Antimicrobial activity of citric acid functionalized iron oxide nanoparticles –Superparamagnetic effect

Journal Pre-proof Antimicrobial activity of citric acid functionalized iron oxide nanoparticles – Superparamagnetic effect Sidra Khan, Zaheer H. Shah, Saira Riaz, Naveed Ahmad, Shumaila Islam, M. Akram Raza, Shahzad Naseem PII:

S0272-8842(20)30110-3

DOI:

https://doi.org/10.1016/j.ceramint.2020.01.109

Reference:

CERI 24041

To appear in:

Ceramics International

Received Date: 23 September 2019 Revised Date:

28 December 2019

Accepted Date: 12 January 2020

Please cite this article as: S. Khan, Z.H. Shah, S. Riaz, N. Ahmad, S. Islam, M.A. Raza, S. Naseem, Antimicrobial activity of citric acid functionalized iron oxide nanoparticles –Superparamagnetic effect, Ceramics International (2020), doi: https://doi.org/10.1016/j.ceramint.2020.01.109. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. 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. © 2020 Published by Elsevier Ltd.

Antimicrobial Activity of Citric Acid Functionalized Iron Oxide Nanoparticles –Superparamagnetic Effect Sidra Khan, Zaheer H Shah1, Saira Riaz1*, Naveed Ahmad2, Shumaila Islam1,3, M Akram Raza1, and Shahzad Naseem1,* 1

Centre of Excellence in Solid State Physics, University of the Punjab, Lahore 54590, Pakistan 2 Department of Physics, University of Education, Lahore, Pakistan 3) Laser Centre, Ibnu-Sina Institute for Scientific and Industrial Research, Universiti Teknologi Malaysia, Skudai, Johor 81310 Malaysia Corresponding author: [email protected] Abstract: Magnetic nanoparticles offer several advantages for various applications including biomedical. However, the surface of nanoparticles must be functionalized. For biocompatibility of magnetic nanoparticles, they must possess superparamagnetic behavior with high saturation magnetization (Ms). For this purpose, we here report an application-oriented sol-gel route for the synthesis of iron oxide nanoparticles i.e. magnetite (Fe3O4). These magnetic nanoparticles are functionalized by different concentrations of citric acid such as 0.1 M, 0.2 M, 0.3 M, 0.4 M and 0.5 M. Typically, citric acid gets adsorbed on the surface of nanoparticles with one or two carboxylate groups leaving at least one carboxylic acid group on the surface making them hydrophilic to prevent agglomeration that allows conjugation with specific drugs. XRD results confirm the magnetite phase of iron oxide nanoparticles (un-functionalized). Magnetite phase and maghemite phase are observed within different concentrations 0.1 M, 0.2 M, 0.4 M and 0.5 M of citric acid functionalized iron oxide nanoparticles. FTIR analysis confirms the attachment/capping of citric acid on nanoparticles’ surface through the band 1710cm-1 corresponding to C=O of COOH group. Moreover, Raman analysis confirms the presence of characteristic band at 670 cm-1 attributed to Fe3O4 (Magnetite). Particle size analyzer indicates the hydrodynamic diameter size of ~ 25 nm after 0.3 M citric acid functionalized nanoparticles and zeta-potential analysis also confirms the functionalization of nanoparticles with citric acid. Citric acid functionalized nanoparticles show superparamagnetic behavior with saturation magnetization of 85emu/g (citric acid concentration 0.3 M). Magnetic properties are also studied at low temperatures (5K to 300K at Max. field 50KOe) to confirm the presence of superparamagnetic behavior with negligible coercivity (Hc) and increased saturation magnetization (100emu/g at 5K). 0.3 M citric acid functionalized nanoparticles (magnetite, superparamagnetic) appeared to be beneficial for antibacterial activity with zone of inhibition of 36 mm. The experimental analyses confirm that 0.3 M citric acid functionalized nanoparticles are promising candidate for biomedical applications.

1

Index Terms— Citric acid, Magnetite nanoparticles, Superparamagnetic behavior, Biomedical applications 1. INTRODUCTION

Recently, superparamagnetic iron oxide nanoparticles have gained global attraction in biomedical applications including magnetic cell labeling, targeted drug delivery, cancer treatment, magnetic hyperthermia and MRI, due to their high saturation magnetization along with superparamagnetic behavior [1-6], enhanced proton relaxation, and biocompatibility [2-10]. Bare superparamagnetic iron oxide nanoparticles suffer particle agglomeration and large particle diameter due to the presence of van der Waals and/or electrostatic forces [5-13]. Therefore, nanoparticles are mostly functionalized with various inorganic and organic materials to prevent agglomeration and oxidation. However, interaction of superparamagnetic iron oxide nanoparticles with large chain organic molecules and/or surfactants leads to weak binding ability, which can be overcome with the help of smaller molecules [10]. Citric acid is well known short-chained molecule, containing three carboxyl functional groups, one or two carboxyl groups absorbed on the nanoparticles surface and at least one remaining free, suitable for facile functionalization; this helps to avoid agglomeration of superparamagnetic iron oxide nanoparticles [1, 14]. Several researchers have reported synthesis and functionalization of iron oxide nanoparticles for biomedical applications by different methods such as Habibi [15] prepared magnetite-carboxymethyl cellulose nanocomposite by a modified co-precipitation method for drug delivery application. Habibi [16] synthesized magnetite–cellulose nano-composite functionalized with amino celluloses. Qua et al. [17] prepared magnetite (Fe3O4)-chitosan nanoparticles by crosslinking method with core-shell structure. Oleic acid modified Fe3O4 nanoparticles were firstly prepared by co-precipitation then chitosan was added to functionalize the surface of Fe3O4 nanoparticles by physical absorption for hyperthermia. Li et al. [1] studied the co-precipitation method based citric acid coated iron oxide nanoparticles at different coating temperatures. Magnetic behavior was observed for 9 nm and 25 nm sized particles. Behdadfar et al. [14] reported the effect of citric acid concentration using hydrothermal method that required long processing hours. Sathish et al. [18] prepared magnetite nanoparticles by polyol method using mercaptopropionic acid (MPA) and ethylene diamine (EDA) as capping agents at calcination temperatures of 200 °C and 300 °C for 2h under vacuum condition. MPA enhanced the crystallinity and saturation magnetization behavior, whereas EDA showed non saturation behavior. Arsalanai et al. [19] used chemical co-precipitation method for the preparation of magnetite nanoparticles coated with natural rubber latex as capping agent and found synthesized nanoparticles to be effective as contrast agent for MRI applications. Spinel structure of 2

magnetite with size in the range of 9-14 nm with paramagnetic behavior was observed. Fatima et al. [20] reported shape-controlled magnetite nanoparticles in the presence of KOH/NH4Ac as capping agents. The solution mixture was maintained in Teflon-lined autoclave cell and maintained at 200 °C for 24 h. The synthesized nanoparticles showed FCC structure of magnetite with particle size in the range of 200 nm-300 nm with superparamagnetic behavior. In this research work, magnetic nanoparticles (MNPs) were synthesized at 80oC using sol-gel process. These nanoparticles were functionalized with different concentrations (0.1M to 0.5M) of citric acid for biomedical applications. The goal of this work was to obtain less aggregated biocompatible magnetic nanoparticles (MNPs) at low temperatures with superparamagnetic behavior. Superparamagnetic behavior was observed for 0.3M citric acid concentration having potential in antimicrobial activity.

2. Experimental Details For synthesis, 2.2g of iron nitrate (as precursor) was dissolved in 10 ml deionized (DI) water at room temperature under continuous stirring for 30 mins. 2 ml of ethylene glycol was added in the reaction mixture, as solvent, and it was heated at 80˚C. During sol synthesis, color of solution changed from reddish orange to black. Sol was further heat treated at 100˚C to obtain powder form of nanoparticles. Detailed synthesis of iron oxide has been reported earlier [21, 22]. The dried nanoparticles were then immersed in different concentrations of citric acid ranging from 0.1M to 0.5M, while ultrasonically agitated at room temperature for 60 mins. After ultrasonication, nanoparticles were magnetically separated and dried at room temperature to obtain citric acid functionalized magnetic nanoparticles [Fig. 1(a, b)]. After ultrasonication process MNPs were washed several times to remove the unbound citric acid between MNPs. Fig. 1(a, b) shows digital photo of nanoparticles before and after placing the magnet, where functionalized magnetic nanoparticles (MNPs) are shown. (a)

(b)

Figure 1 Digital photo of citric acid functionalized MNPs (a) before placing a magnet (b) after placing a magnet

3. Characterizations: Structural characterization and phase identification of un-functionalized and citric acid functionalized MNPs were observed using Bruker D8 Advance X-ray Diffractometer. Shimadzu IR Tracer – 100 Fourier 3

transform infrared spectrophotometer was used to observe the vibrations/stretching of un-functionalized and functionalized nanoparticles within the range of 650 cm-1 – 4000 cm-1. RAMAN spectra were measured using Renishaw RAMAN spectrometer. Magnetic properties were studied using Lake Shore’s 7407 Vibrating Sample Magnetometer (VSM). The hydrodynamic diameter size, polydispersive index (PDI), and zeta potential within different pH 1-12 for un-functionalized and functionalized MNPs were observed by laser analyzer (Zetasizer Nanoseries, Malvern).

3. Results and Discussion

Figure 2(a-f) shows XRD patterns of un-functionalized and 0.1 M -0.5 M citric acid functionalized magnetic nanoparticles (MNPs). Figure 2(a) illustrates the diffraction peaks corresponding to (220), (311), (331), (511), (440) and (622) planes (according to JCPDS card no. 01-089-0950), indicating the formation of pure magnetite phase of iron oxide. (a)

Figure 2 XRD patterns of (a) un-functionalized and at (b) 0.1 M (c) 0.2 M (d) 0.3 M (e) 0.4 M, (f) 0.5 M concentrated citric acid functionalized MNPs The prominent peaks at 18.15°, 30.09°, 35.62°, 43.28°, 53.76°, 57.16°, 62.70° and 75.2° corresponding to magnetite were observed after functionalization [Figure 2(b-f)]. Some low intensity peaks corresponding to maghemite (γ- Fe2O3) at 26.82° and 38.47°, for 0.1 M and 0.2 M citric acid functionalized nanoparticles were also observed [Figure 2(b, c)]. However, these peaks diminished with the increase of citric acid concentration up to 0.3 M. Further increase of citric acid concentration to 0.4 M & 0.5 M resulted in reappearance of maghemite phase along with magnetite due to increase in number of interaction particles beyond certain limit thus giving rise to relatively reduced crystallinity along with smaller crystallite size. 4

By disintegration of precursor, metal cations are produced in the solution with dissolution of intermediate phases. When ions’ concentration in the solution exceeds the super-saturation limit, nucleation process starts and results in elimination of secondary phases. Thus, elimination of secondary phase was observed at citric acid concentrations of 0.3 M. The phase purity at low temperature might be due to increase in intermolecular probability compared to intramolecular reaction in metal-oxygen polymeric networks. At 0.5 M concentration, decrease in crystallinity of MNPs and presence of hump at low diffraction angle is due to low scattering power of citric acid, as documented in the literature [6]. Magnetite belongs to cubic space group Fd3m representing an inverse spinel ferrite. According to the literature [23], as cubic inverse spinel oxides, both phases Fe3O4 and γ -Fe2O3 share the same close-packed face centered cubic (fcc) oxygen sublattice, but in the Fe3O4, Fe2+ cations at octahedral interstitial sites are substituted by vacancies and Fe3+ cations in γ -Fe2O3. The parameters such as structural model, cell parameters, sites, positional coordinates and R-factor of functionalized nanoparticles are summarized in Table I.

Table I Rietveld refined structural parameters of citric acid (CA) functionalized MNPs Structural Model Fe3O4 (0.1M CA functionalized) Cubic (Fd3m)227

Fe3O4 (0.2M CA functionalized)

Fe3O4 (0.3M CA functionalized)

Fe3O4 (0.4M CA functionalized)

Cell Parameters

Sites

Positional Coordinates

R factors

a

8.37 (Å)

Fe1

x 0.37

y 0.37

z 0.37

Rexp

4.64

b c

8.37 (Å) 8.37 (Å)

Fe2 O

0 0.24

0 0.24

0 0.24

Rp Rwp

0.86 1.07

V

586.57 (Å3)

Rb ϰ2

23.64 0.05

a

8.36 (Å)

Fe1

0.37

0.37

0.37

Rexp

4.23

b c V

8.36 (Å) 8.36 (Å) 586.01 (Å3)

Fe2 O

0 0.24

0 0.24

0 0.24

Rp Rwp Rb ϰ2

0.68 0.90 44.27 0.04

a

8.36 (Å)

Fe1

0.37

0.37

0.37

Rexp

4.06

b c V

8.36 (Å) 8.36 (Å) 586.33 (Å3)

Fe2 O

0 0.24

0 0.24

0 0.24

Rp Rwp Rb ϰ2

0.85 1.01 44.56 0.062

a

8.37 (Å)

Fe1

0.37

0.37

0.37

Rexp

3.82

b c V

8.37 (Å) 8.37 (Å) 587.73 (Å3)

Fe2 O

0 0.24

0 0.24

0 0.24

Rp Rwp Rb

1.44 2.29 31.26

5

Fe3O4 (0.5M CA functionalized)

ϰ2

0.36

a

8.38 (Å)

Fe1

0.37

0.37

0.37

Rexp

4.81

b c V

8.38 (Å) 8.38 (Å) 589.40 (Å3)

Fe2 O

0 0.24

0 0.24

0 0.24

Rp Rwp Rb ϰ2

1.28 1.54 23.44 0.10

Crystallite size and dislocation density of unfunctionalized and citric acid functionalized MNPs were calculated using Eq. (1) & (2) [24]. =

( .

)

=

(1) (2)

Here λ is the wavelength of X-ray Cu Kα used, β is FWHM taken in radians, ɵ is the Bragg angle and n is the factor which is equal to 1 when dislocation density is minimum. Variation in crystallite size and dislocation density is shown in Fig. 3. Crystallite size of ~ 25.86 nm is observed for un-functionalized MNPs. Decrease in crystallite size (~21-22 nm) is observed with functionalization of 0.1-0.2M citric acid. Decreased values might have been observed because of the restructuring processes occurring due to the inclusion of citric acid [25]. Such re-structuring process results in the appearance of two competing iron oxide phases, i.e. maghemite and magnetite as was observed in XRD results [Fig. 2 (b-c)]. Increase in crystallite size was observed after functionalization with 0.3 M citric acid concentration due to the appearance of phase pure magnetite [Fig. 2(d)]. However, with further increase of citric acid concentration up to 0.4-0.5 M, crystallite size decreased probably due to increased number of particles beyond certain limit giving rise to mixed iron oxide phases [26]. Mostly, crystallite size strongly depends on strength and stability of phase at particular concentration, as reported previously [21].

6

Figure 3 Variations in crystallite size and dislocation density as a function of citric acid functionalization Figure 4(a-g) shows the FTIR patterns of pure citric acid, un-functionalized and citric acid functionalized MNPs. The FTIR spectrum of pure citric acid [Fig. 4(a)] has dispersive and broadened O-H stretching vibration at 3000 cm-1, and (C=O) stretching vibration of (R-COOH) group at 1739 cm-1. Asymmetrical and symmetrical stretching vibrations of carboxylate group were observed at 1547 and 1386 cm-1, respectively, as reported earlier [27]. A small feature at ~ 663 cm-1 is ascribed to the Fe-O bonding in the trace of un-functionalized MNPs shown in Fig. 4(b). Moreover, in un-functionalized nanoparticles, the sharp bands at ~ 870 cm-1, 1038 cm-1, 1080 cm-1, and 1381 cm-1, are assigned to the symmetric and asymmetric stretching of C–O–C group from ethylene glycol, as reported previously [27], which became less intense after functionalization as shown in Fig. 4(c-g). A feature appearing at 1265 cm-1 is assigned to the OH group of citric acid as reported by Nigam et al. [28]. The band at 1709 cm-1 is assigned to asymmetric stretching of C=O vibration from COOH group of citric acid which is not detected in unfunctionalized nanoparticles. The small band around 1651 cm-1 from un-functionalized MNPs shifts to 1640 cm-1 after functionalization with different concentrations revealing binding of a citric acid species radical to the surface of nanoparticles through chemisorptions of carboxylate (citrate) ions, as documented in the literature [28, 29]. Nigam et al. [28] reported that this band stretching frequency shifting to lower value was probably due to the weakening of C=O bond, because carboxylate species from citric acid form the complexes with Fe atoms on the iron oxide surface interpreting partial single bond character to the C=O bond. Inset shows the schematic representation of carboxylic acid adsorption onto the surface of nanoparticles (Fe3O4). A small feature ~ 2607 cm-1 in functionalized nanoparticles whilst features around 1286 cm-1 and 2946 cm−1 in un-functionalized nanoparticles are attributed to CH2 stretching probably due to the organic species i.e. citric acid [27]. A narrow band ~ 3288 cm-1 in un-functionalized nanoparticles are attributed to the OH groups but after citric acid assistance this band becomes broader specifically at 0.3 M concentration, suggesting that citric acid binds well to the magnetic surface by carboxylate at this concentration and turns the MNPs into hydrophilic nature which is beneficial for biomedical applications.

7

Figure 4 FTIR spectra of (a) pure citric acid (b) un-functionalized and (c) 0.1 M (d) 0.2 M (e) 0.3 M (f) 0.4 M (g) 0.5 M concentrated citric acid functionalized MNPs; Inset shows the schematic representation of carboxylic acid adsorption onto the surface of MNPs Raman spectra of un-functionalized and 0.1M - 0.5 M citric acid functionalized magnetic nanoparticles (MNPs) were measured using laser of wavelength 514 nm and shown in Figure 5(a-f). In un-functionalized and functionalized MNPs, prominent peak at 670 cm−1 is ascribed as A1g mode of Fe3O4 known as characteristic band. Whereas, features at 538 cm− 1 and 298 cm− 1 are attributed to the (T2g) and (Eg) mode of Fe3O4, respectively. The same Raman bands were observed after functionalization with 0.1M0.5M concentration of citric acid. However, at 0.3M-0.4 M concentration, the band at 298 cm− 1 became wider and almost vanished at 0.5M. The clear change can be observed at 670 cm− 1 Raman band width at 0.1M-0.5 M concentration. These variations are probably due to phase transition or formation of pure magnetite phase, as documented in the literature [23], and also discussed above in XRD analysis.

8

Figure 5 RAMAN spectra of (a) un-functionalized and (b) 0.1 M (c) 0.2 M (d) 0.3 M (e) 0.4 M, (f) 0.5 M concentrated citric acid functionalized MNPs Fig. 6(a, b) shows the magnetization curves of un-functionalized and citric acid functionalized sols, respectively. These sols show small hysteresis indicating the presence of uncompensated magnetic moments.

Figure 6 Magnetization curves of (a) un-functionalized (b) citric acid functionalized iron oxide sols Fig. 7(a) shows M-H curves of un-functionalized magnetic nanoparticles (MNPs). These MNPs show superparamagnetic behavior with

high value of

saturation

magnetization (~90.23emu/g).

In

superparamagnetic behavior concept of single domain is very important for understanding its magnetization response [30].

9

Figure 7 M-H curves of (a) un-functionalized and (b) citric acid functionalized MNPs at 0.1M-0.5M concentrations; inset shows clear view of superparamagnetic nature of 0.3M functionalized MNPs

Figure 7(b) exhibited that saturation magnetization of citric acid functionalized nanoparticles is less as compared with un-functionalized MNPs [Figure 7(a)]. Decreased saturation magnetization at 0.1M-0.2M citric acid concentration is attributed to structural phase transition from pure magnetite phase to mixed maghemite-magnetite phases [Figure 2(a, b)], leading to a magnetic transition from superparamagnetic to soft ferromagnetic material. Variation in saturation magnetization and coercivity are shown in Figure 8(a, b). Pure magnetite phase leading to a superparamagnetic behavior, with increased saturation magnetization, is observed at 0.3M citric acid concentration. However, decrease in saturation magnetization with further increase in citric acid concentration to 0.4-0.5M is in agreement with the structural re-arrangement leading to mixed maghemite-magnetite phase [Figure 2(b)]. Such variation in magnetization is due to the presence / absence of Fe3+ and Fe2+ cations; Fe3O4 contains Fe2+ and Fe3+ cations present on octahedral and tetrahedral sites (Table 1). Whereas, maghemite possesses vacancies in cationic sub lattice that results in decreased value of saturation magnetization at 0.1-0.2M citric acid concentration. In addition, at 0.4-0.5M citric acid concentration number of interacting particles increased beyond the certain solubility limit giving rise to deterioration in magnetic properties. Thus, decreased saturation magnetization was observed because of the effective presence of Fe2+ cations, responsible for the appearance of maghemite phase. At 0.3M citric acid concentration elimination of secondary phase, i.e. maghemite, was observed resulting in superparamagnetic behavior with increased saturation magnetization. Jahn Teller effect can be used to explain the increased saturation magnetization value observed at 0.3M citric acid concentration [31]. According to this effect structural distortion may influence the cationic interaction by suppressing Fe2+ spin as compared to the canting of Fe3+ spin in unit cell. Magnetic moment per formula unit (µ f.u.) in Böhr magneton for citric acid functionalized MNPs was calculated using Eq. 3 given below [32]: µ f.u. (µ B) = (Ms x mol. weight / µ B x NA) x10-3

(3)

Where, Ms is measured saturation magnetization in emu/g, mol. W is molecular weight in g, µ B is Böhr magneton = 9.274 × 10-24 J/T, NA is Avogadro constant = 6.022 × 1023 mol-1. In case of magnetite Fe2+ cations are present on octahedral site and Fe3+ cations take position on both 10

octahedral and tetrahedral sites. Based on Hund’s rule, because of antiparallel arrangement of cations on sublattices, the magnetization of magnetite is 4.0µB/f.u. Un-functionalized MNPs exhibit magnetization of 3.82 µB/f.u. [Figure 8]. High value of µB/f.u. indicates formation of magnetite phase. Reduction in µB/f.u. in case of functionalized nanoparticles (MNPs) arises from changes in grain size and functionalization of citric acid (non-magnetic material), as observed by other researchers [33].

(a)

Figure 8 Variation in (a) Saturation magnetization and Bohr magnetron (b) Coercivity of citric acid functionalized nanoparticles Magnetization was also checked in the temperature range 5 to 300K [Figure 9]. It has been noticed that room temperature characterized M-H curves (0.3M citric acid functionalized MNPs) exhibited high saturation magnetization. The room temperature saturation magnetization is ~ 85emu/g which is slightly lower than bulk (~92 emu) reported value [33, 34]. This slight low value is associated with small size of NPs (25nm). A noteworthy increase in magnetization has been observed at 5K. Approximately 100 emu/g saturation magnetization has been observed at 5K and its value decreased with increase in temperature. Increased saturation magnetization at 5K is associated with spin glass transition and involvement of quantum effect at low temperature, as observed by other researchers [35-37]. Moreover, MNPs are quantized by spin wave excitation, therefore effect of discrete level is higher. While at higher temperature continuous excitation spectrum occurs and hence saturation magnetization is considered as in bulk. Coercivity is not noticeable at all temperatures due to absence of long-range magnetic dipole.

11

Figure 9 Low temperature M-H curves of 0.3M citric acid functionalized MNPs.

Figure 10 ZFC-FC analysis of 0.3M citric acid functionalized MNPs at different applied fields To obtain the blocking temperature (TB) of the functionalized MNPs, ZFC (zero field cooled) – FC (field cooled) measurements were performed. ZFC was studied in the temperature range of 4 to 300K. ZFC-FC behavior of MNPs with 0.3M functionalized concentration was observed for various externally applied magnetic fields as shown in Figure 10. Maximum of ZFC shows the blocking temperature (TB) of about 80K at 5Oe [Figure 10]. TB splitting further confirms the small size of MNPs. The transition to super paramagnetic regime (SPM) was maximum at the reduced blocking temperature (TB) as shown in ZFC curves [Figure 10]. Above TB, superposition of the FC with the ZFC curves is observed. Such superposition shows the reversible character of the SPM behavior. Below TB, in the irreversible range, the FC curve separates from the ZFC. Increase in TB value with increased concentration of functionalized material is in agreement with the previously reported experimental and theoretical results [38, 39]. Almost flat curve has been observed in FC which shows the occurrence of canted spins. For bio-medical applications, the functional material adsorption on the surface of MNPs and hydrodynamic diameter size of particles plays a crucial role, therefore, Zeta-potential values for un-functionalized and 12

0.1M-0.5M citric acid functionalized MNPs at different pH (1-11) were calculated. Figure 11(a-f) shows the zeta-potential of MNPs and 0.1 M-0.5 M citric acid functionalized MNPs at different pH values (1-12). Figure 11(b-f) clearly shows that 0.1 M-0.5 M functionalized citric acid particles are adsorbed on the surface of MNPs resulting in the high negative surface charges as compared to un-functionalized MNPs (low negative charge surface), which is in agreement with the literature [24]. Moreover, the high zetapotential negative values of functionalized MNPs confirmed the negatively charged carboxylate groups’ presence on the MNPs surface [as shown in Figure 11(d)-inset]. Furthermore, low electrostatic repulsive force ~ -5 mV at pH 10 for 0.5 M citric acid functionalized MNPs, whereas, high electrostatic repulsive force ~ -17 mV at pH 10 for 0.3 M citric acid functionalized MNPs among the other 0.1 M (~ -14 mV at pH 10), 0.2 M (~ -15 mV at pH 10), and 0.4 M (~ -16 mV at pH 10) functionalized MNPs was observed. High electrostatic repulsive force for 0.3 M citric acid functionalized MNPs suggests their water stability among the negatively charged functionalized MNPs in aqueous suspension. Basically, some carboxylate groups from citric acid adsorbed / coordinate on the surface of MNPs while un-coordinated species prolong into water medium confirming the water stability to MNPs, as documented in the literature [25]. Furthermore, Figure 11 (g) illustrates the average hydrodynamic diameter ~ 62.07 nm of un-functionalized MNPs with 0.8 PDI (polydispersityindex). Whereas, average hydrodynamic diameter ~ 52 nm of 0.1 M, 76 nm of 0.2 M, 87 nm of 0.3 M, 102 nm of 0.4 M, and 66 nm of 0.5 M functionalized citric acid MNPs [Fig.11 (h-l)] was observed with 0.8 PDI.

13

(g)

(h)

(b)

(i)

(c)

(d)

(j)

(e)

(k)

(f)

(l)

14

Figure 11. Zeta-potential at different pH values of (a) MNPs, whereas, citric acid functionalized MNPs correspond to (b) 0.1 M (c) 0.2 M (d) 0.3 M (e) 0.4 M (f) 0.5 M, Inset of Fig.11(d) shows the possible schematic representation of citric acid capping on the MNPs surface. Whilst, (g-l) attributed the hydrodynamic diameter of (a-f), respectively. Antibacterial analysis for citric acid functionalized MNPs was performed against Escherichia coli (E. Coli) gram-negative bacteria and Bacillus Subtilis (Baci) gram positive bacteria. Agar well diffusion method was used to analyze the antibacterial activity using Muller-Hinton agar plates. 20 mL autoclaved Muller-Hinton agar was poured into petri dishes and was allowed to solidify. Streaking of bacteria to be studied was done uniformly onto this agar. Citric acid functionalized MNPs were sonicated in distilled water and were poured into these wells. All these plates were then incubated for 24h at 37 oC. Clear zone surrounding the loaded well after incubation was zone of inhibition mostly expressed in millimeters, measured by a meter ruler. Zone of inhibition provided the antibacterial strength of citric acid functionalized MNPs against E. Coli and Bacillus Subtillis bacterial strains.

Figure 12. Antibacterial activity of (a) un-functionalized MNPs (b) 0.3M citric acid functionalized MNPs against Bacillus bacterial strains

15

TABLE II Antibacterial activity of un-functionalized and citric acid functionalized iron oxide nanoparticles Zone of inhibition (ZOI) (mm) Concent ration (µg/mL)

Un-functionalized Bacillus E.Coli

0.1M Bacillus E.Coli

0.2M Bacillus E.Coli

0.3M Bacillus E.Coli

0.4M Bacillus E.Coli

0.5M Bacillus E.Coli

5 10 15 20

23 25 26 27

24 23 25 25

23 25 28 29

32 31 33 36

25 24 21 22

24 26 25 28

19 20 21 22

21 29 22 24

26 29 25 23

29 22 23 26

24 26 27 21

22 24 27 29

Un-functionalized MNPs showed inhibition zone of 27 mm as shown in Figure 12 (a). Whereas, with addition of citric acid inhibition zone increased due to increase of reactive oxygen species (ROS). ROS was increased in functionalized MNPs because of increased number of electron hole pairs generation. Moreover, surface defects such as oxygen defects generated ROS that contributed towards the decay of bacteria. Antibacterial assessment against Bacillus bacterial stain is performed for citric acid functionalized MNPs as shown in Figure 12(b). 0.3M citric acid functionalized MNPs with superparamagnetic properties were found to be effective against abovesaid bacteria. The antibacterial activity for all citric acid concentrations is listed in Table II. It is worth mentioning here that during synthesis of nanoparticles no chemical based antibacterial agent was utilized for magnetite functionalization. Zone of inhibition (36 mm) showed that 0.3M citric acid functionalized MNPs interrupted cell wall synthesis process of bacterium, inhibited growth biosynthesis, interfered the DNA transcription process and disturbed the metabolic pathways of all the chain reactions occurring in bacterium cell. 5. CONCLUSIONS Iron oxide (magnetite phase) nanoparticles were synthesized at low-temperature by sol-gel method. In order to provide additional functional groups on surface of un-functional magnetic nanoparticles (MNPs) for bio-medical application, these MNPs were functionalized with citric acid. Concentration of citric acid was varied as 0.1M, 0.2M, 0.3M, 0.4M and 0.5M. XRD results indicated the formation of magnetite (Fe3O4) phase for un-functionalized and 0.3M citric acid functionalized MNPs. FTIR bands corresponding to C-O and C=O from COOH group of citric acid indicated successful attachment of functional groups on MNPs surface. Un-functionalized and citric acid functionalized MNPs at 0.3 M citric acid concentration showed super paramagnetic behavior. MNPs functionalized with citric acid concentration of 0.3M resulted in high saturation magnetization of 85emu/g with hydrodynamic diameter size ~ 25 nm, thus making them a potential candidate for biomedical applications. Moreover, zeta-potential analysis also confirmed the functionalization of MNPs with citric acid and less aggregation of MNPs than un-functionalized MNPs. A 16

noticeable increase in saturation magnetization was observed at 5K with negligible coercivity. ZFC-FC study showed the blocking temperature ~80K at 5Oe applied magnetic field. Absence of coercivity at very low temperature further confirmed the presence of superparamagnetic behavior. Super paramagnetic, 0.3M concentrated citric acid functionalized MNPs proved to be successful candidate as antibacterial agent with zone of inhibition of ~36mm against Bacillus bacterial stain. Acknowledgement:

Authors are thankful to Higher Education Commission Pakistan (HEC) for financial support through research grant no. HEC/CSSP/2017-18.

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Highlights

1. Citric acid functionalized magnetite nanoparticles (MNPs) were synthesized by low-temperature solgel method. 2. XRD results indicated the formation of magnetite (Fe3O4) phase at 0.3M citric acid concentration. 3. FTIR bands of C-O and C=O from COOH group from citric acid indicated successful attachment of functional groups on MNPs surface. 4. 0.3M functionalized MNPs resulted in high saturation magnetization of 85emu/g with size less than 25nm. 5. 0.3M citric acid functionalized MNPs proved to be successful candidate as antibacterial agent with zone of inhibition 36mm.

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Respected Sir!

All authors don’t have any conflict of interest.

Regards! Sidra Khan Ph.D. Scholar!