation of Methylene Blue Using
CE
Biologically. Bioinorg Chem Appl 2014:1–8. doi: 10.1155/2014/742346
AC
Viswanathan B, Raj KJA (2009) Effect of surface area, pore volume and particle size of P25 titania on the phase transformation of anatase to rutile. Indian J Chem - Sect A Inorganic, Phys Theor Anal Chem 48:1378–1382. Wang Y, Ding X, Chen Y, et al (2016) Antibiotic-loaded, silver core-embedded mesoporous silica nanovehicles as a synergistic antibacterial agent for the treatment of drug-resistant infections. Biomaterials 101:207–216. doi: 10.1016/j.biomaterials.2016.06.004 Wankhede YB, Kondawar SB, Thakare SR, More PS (2013) Synthesis and characterization of
ACCEPTED MANUSCRIPT silver nanoparticles embedded in polyaniline nanocomposite. Adv Mater Lett 4:89–93. doi: 10.5185/amlett.2013.icnano.108 Wilkinson LJ, White RJ, Chipman JK (2016) Silver and nanoparticles of silver in wound dressings: A review of efficacy and safety. J Wound Care 20:543–549. doi:
IP
T
10.12968/jowc.2011.20.11.543
CR
Yağci R, Oflu Y, Dinçel a, et al (2007) Penetration of second-, third-, and fourth-generation topical fluoroquinolone into aqueous and vitreous humour in a rabbit endophthalmitis
US
model. Eye (Lond) 21:990–4. doi: 10.1038/sj.eye.6702414
AN
Yoksan R, Chirachanchai S (2009) Silver nanoparticles dispersing in chitosan solution: Preparation by ??-ray irradiation and their antimicrobial activities. Mater Chem Phys
M
115:296–302. doi: 10.1016/j.matchemphys.2008.12.001
ED
Youssef AM, Abdel-Aziz MS, El-Sayed SM (2014) Chitosan nanocomposite films based on Ag-
PT
NP and Au-NP biosynthesis by Bacillus Subtilis as packaging materials. Int J Biol Macromol 69:185–191. doi: 10.1016/j.ijbiomac.2014.05.047
CE
Zodrow K, Brunet L, Mahendra S, et al (2009) Polysulfone ultrafiltration membranes
AC
impregnated with silver nanoparticles show improved biofouling resistance and virus removal. Water Res 43:715–723. doi: 10.1016/j.watres.2008.11.014
ACCEPTED MANUSCRIPT
Table I. Surface area, pore volume and pore size calculated by different methods
28.69
0.00639 Adsorption avg. pore width (4V/A by BET) 12.97
BJH adsorption cumulative
21.92 24.98 BJH adsorption cumulative 0.00879 BJH cumulative
AN M ED PT CE AC
t-Plot (ext.)
T
12.74 16.89 Single point BET
Langmuir
US
Pore size (Å)
BET
IP
Pore volume (cm3/g)
Single point BET
CR
Surface area (m2/g)
15.19
ACCEPTED MANUSCRIPT Table II. Antibacterial activity (measure in millimeter (mm)) of pure and loaded Ag2O NPs Samples
Gram positive bacteria
Gram negative bacteria
Fungal species
S. Aureus
E. Coli
P. Aeruginosa
C. Albicans
A. Niger
NC-40
0.00
0.00
0.00
0.00
0.00
0.00
S-40
2.5
2.6
2.9
2.6
S-60
5.4
5.6
5.5
5.6
SL-40
5.8
5.3
6.6
7.8
SL-60
8.3
8.5
9.2
PC-40
13.8
13.9
15.1
T
B. Subtilis
1.9
4.9
3.8
5.5
3.8
9.25
7.3
5.8
14.4
13.7
13.3
US
CR
IP
2.2
AC
CE
PT
ED
M
AN
Where, S-40 = pure Ag2O NPs, SL= drug loaded Ag2O NPs, NC = negative control, PC = positive control, 40 and 60 = volume of solution in microliter (µl).
ACCEPTED MANUSCRIPT Highlights Mixed phase Ag2O NPs i.e. cubic and hexagonal were prepared.
98.56 % rhodamine 6G was degrade in 330 min.
Significant activity against microorganisms was examined.
AC
CE
PT
ED
M
AN
US
CR
IP
T
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7
Sirajul Haq, Wajid Rehman, Muhammad Waseem, Vera Meynen, Saif Ullah Awan, Shaukat Saeed, Naseem Iqbal PII: DOI: Reference:
S1011-1344(18)30564-5 doi:10.1016/j.jphotobiol.2018.07.011 JPB 11297
To appear in:
Journal of Photochemistry & Photobiology, B: Biology
Received date: Revised date: Accepted date:
23 May 2018 3 July 2018 11 July 2018
Please cite this article as: Sirajul Haq, Wajid Rehman, Muhammad Waseem, Vera Meynen, Saif Ullah Awan, Shaukat Saeed, Naseem Iqbal , Fabrication of pure and moxifloxacin functionalized silver oxide nanoparticles for photocatalytic and antimicrobial activity. Jpb (2018), doi:10.1016/j.jphotobiol.2018.07.011
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Fabrication of pure and moxifloxacin functionalized silver oxide nanoparticles for Photocatalytic and antimicrobial activity Sirajul Haq1,3, Wajid Rehman1, Muhammad Waseem*2, Vera Meynen3, Saif Ullah Awan4,
Department of Chemistry, Hazara University, Mansehra Pakistan
IP
1
T
Shaukat Saeed5 and Naseem Iqbal6
Department of Chemistry, COMSATS University Islamabad (CUI), Islamabad Pakistan
3
Laboratory of Adsorption and Catalysis, University of Antwerp, Universiteitsplein 1, B-
CR
2
Department of Electrical Engineering, NUST College of Electrical and Mechanical
AN
4
US
2610 Wilrijk, Belgium
Department of Metallurgy and Materials Engineering, Pakistan Institute of Engineering and
PT
US-Pakistan Centre for Advanced Studies in Energy, NUST Islamabad, Pakistan Email: [email protected] (Muhammad Waseem)
CE
6
ED
Applied Sciences, Islamabad, Pakistan
AC
5
M
Engineering, Islamabad, Pakistan
ACCEPTED MANUSCRIPT ABSTRACT This paper reports the synthesis of silver oxide (Ag2O) and moxifloxacin functionalized silver oxide (M-Ag2O) nanoparticles for photocatalytic and antimicrobial activity. The Ag2O nanoparticles were synthesized by using 2 dimethyl amino ethanol as reducing agent. The BET
IP
T
surface area measured from N2 adsorption method was found to be 16.89 m2/g. The mix (cubic and hexagonal) phase of silver oxide (Ag2O) nanoparticles was confirmed by X-rays diffraction
CR
(XRD). The extra diffracted peaks were observed after moxifloxacin fictionalization. The
US
scanning electron micrographs display spherical shaped particles of different sizes. The elemental composition and weight percent of both samples were studied by energy dispersive X-
AN
ray (EDX). The decrease in the weight percent of silver with the subsequent increase in the
M
weight percent of carbon and oxygen revealed the successful loading of moxifloxacin onto Ag2O NPs. The two stages of weight loss due to the removal of physisorbed and chemisorbed water
ED
was examined during thermogravimetric analysis (TGA). The optical band gap derived from the
PT
diffuse reflectance spectrum (DRS) was 1.83 eV, which corresponds to the transmittance edge of 676 nm. The Fourier transform infrared (FTIR) band at 668.56 cm-1 confirms the successful
CE
synthesis of moxifloxacin functionalized silver oxide (Ag2O) nanoparticles. The pure Ag2O
AC
nanoparticles were used for the degradation of rhodamine 6G and 98.56 % dye was degraded in 330 minutes. The bacterial species selected for the present study were Bacillus subtilis, Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Candida albicans and Aspergillus Niger. Both pure and functionalized Ag2O NPs were screened against selected bacterial and fungal species and they showed improved activity with the volume of samples taken in wells. However, the activity of Ag2O NPs against fungi was found less effective than bacteria which may be due to the difference in the composition of the cell wall. Further gram-
ACCEPTED MANUSCRIPT positive bacteria showed more resistance toward both samples as compared to the gram-negative bacteria. It was concluded that Ag2O NPs upon conjugation with moxifloxacin displayed promising antimicrobial activity.
IP
T
Keywords: Antimicrobial; Characterization; Moxifloxacin; Nanoparticles; Photocatalytic; Silver
AC
CE
PT
ED
M
AN
US
CR
Nanoparticles.
ACCEPTED MANUSCRIPT
1.
INTRODUCTION
T
Noble metals have attracted much attention due to their technological and medical
IP
applications (Khodashenas, Bahareh 2005; Kohl et al. 2011). Among these metals, silver is one
CR
of the most famous therapeutic agent, used since last century. It shows promising antimicrobial activity due to high thermal stability and low toxicity to the cell (Poon and Burd 2004; Chen and
US
Schluesener 2008; Rai et al. 2012). Silver Nanoparticles (AgNPs) and other silver containing
AN
compounds are widely used as an antimicrobial agent in cosmetic and hygienic products (Edwards-Jones 2009; Wilkinson et al. 2016) as well as for the elimination of microorganisms
M
(Kim et al. 2007; Hebeish et al. 2014). Silver oxide nanoparticles (Ag2O NPs) are not limited to
ED
bacteria and fungi but also have significant antiviral and larvicidal effects (Zodrow et al. 2009; Salunkhe et al. 2011). The silver oxide nanoparticles are also applicable to drug delivery, nano-
PT
electronics, data storage, catalytic and antimicrobial activities (Soltani et al. 2016).
CE
In literature, AgNPs were assimilated with some organic compounds to develop new
AC
therapeutic agents (Li et al. 2005; Kong and Jang 2008; Wang et al. 2016). Recently, Ag and Ag2O NPs were incorporated with chitosan-based matrix (Alananbeh et al. 2017a), doxycycline (Lalit et al. 2015), chloramphenicol (Haghighi Pak et al. 2016), carboxymethyl chitosan (Pei et al. 2015), curcumin (Ravindra et al. 2012), doxorubicin and alendronate (Benyettou et al. 2015). These conjugates possess broad-spectrum antimicrobial activity and thus offering an opportunity to develop new generation antibiotics (Morones and Elechiguerra 2005; Yoksan and Chirachanchai 2009; Rai et al. 2012; Mohamed and Sabaa 2014; Youssef et al. 2014;
ACCEPTED MANUSCRIPT Alananbeh et al. 2017a). Moxifloxacin is one of the fourth-generation broad-spectrum fluoroquinolone antibiotics, usually used for the treatment of ocular infection (Speciale et al. 2002). The moxifloxacin has been reported to have excellent antimicrobial activity than ciprofloxacin and ofloxacin. It was found more effective against gram-negative bacteria due to
T
better penetration as compared to ciprofloxacin, levofloxacin and ofloxacin (Dajcs et al. 2004;
IP
Yağci et al. 2007). In some earlier studies, moxifloxacin loaded chitosan-dextran and PLG
CR
nanoparticles were developed for antimicrobial activity against Staphylococcus aureus, Pseudomonas aeruginosa and Mycobacterium tuberculosis respectively (Ahmad et al. 2013;
US
Kaskoos 2014). Therefore, moxifloxacin encapsulated nano silver oxide could be a better choice
AN
for a bactericidal activity.
M
The selection of microorganisms in the present study were purely based on their abundance in nature and numerous toxic effects toward human health. The microorganisms
ED
usually contaminate food and water. The Bacillus subtilis is rod shaped Gram-positive bacteria,
PT
usually found in soil, vegetation and contaminate food-stuff (Stein et al. 2005). Staphylococcus aureus is round shaped Gram-positive bacteria found in human body and cause skin infection
CE
including abscesses and food poisoning (Mack et al.; Dajcs et al. 2004). The Escherichia coli and
AC
Pseudomonas aeruginosa are rod shaped Gram-negative bacteria; the former one cause serious food poisoning while the later one associated with serious diseases i.e. pneumonia and various sepsis. The Candida albicans is an opportunistic pathogenic yeast and a cause of infection in human known as candidiasis while Aspergillus niger is fungus and cause black mould that causes mental impairment, breathing problems as well as damage to internal organs (Romani 2000; Dijck 2002).
ACCEPTED MANUSCRIPT The present study is focused on the synthesis of silver oxide nanoparticles (Ag2O NPs) by modified chemical precipitation method. The silver oxide and moxifloxacin were selected on the basis of their excellent antimicrobial efficacy. Individually, both systems were found effective against many bacterial strains. However, based on literature review, to the best of our
T
knowledge, the combined effects of Ag2O and moxifloxacin functionalized Ag2O has never been
IP
reported. The samples were characterized by N2 adsorption method, X-rays diffraction (XRD),
CR
scanning electron microscopy (SEM), energy dispersive X-ray (EDX), thermogravimetric analysis (TGA), diffuse reflectance spectroscopy (DRS) and Fourier transform infrared (FTIR)
US
spectroscopy. The pure Ag2O NPs were used the degradation of rhodamine 6G in the presence of
AN
solar light source. Both pure and moxifloxacin functionalized samples (M-Ag2O) were tested against selected bacteria and fungi species. The selection of microorganisms was because of their
EXPERIMENTAL
2.1.
Materials
PT
2.
ED
M
abundance in nature and numerous toxic effects toward human health.
CE
Silver nitrate (99.99%), ammonium hydroxide (33.33%), 2-dimethyl amino ethanol (99.5%) and rhodamine 6G (95%) were purchased from Merck and used as received. The
AC
Moxifloxacin was obtained from Santa Cruz Biotechnology and tryptic soya agar was obtained from Sigma-Aldrich. All the solutions were prepared in deionized water. 2.2.
Synthesis of silver oxide nanoparticles Stock solution of 3 mM silver nitrate was prepared and 85 mL of which was mixed
slowly with 15 mL of 2-dimethyl amino ethanol. The dropwise mixing of these contents was performed on hot plate at a stirring speed of 200 rpm under constant heating of 55 °C. After 15
ACCEPTED MANUSCRIPT minutes, it was observed that the colorless solution turned completely brown. At this stage, the contents were agitated further for 45 minutes and the heating was switched off. Afterward, the suspension was aged for 10 h and the particles thus obtained were separated from the solvent by centrifuging at 4000 rpm for 20 minutes. The Ag2O NPs were then washed three times each with
polymeric bottles for further investigations. Moxifloxacin loading on silver oxide nanoparticles
CR
2.3.
IP
T
ethanol and water. The final product was dried at 50 °C in an oven for 10 h and then stored in
US
For 25 % drug loading (250 mg per gram), 0.25 g moxifloxacin was first dissolved in ethanol and then transferred into burette. On the other hand, 1g Ag2O NPs were dispersed in 20
AN
mL ethanol which has already been taken in conical flask. The particles were mixed at room
M
temperature with the stirring speed of 200 rpm for 10 minutes. To this, moxifloxacin solution
ED
was added in dropwise manner at 55 °C under stirring speed of 200 rpm for 20 minutes. The reaction mixture was then irradiated ultrasonically (Model W-225) at room temperature (20 °C)
PT
for more 15 minutes and then aged for 1 h. Finally, the filtrate was dried at 50 °C in drying oven
Characterization
AC
2.4.
CE
and stored in polyethylene bottle for further use.
The surface area and pore size of Ag2O NPs were measured by surface area analyzer Micromeritics model Gemini VII 2390i. The X-rays diffraction (XRD) pattern was acquired by X-ray diffraction model Panalytical X-Pert Pro equipped with Cu-K?? radiation (?? = 1.54056 Å) at 40 kV voltage and the current was 30 mA. The field emission scanning electron microscope (SEM) model JED-2300F JEOL was used to study the morphology and composition at 20 kV with an EDX detector. The thermal stability of particles was tracked by TG/DTA analyzer model
ACCEPTED MANUSCRIPT Perkin Elmer model 6300. The sample was heated till 1000 °C with heating rate 10 °C/min. The optical property of Ag2O NPs was studied by diffuse reflectance spectroscopy (DRS) model UVVIS/ NIR spectrometer lambda 950 with integrating sphere of 200-2500 nm. The FTIR spectra were recorded by Nicolet 6700 (USA) in the wavenumber range 4000-500 cm-1.
T
Photocatalytic activity
IP
2.5.
CR
The photocatalytic study of Ag2O NPs was conducted to degrade rhodamine 6G in aqueous solution. The photocatalytic activity was carried out in a Pyrex beaker in the presence of
US
Ag2O NPs and solar light source (US-800 (250 W)). 50 ml rhodamine 6G solution (15 ppm) was transferred into a beaker containing 20 mg of the Ag2O NPs (0.4 g/L), covered with aluminum
AN
foil to avoid the interaction of solar light. The solution mixture was stirred for 30 minutes in dark
M
to established adsorption/desorption equilibrium and was then exposed to simulated solar light
ED
irradiation. After a specific interval, (i.e. 5, 15, 30, 50, 75, 105, 140, 180, 225, 275 and 330 minutes) 3 mL of the sample was centrifuged at 4000 rpm for 4 minutes to remove the catalyst. centrifuged
sample
was
spectrophotometrically
examined
using
double
beam
PT
The
CE
spectrophotometer (Thermo Spectronic UV 500) and the absorbance maxima at 526 nm were noted in function of time. Bioactivity assay
AC
2.6.
The antimicrobial activity of pure and drug loaded Ag2O NPs was evaluated by using agar well diffusion methods (Ahmad et al. 2013). The microorganisms selected for antimicrobial screening were Bacillus subtilis and Staphylococcus aureus as Gram-positive bacteria, Escherichia coli and Pseudomonas aeruginosa as Gram-negative bacteria and Candida albicans and Aspergillus Niger as fungal species. The stock solution of pure and drug loaded Ag2O NPs
ACCEPTED MANUSCRIPT were prepared by dissolving 1mg of each sample in 1mL of Milli Q water. The effect of concentration on the antimicrobial activity, each well was loaded independently with different volume of prepared solutions i.e. 40 µL and 60 µL. The bacterial and fungal inoculum were prepared by culturing onto tryptic soya agar and then incubated at 37 °C for 24 h and at 25 °C for
3.
RESULTS AND DISCUSSION
3.1.
Surface area and pore size distribution
CR
IP
T
4 days respectively. The zone of inhibition was measured and shown in the unit of millimeter.
US
The N2 adsorption study of the silver oxide nanoparticles was carried out to measure the
AN
surface area by applying single and multipoint BET, Langmuir, t-plot external surface area and BJH adsorption cumulative surface area. The BET surface area (SBET) of the Ag2O NPs was
M
found to be 16.89 m2/g. The surface area measured by different methods are summarized in
ED
Table I. Both BET and BJH cumulative methods were used to measure pore volume and pore size which were found in the range 0.000639-0.00879 cm3/g and 12.97-15.19 Å respectively.
PT
The variation occurred in the surface area of Ag2O NPs was due to the difference in geometry
CE
and crystallites size. The results further confirmed that the particles were mesoporous in nature with high surface area. This anomaly in the surface area and porosity are correlated with
AC
different morphological nature of the silver oxide crystals as given by the equation 1(Alananbeh et al. 2017a).
(1)
Where Ksv is the shape factor, Sm is the surface area, p is the density and Dsv is the diameter of the particle. The equation shows that the surface area varies directly with the morphology of the particles, as the difference in geometrical shapes was confirmed by XRD analysis. The average
ACCEPTED MANUSCRIPT particles size was estimated from BET surface area of the particles by utilizing equation 2 (Viswanathan and Raj 2009). (2)
T
Where D is the particle size in nm, SBET is the surface area (m2/g) and p is the density of
IP
particles (silver oxide =7.14 g/ml). The particle size derived from the surface area was 49.76 nm,
CR
which is almost similar with average crystallite size calculated by using Debye-Scherer’s equation (Haq et al. 2018). Therefore, the particle size calculated from the surface area was
X-rays diffraction (XRD) analysis
AN
3.2.
US
found in good agreement with the average crystallite size derived from XRD data.
M
Fig. 1 represents the XRD patterns of Ag2O and (M-Ag2O) NPs. The XRD pattern displays diffraction peaks at 2θ values with corresponding hkl planes 32.84o (111), 38.07o (200),
ED
55.07o (220), 65.59o (311) and 68.82o (222). These planes were matched with reference card 00-
PT
041-1104, corresponding to the cubic geometry. Whereas the diffraction peak at 56.06 o (003) was associated with the hexagonal geometry (Reference card no 00-042-0874 and 00-019-1155).
CE
Debye-Scherer’s equation was used to measure the crystallite size calculated from the full width
AC
at half maxima (FWHM). The crystallite sizes were found to be 51.26 nm for cubic and 49.75 nm for hexagonal phase respectively. The XDR data therefore, confirms the presence of mixed phase in the sample. After drug loading, the new reflection was detected at 2θ values 21.62o, 24.82o and 29.61o which can be associated to the moxifloxacin. Mudgil and Pawar found reflections at 2θ values 8.0o, 8.4o, 10.0o, and 17.3o while studying the diffraction pattern of moxifloxacin (Mudgil and Pawar 2013). In this manuscript, the diffractions were observed at relatively higher intensities which may be due to the presence of Ag2O in the core material. The
ACCEPTED MANUSCRIPT crystallite size after functionalization has been increased to 53.26 nm for cubic and 50.87 nm for hexagonal phase respectively. This increase further supports the surface coverage of Ag2O NPs with moxifloxacin. Scanning electron microscopy (SEM) Energy dispersive X-rays (EDX) and mapping
T
3.3.
IP
The SEM micrograph, EDX and mapping studies are shown in Figs. 2 and 3 respectively.
CR
The micrograph shows that the particles before drug loading (Fig. 2 a) were irregularly distributed with little agglomeration. Most of the particles have distinct boundaries with various
US
morphological shapes and sizes. However, after drug loading, the distinct boundaries were diminished, resulting in the particles covered with moxifloxacin (Fig. 2 b). The EDX analysis
AN
shows that the weight percent of silver and oxygen in Ag2O NPs was 89.87 % and 9.18 %
M
respectively. A little amount of sodium (0.95 %) has also been detected as an impurity (Fig. 2 c
ED
and d). The EDX mapping micrographs for silver, oxygen and sodium were shown in the form of respective green, red and blue color in the Fig. 3 (a-c). In the drug loaded sample, the signals for
PT
the C atoms are prominent in both EDX (Fig 2 d) and mapping micrograph (Fig 3 d). For a drug
CE
loaded sample, the respective percent weight of silver, oxygen, carbon and sodium was found to be 83.11%, 12.55%, 3.11% and 1.22 % respectively. The decrease in the weight percent of silver
AC
and the subsequent increase in the weight percent of carbon and oxygen points towards the successful loading of moxifloxacin onto Ag2O NPs. 3.4.
Thermal analysis and diffuse reflectance (DRS) studies The TGA/DTG curves (Fig. 4 A) of Ag2O NPs shows two stages of weight loss. The first
weight loss was associated with the removal of solvent molecules while the second was linked to the loss of chemisorbed water. The DTG curve has a sharp endothermic peak 70 °C and small
ACCEPTED MANUSCRIPT one at 110 °C which is due to the loss of ethanol traces and moisture absorbed by the sample while a board endothermic peak in the range 380-530 °C corresponds to slow weight loss of chemisorbed water. The heat treatment shows that Ag2O NPs was stable after 530 °C as it displays no significant weight loss after the release of absorbed water.
IP
T
The band gap energy of a semiconductor is the minimum amount of energy to excite an outer most electron from the valance shell to the conduction band. The diffuse reflectance
CR
spectrum is the most important tool to calculate the band gap energy (Eg) of semiconductor
US
(Ullah et al. 2016). The Fig. 4 B represents the reflectance spectrum from which the transmittance edge and band gap energy of Ag2O NPs were calculated. The transmittance edge
AN
was calculated by extrapolating the sharp raising edge of the spectrum with a horizontal portion
M
and was found to be 676 nm. Based on transmittance edge, the calculated band gap was 1.83 eV
3.5.
ED
(Kuzhalosai et al. 2013; Vaida et al. 2016).
Fourier transform infrared (FTIR) spectroscopy
PT
The FTIR spectra of Ag2O NPs are given in Fig. 5. A broad band around 3319-3080 cm-1
CE
and 1588 cm-1 are due to the water molecules (Sripriya et al. 2013). The band at 1038 cm-1 is attributed to O-Ag-O (Haghighi Pak et al. 2016) while a couple of bands at 878 and 708 cm-1 are
AC
due to stretching vibrations of Ag-O-Ag bond (Vanaja et al. 2014), whereas two small bands positioned at 678 and 618 cm-1 are linked to Ag-O (Kumar and Rani 2013; Wankhede et al. 2013). The bands appeared in the spectrum of the drug loaded sample at 3538 and 3468 cm-1 are attributed to starching vibrations of N-H and O-H respectively (Lalit et al. 2015). The bands at 1733 and 1618.89 cm-1 are assigned to vC=O in COOH and C=C vibration respectively. The other bands appeared at 1698.38, 1643.04 and 1516.75 cm-1 are due to N-H and C-N stretching vibration of amine. The band centered at 1456.84 cm-1 is due to the stretching vibration C-O-C
ACCEPTED MANUSCRIPT while the band at 1323.28 cm-1 is assigned to symmetric vibration of -C-O functionality of alcohol, ether or carboxylic acid. The transmittance bands at 1043.15 and 944.89 cm-1 are due to C=C and C-F stretching respectively. The disappearance of O-H band and the appearance of a new band at 668.56 cm-1 is associated with Ag-O bond which confirms the incorporation of
T
silver into moxifloxacin. The FTIR study revealed that Ag2O NPs and drug had formed a
Photocatalytic activity
CR
3.6.
IP
bridging complex at phenolic oxygen of the moxifloxacin.
US
The photocatalytic activity of Ag2O NPs was examined against rhodamine 6G in the presence of the solar light source. A clear decrease in absorbance maxima was observed after
AN
solar light exposure and directly related to time. The % degradation and reaction rate constant
M
were calculated by the equations 3 and 4 respectively. The rate constant calculated is
ED
0.0128/min, proposed that 98.56 % rhodamine 6G was degraded in 330 minutes. The Fig. 6 (b and d) also demonstrated that % degradation increases while C/Co decreases with solar light
)
(3)
(4)
AC
(
CE
PT
exposure time.
The rhodamine 6 g was degraded in the presence of Ag2O NPs and solar light source, as the Ag2O NPs ability to oxidize the organic dye depends on the intensity of light. When light beam strike on the surface of Ag2O NPs crystal, the electron from an inner (4d) shell excited into outer shell (5sp) having high energy. The hole (h+) generated in the inner d shell have ability to capture an electron from the adsorbed dye (rhodamine 6G) and allow the dye to degrade (Chen et al. 2010).
ACCEPTED MANUSCRIPT 3.7.
Antimicrobial activity Antimicrobial activity of pure and drug loaded Ag2O NPs was carried out against
selected gram positive, gram negative bacterial and fungal species, utilizing agar well diffusion method as shown in Fig. 7. The standard drugs used were Levofloxacin for bacteria and
IP
T
Fluconazole for fungi. The clear inhibition zones were measured as the activity of pure and loaded silver oxide nanoparticles against microorganisms and compiled in Table II. The
CR
experiment shows that the activity of both pure and loaded Ag2O NPs increases with increasing
US
volume of prepared solutions (from 40µL to 60µL). Both samples have effectively inhibited the growth of microorganisms and the results are in agreement with those reported earlier
AN
(González-Sánchez et al. 2015; Haq et al. 2018). Similarly silver and silver oxide nanoparticles
M
were utilized for antibacterial and antifungal activities (Alananbeh et al. 2016, 2017b). The silver nanoparticles dispersed in chitosan solution showed larger inhibition zone against Gram-negative
ED
bacteria as compared to Gram-positive bacteria (Yoksan and Chirachanchai 2009). Silver
PT
nanoparticles and its composite with chitosan were also screened for antifungal activity, which suggest that the incorporation of silver into chitosan matrix enhances its antifungal activity
CE
(Alananbeh et al. 2017a).
AC
The surface of Ag2O NPs can release silver cation (Ag+), so even simple colloidal solution contains three forms of silver, i.e. solid silver, free silver ion (Ag+) or silver complexes and surface- adsorbed silver cation (Ag+) (González-Sánchez et al. 2015). The activity was high against gram-negative bacteria as compared to gram-positive bacteria. This is due to the difference in the composition of the cell wall of gram-positive and gram-negative bacteria. The cell wall of gram-positive bacteria is composed of teichoic acid which is negatively charged due to phosphate in their structure while outer membrane of gram-negative bacteria has
ACCEPTED MANUSCRIPT phospholipids and lipopolysaccharide which impart a strong negative charge to the bacterial surface. The less resistivity of gram-negative bacteria toward Ag2O NPs are due to the interaction of its strong surface negative charge with Ag+ ions. The strong growth inhibiting effects of both samples toward gram-negative bacteria than gram-positive bacteria are possibly
T
due to the difference in peptidoglycan layer of the cell wall and presence of porine (Prameela
IP
Devi et al. 2013). Gram-positive bacteria contain a thick and rigid peptidoglycan layer than a
CR
gram-negative bacteria, which provide extra strength to the cell wall and hence shows more resistance. However, both pure and Moxifloxacin loaded Ag2O NPs show less activity against
US
fungi as compared to the bacteria. This is due to the difference in electrostatic charges and
AN
cellular structure of bacteria and fungi. The fungus cell wall is more complex followed by cell membrane which provides more strength and shows more resistance to upcoming penetrating
M
agents (therapeutic agents). The Ag2O NPs have the ability to generate free radical which
ED
interact with negatively charges outer membrane and disturbed its functions. Bacterial cell wall possesses strong negative charge on their surface which can easily interact with Ag+ ions. The
PT
Ag+ have not only the ability to attach with the outer surface but also can penetrate inside the
CE
cell, interacting the phosphorous containing compounds like DNA, and hence can interrupt the replication process (Prabhu and Poulose 2012). On the other hand, the fungus cell wall due to the
AC
weak electrostatic force of attraction and provides a weak binding site for Ag+ ions. In the present study, the antimicrobial activity of M-Ag2O NPs has been found more active than the pure Ag2O NPs. This is due to the coordination of Ag2O NPs with moxifloxacin, which enhances the lipophilic character of the bridging complex. The enhanced activity of functionalized samples was attributed to the additive effect of both Ag+ and moxifloxacin. The
ACCEPTED MANUSCRIPT possible chemical reaction for the functionalization of Ag2O NPs with moxifloxacin is given in
US
CR
IP
T
the form of the following reaction.
The Ag2O NPs after hydration result in the formation of AgOH, which in turn, will react with
AN
moxifloxacin to form a bridging complex at phenolic oxygen along with the removal of H2O. This has also been confirmed from the FTIR analysis, where the absence of a band at 3468 cm-1
M
and the appearance of 668.56 cm-1 were associated with O-H and O-Ag moieties respectively.
ED
After the incorporation of Ag2O into moxifloxacin structure, it is expected to be present in the
PT
form of silver ion in the moxifloxacin structure. Therefore, the enhanced activity was associated with the additive effect of both silver ion and moxifloxacin.
CE
The increase in the activity M-Ag2O NPs may be explained by overtone’s concept and
AC
Tweede’s chelation theory (Johnson and Sivasankaran 2013; Emam et al. 2017). According to overtone’s concept of cell permeability, the lipid membrane that surrounds the cell favors the passage of only the lipid-soluble materials due to liposolubility which is an important factor to enhance the activity of the sample. According to Tweede’s theory, chelation considerably reduced the polarity of the metal ion because of the partial sharing of its positive charge with the donor groups and the electron delocalization over the whole chelate ring. Such chelation could enhance the lipophilic character of the Ag2O NPs, which subsequently favors its
ACCEPTED MANUSCRIPT permeation through the lipid layer of the cell membrane. The order of antimicrobial activity was noted as gram-negative bacteria > gram-positive bacteria > fungi. 4.
Conclusion
T
Finally, it can be concluded that we have developed mixed phase Ag2O NPs i.e. cubic
IP
and hexagonal phases by using 2-dimethyl amino ethanol as reducing and precipitating agents.
CR
The phase confirmation and crystallite size of Ag2O NPs was confirmed by XRD analysis. The functionalization of Ag2O NPs with moxifloxacin was confirmed by FTIR spectroscopy. The
US
photocatalytic experiment shows that 98.56 % rhodamine 6G was degraded in 330 min with the degradation rate of 1.28 × 102 min. Both pure and loaded samples were found to have significant
AN
activity against examined microorganisms, although they were found less active than standard
M
drugs (Levofloxacin for bacteria and Fluconazole for fungi).
ED
Conflict of interest
AC
CE
PT
The authors declare that they have no conflict of interest.
ACCEPTED MANUSCRIPT REFERENCES Ahmad T, Wani IA, Manzoor N, et al (2013) Biosynthesis, structural characterization and antimicrobial activity of gold and silver nanoparticles. Colloids Surfaces B Biointerfaces
T
107:227–234. doi: 10.1016/j.colsurfb.2013.02.004
IP
Alananbeh KM, Al-Qudah Z, El-Adly A, Al Refaee WJ (2016) Impact of Silver Nanoparticles
CR
on Bacteria Isolated From Raw and Treated Wastewater in Madinah, KSA. Journal, Arab Sci F O R Arab Saudi. doi: 10.1007/s13369-016-2133-3
US
Alananbeh KM, Al-Refaee WJ, Al-Qodah Z (2017a) Antifungal Effect of Silver Nanoparticles
AN
on Selected Fungi Isolated from Raw and Waste Water. Indian J Pharm Sci 79:559–567. doi: 10.4172/pharmaceutical-sciences.1000263
M
Alananbeh KM, Al-Refaee WJ, Al-Qodah Z (2017b) Antifungal Effect of Silver Nanoparticles
ED
on Selected Fungi Isolated from Raw and Waste Water. Indian J Pharm Sci 79:559–567.
PT
doi: 10.4172/pharmaceutical-sciences.1000263 Benyettou F, Rezgui R, Ravaux F, et al (2015) Synthesis of silver nanoparticles for the dual
CE
delivery of doxorubicin and alendronate to cancer cells. J Mater Chem B 3:7237–7245. doi:
AC
10.1039/C5TB00994D
Chen X, Schluesener HJ (2008) Nanosilver: A nanoproduct in medical application. Toxicol Lett 176:1–12. doi: 10.1016/j.toxlet.2007.10.004 Chen X, Zheng Z, Ke X, et al (2010) Supported silver nanoparticles as photocatalysts under ultraviolet and visible light irradiation. Green Chem 12:414–419. doi: 10.1039/b921696k Dajcs JJ, Thibodeaux BA, Marquart ME, et al (2004) Effectiveness of ciprofloxacin,
ACCEPTED MANUSCRIPT levofloxacin, or moxifloxacin for treatment of experimental Staphylococcus aureus keratitis. Antimicrob Agents Chemother 48:1948–1952. doi: 10.1128/AAC.48.6.19481952.2004 Dijck PWM Van (2002) On the safety of Aspergillus niger – a review. 426–435. doi:
IP
T
10.1007/s00253-002-1032-6
CR
Edwards-Jones V (2009) The benefits of silver in hygiene, personal care and healthcare. Lett Appl Microbiol 49:147–152. doi: 10.1111/j.1472-765X.2009.02648.x
US
Emam SM, El-Tabl AS, Ahmed HM, Emad EA (2017) Synthesis, structural characterization,
AN
electrochemical and biological studies on divalent metal chelates of a new ligand derived from pharmaceutical preservative, dehydroacetic acid, with 1,4-diaminobenzene. Arab J
M
Chem 10:S3816–S3825. doi: 10.1016/j.arabjc.2014.05.019
ED
González-Sánchez MI, Perni S, Tommasi G, et al (2015) Silver nanoparticle based antibacterial
PT
methacrylate hydrogels potential for bone graft applications. Mater Sci Eng C 50:332–340. doi: 10.1016/j.msec.2015.02.002
CE
Haghighi Pak Z, Abbaspour H, Karimi N, Fattahi A (2016) Eco-Friendly Synthesis and
AC
Antimicrobial Activity of Silver Nanoparticles Using Dracocephalum moldavica Seed Extract. Appl Sci 6:69. doi: 10.3390/app6030069 Haq S, Rehman W, Waseem M, et al (2018) Effect of heating on the structural and optical properties of TiO2 nanoparticles: antibacterial activity. Appl Nanosci 8:11–18. doi: 10.1007/s13204-018-0647-6 Hebeish A, El-Rafie MH, EL-Sheikh MA, et al (2014) Antimicrobial wound dressing and anti-
ACCEPTED MANUSCRIPT inflammatory efficacy of silver nanoparticles. Int J Biol Macromol 65:509–515. doi: 10.1016/j.ijbiomac.2014.01.071 Johnson A, Sivasankaran N (2013) Synthesis, characterization and biological studies on some metal complexes with Schiff base ligand containing pyrazolone moiety. J Saudi Chem Soc
IP
T
20:1–8. doi: 10.1016/j.jscs.2013.04.007
CR
Kaskoos RA (2014) Investigation of moxifloxacin loaded chitosan-dextran nanoparticles for topical instillation into eye: In-vitro and ex-vivo evaluation. Int J Pharm Investig 4:164–73.
US
doi: 10.4103/2230-973X.143114
AN
Khodashenas, Bahareh G (2005) Synthesis of silver nanoparticles with different shapes. Mater Lett 59:1760–1763. doi: 10.1016/j.matlet.2005.01.061
M
Kim JS, Kuk E, Yu KN, et al (2007) Antimicrobial effects of silver nanoparticles. Nanomedicine
ED
Nanotechnology, Biol Med 3:95–101. doi: 10.1016/j.nano.2006.12.001
PT
Kohl Y, Kaiser C, Bost W, et al (2011) Preparation and biological evaluation of multifunctional PLGA-nanoparticles designed for photoacoustic imaging. Nanomedicine Nanotechnology,
CE
Biol Med 7:228–237. doi: 10.1016/j.nano.2010.07.006
AC
Kong H, Jang J (2008) Antibacterial properties of novel poly(methyl methacrylate) nanofiber containing silver nanoparticles. Langmuir 24:2051–2056. doi: 10.1021/la703085e Kumar H, Rani R (2013) Structural Characterization of Silver Nanoparticles Synthesized by Micro emulsion Route. Int J Eng Innov Technol 3:344–348. Kuzhalosai V, Subash B, Senthilraja A, et al (2013) Synthesis, characterization and photocatalytic properties of SnO 2-ZnO composite under UV-A light. Spectrochim Acta -
ACCEPTED MANUSCRIPT Part A Mol Biomol Spectrosc 115:876–882. doi: 10.1016/j.saa.2013.06.106 Lalit M, Chanan R, Singh N (2015) Drug Loaded Cyanobacterial Nano-formulation : Preparation , Characterization and Bioactivity Evaluation. 5:423–430.
T
Li P, Li J, Wu C, et al (2005) Synergistic antibacterial effects of β-lactam antibiotic combined
IP
with silver nanoparticles. Nanotechnology 16:1912–1917. doi: 10.1088/0957-4484/16/9/082
CR
Mack D, Davies AP, Harris LG, et al Staphylococcus epidermidis in Biomaterial-Associated
US
Infections.
Mohamed RR, Sabaa MW (2014) Synthesis and characterization of antimicrobial crosslinked
M
doi: 10.1016/j.ijbiomac.2014.05.025
AN
carboxymethyl chitosan nanoparticles loaded with silver. Int J Biol Macromol 69:35–99.
Morones JR, Elechiguerra JL (2005) The bactericidal effect of silver nanoparticles.
ED
Nanotechnology 16:2346–53. doi: 10.1088/0957-4484/16/10/059
PT
Mudgil M, Pawar PK (2013) Preparation and in Vitro/Ex Vivo evaluation of moxifloxacin-
CE
loaded PLGA nanosuspensions for ophthalmic application. Sci Pharm 81:591–606. doi: 10.3797/scipharm.1204-16
AC
Pei Z, Sun Q, Sun X, et al (2015) Preparation and characterization of silver nanoparticles on silk fibroin/carboxymethylchitosan composite sponge as anti-bacterial wound dressing. Biomed Mater Eng 26:S111–S118. doi: 10.3233/BME-151296 Poon VKM, Burd A (2004) In vitro cytotoxity of silver: Implication for clinical wound care. Burns 30:140–147. doi: 10.1016/j.burns.2003.09.030 Prabhu S, Poulose EK (2012) Silver nanoparticles: mechanism of antimicrobial action, synthesis,
ACCEPTED MANUSCRIPT medical applications, and toxicity effects. Int Nano Lett 2:32. doi: 10.1186/2228-5326-2-32 Prameela Devi T, Kulanthaivel S, Kamil D, et al (2013) Biosynthesis of silver nanoparticles from Trichoderma species. Indian J Exp Biol 51:543–547. doi: 10.1007/s13205-013-0138-0
T
Rai MK, Deshmukh SD, Ingle AP, Gade AK (2012) Silver nanoparticles: The powerful
IP
nanoweapon against multidrug-resistant bacteria. J Appl Microbiol 112:841–852. doi:
CR
10.1111/j.1365-2672.2012.05253.x
Ravindra S, Mulaba-Bafubiandi AF, Rajinikanth V, et al (2012) Development and
US
Characterization of Curcumin Loaded Silver Nanoparticle Hydrogels for Antibacterial and
AN
Drug Delivery Applications. J Inorg Organomet Polym Mater 22:1254–1262. doi: 10.1007/s10904-012-9734-4
M
Romani L (2000) Innate and adaptive immunity in Candida albicans infections and
ED
saprophytism. 68:175–179.
PT
Salunkhe RB, Patil S V., Patil CD, Salunke BK (2011) Larvicidal potential of silver nanoparticles synthesized using fungus Cochliobolus lunatus against Aedes aegypti
CE
(Linnaeus, 1762) and Anopheles stephensi Liston (Diptera; Culicidae). Parasitol Res
AC
109:823–831. doi: 10.1007/s00436-011-2328-1 Soltani M, Jamali-sheini F, Yousefi R (2016) Effect of growth condition on structure and optical properties
of
hybrid
Ag-CuO
nanomaterials.
Adv
Powder
Technol.
doi:
10.1016/j.apt.2016.08.004 Speciale A, Musumeci R, Blandino G, et al (2002) Minimal inhibitory concentrations and timekill determination of moxifloxacin against aerobic and anaerobic isolates. Int J Antimicrob
ACCEPTED MANUSCRIPT Agents 19:111–118. doi: 10.1016/S0924-8579(01)00486-1 Sripriya J, Anandhakumar S, Achiraman S, et al (2013) Laser receptive polyelectrolyte thin films doped with biosynthesized silver nanoparticles for antibacterial coatings and drug delivery applications. Int J Pharm 457:206–213. doi: 10.1016/j.ijpharm.2013.09.036
IP
T
Stein T, Mikrobiologie I, Goethe- JW (2005) MicroReview Bacillus subtilis antibiotics :
CR
structures , syntheses and. 56:845–857. doi: 10.1111/j.1365-2958.2005.04587.x Ullah S, Hasanain SK, Aftab M (2016) In fl uence of Li 1 þ co-doping defects on luminescence
US
and bandgap narrowing of ZnO : Co 2 þ nanoparticles due to band tailing effects. J Lumin
AN
172:231–242. doi: 10.1016/j.jlumin.2015.12.022
M
Vaida M, Duteanu N, Grozescu I (2016) The effects of doping on the structural, optical and
ED
electric properties of Zn4Sb3 material（吸收谱，电子带隙）. J Serbian Chem Soc 81:4– 4. doi: 10.2298/JSC150918004V
PT
Vanaja M, Paulkumar K, Baburaja M, et al (2014) Degradation of Methylene Blue Using
CE
Biologically. Bioinorg Chem Appl 2014:1–8. doi: 10.1155/2014/742346
AC
Viswanathan B, Raj KJA (2009) Effect of surface area, pore volume and particle size of P25 titania on the phase transformation of anatase to rutile. Indian J Chem - Sect A Inorganic, Phys Theor Anal Chem 48:1378–1382. Wang Y, Ding X, Chen Y, et al (2016) Antibiotic-loaded, silver core-embedded mesoporous silica nanovehicles as a synergistic antibacterial agent for the treatment of drug-resistant infections. Biomaterials 101:207–216. doi: 10.1016/j.biomaterials.2016.06.004 Wankhede YB, Kondawar SB, Thakare SR, More PS (2013) Synthesis and characterization of
ACCEPTED MANUSCRIPT silver nanoparticles embedded in polyaniline nanocomposite. Adv Mater Lett 4:89–93. doi: 10.5185/amlett.2013.icnano.108 Wilkinson LJ, White RJ, Chipman JK (2016) Silver and nanoparticles of silver in wound dressings: A review of efficacy and safety. J Wound Care 20:543–549. doi:
IP
T
10.12968/jowc.2011.20.11.543
CR
Yağci R, Oflu Y, Dinçel a, et al (2007) Penetration of second-, third-, and fourth-generation topical fluoroquinolone into aqueous and vitreous humour in a rabbit endophthalmitis
US
model. Eye (Lond) 21:990–4. doi: 10.1038/sj.eye.6702414
AN
Yoksan R, Chirachanchai S (2009) Silver nanoparticles dispersing in chitosan solution: Preparation by ??-ray irradiation and their antimicrobial activities. Mater Chem Phys
M
115:296–302. doi: 10.1016/j.matchemphys.2008.12.001
ED
Youssef AM, Abdel-Aziz MS, El-Sayed SM (2014) Chitosan nanocomposite films based on Ag-
PT
NP and Au-NP biosynthesis by Bacillus Subtilis as packaging materials. Int J Biol Macromol 69:185–191. doi: 10.1016/j.ijbiomac.2014.05.047
CE
Zodrow K, Brunet L, Mahendra S, et al (2009) Polysulfone ultrafiltration membranes
AC
impregnated with silver nanoparticles show improved biofouling resistance and virus removal. Water Res 43:715–723. doi: 10.1016/j.watres.2008.11.014
ACCEPTED MANUSCRIPT
Table I. Surface area, pore volume and pore size calculated by different methods
28.69
0.00639 Adsorption avg. pore width (4V/A by BET) 12.97
BJH adsorption cumulative
21.92 24.98 BJH adsorption cumulative 0.00879 BJH cumulative
AN M ED PT CE AC
t-Plot (ext.)
T
12.74 16.89 Single point BET
Langmuir
US
Pore size (Å)
BET
IP
Pore volume (cm3/g)
Single point BET
CR
Surface area (m2/g)
15.19
ACCEPTED MANUSCRIPT Table II. Antibacterial activity (measure in millimeter (mm)) of pure and loaded Ag2O NPs Samples
Gram positive bacteria
Gram negative bacteria
Fungal species
S. Aureus
E. Coli
P. Aeruginosa
C. Albicans
A. Niger
NC-40
0.00
0.00
0.00
0.00
0.00
0.00
S-40
2.5
2.6
2.9
2.6
S-60
5.4
5.6
5.5
5.6
SL-40
5.8
5.3
6.6
7.8
SL-60
8.3
8.5
9.2
PC-40
13.8
13.9
15.1
T
B. Subtilis
1.9
4.9
3.8
5.5
3.8
9.25
7.3
5.8
14.4
13.7
13.3
US
CR
IP
2.2
AC
CE
PT
ED
M
AN
Where, S-40 = pure Ag2O NPs, SL= drug loaded Ag2O NPs, NC = negative control, PC = positive control, 40 and 60 = volume of solution in microliter (µl).
ACCEPTED MANUSCRIPT Highlights Mixed phase Ag2O NPs i.e. cubic and hexagonal were prepared.
98.56 % rhodamine 6G was degrade in 330 min.
Significant activity against microorganisms was examined.
AC
CE
PT
ED
M
AN
US
CR
IP
T
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7