Antibacterial activity of PLAL synthesized nanocinnamon

Materials and Design 132 (2017) 486–495

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Antibacterial activity of PLAL synthesized nanocinnamon Ali Aqeel Salim a, Noriah Bidin a,⁎, Ahmad Shehab Lafi b,c, Fahrul Zaman Huyop b a b c

Laser Center, Ibnu Sina Institute for Scientific and Industrial Research (ISI-SIR), Universiti Teknologi Malaysia, 81310 Johor Bahru, Johor, Malaysia Department of Biotechnology and Medical Engineering, Faculty of Bioscience and Medical Engineering, Universiti Teknologi Malaysia, 81310 Johor Bahru, Johor, Malaysia Center of Desert Studies, University of Anbar, Ramadi, IRAQ

H I G H L I G H T S

G R A P H I C A L

A B S T R A C T

• Highly crystalline cinnamon nanoparticles (CNPs) were prepared via PLAL method in methanol. • The morphology of CNPs was controlled by varying the laser ablation energy. • Effects of varying laser ablation energies on the properties of CNPs were examined. • Elliptical CNPs displayed strong antibacterial activity against various bacterial strains.

a r t i c l e

i n f o

Article history: Received 6 May 2017 Received in revised form 3 July 2017 Accepted 6 July 2017 Available online 19 July 2017 Keywords: Nanocinnamon Laser ablation Structure Morphology Antibacterial activity

a b s t r a c t Natural cinnamon containing polyphenolic compound is well known for diverse biological activities, broad range of pharmacological and therapeutic properties. However, the potential of nanocinnamon for antibacterial usage was not widely explored. Highly crystalline elliptical shaped cinnamon nanoparticles (CNPs) were prepared via pulse laser ablation in liquid (PLAL) by immersing a cinnamon target in methanol. Effects of varying laser fluence on the structure, morphology and optical properties of as-grown CNPs were determined. Samples were characterized via UV-Vis, FTIR, XRD, TEM, HRTEM, SAED, EDX, DLS and HPLC measurements. Methanol was found to be favorable for the growth of CNPs at optimum fluence of 5.73 J/cm2. These CNPs revealed robust antibacterial activity against Gram-negative and Gram-positive bacterial strains including Escherichia coli, Pseudomonas aeruginosa, Bacillus subtilis and Staphylococcus aureus. Antibacterial activity of CNPs was evaluated via agar well diffusion assay and optical density (OD600) tests. It was established that the PLAL may constitute a basis for the production CNPs with desired size distribution potential for nanomedicinal applications. © 2017 Elsevier Ltd. All rights reserved.

1. Introduction Nanomedicine being an emerging area of nanotechnology requires precise techniques of growth and characterization of materials at nanoscale with desirable biological, physical and chemical attributes [1–3]. Among various nanostructures especially nanoparticles (NPs) owing ⁎ Corresponding author. E-mail address: [email protected] (N. Bidin).

http://dx.doi.org/10.1016/j.matdes.2017.07.014 0264-1275/© 2017 Elsevier Ltd. All rights reserved.

to their distinctive electronic structure properties show great prospects towards broad arrays of biomedical applications [4,5]. Recently, organic NPs received focused research attention because of their potential applications in pigments, cosmetics, drugs and antibacterial activity [4–6]. Though conventional grinding method can prepare NPs of size around 100 nm but several applications need NPs of size even much smaller than this. Using reprecipitation of organic molecules one can achieve nm-sized particle. In the approach, an organic molecule solution is quickly injected into a poor solvent with or without a surfactant,

A.A. Salim et al. / Materials and Design 132 (2017) 486–495

forming precipitates of the molecule [7,8]. Over the years, diverse techniques were developed to prepare nanoscale structures with desired morphology, size, shape, and compositions dependent properties [9–11]. Following bottom–up liquid precipitation approach small quantities of drug nanosuspensions were prepared [12]. However, the requirement of an appropriate pair of solvent/antisolvent with optimum ratio optimized makes this approach limited for tight drug supply. Meanwhile, the amorphous NPs formed by this process must be eliminated to avoid the toxic effects [13]. In the top–down approach, the two techniques are commonly used for nanocrystal fabrication including media milling and high-pressure homogenization. Media milling process needs five of the six commercial products in well plates or in low volume vials. The presence of wear or deformation of the wells limits its practicability [14], wherein surface adhesion mediated incomplete recovery of the drug from the milling media and inner milling chamber occurs [15]. Compared to ion implantation in solids [16] and nanoemulsion technique [17], laser ablation and fragmentation in liquid are popular one due to their simplicity, cost-effectiveness, rapidity in reactive quenching of ablated species at the interface between the plasma and liquid, eco-friendliness, largescale reproducibility of contaminant free samples and easy production of inorganic and organic NPs [18–21]. In pulse laser ablation in liquid (PLAL), a solid target is irradiated and the ejected material forms NPs inside the immersed liquid media. In laser fragmentation in liquid (LFL), a stirring suspension of microparticles is irradiated to rupture them into NPs [14]. Earlier studies revealed that PLAL is advantageous than LFL because the NPs morphology (size and phase) can be better controlled by adjusting the laser pulse width, fluence, wavelength, and pulse repetition rate [22–26]. It was shown that higher fluence led to smaller NPs, and the size could alter drastically depending on pulse width [25,26]. Besides, laser wavelength and fluence [25] could change the phase of the formed NPs significantly. Frequent laser shots over a short experimental duration (increase in repetition rate) could improve the yield [27]. In short, the overall properties and productivity of NPs via PLAL method can be finely tuned by controlling various laser parameters [22,28]. Tsuji et al. [29] acknowledged that the laser ablation efficiency is strongly wavelength dependent. Actually, the absorption of less energy by the existing NPs and the solvent molecules allows the liquid to act as reactive medium for the compound formation of NPs and thereby provides favorable thermodynamic conditions for their growth [22]. Conversely, Amans et al. [30] showed that in the case of oxides in an aqueous solution of complexion molecules, there is only a slight influence of the laser energy on the size distribution. In-depth analysis on the PLAL based synthesis suggested a very complex growth mechanism which could emerge from the collective phenomena of laser matter interaction and solution chemistry [30–32]. A laser beam upon interacting with a target material leads to the formation of plasma and cavitation bubble where the ablated matter condenses. After the cavitation bubble collapses, NPs are dispersed into the liquid [27]. Regarding the laser fluence and repetition rate, the optical breakdown of the liquid is the major limiting factor. Generally, the cavitation bubble lifetime increases with the laser fluence. For faster pulse interval (repetition rate) the NPs productivity is higher. In this repetitive process, the cavitation bubble induced by the former pulse must be temporally or spatially bypassed [33,27,22]. Small-angle X-ray scattering revealed the dispersion of nanoscale particles inside the cavitation bubble [32,27]. The primary NPs (below 10 nm) exhibited a smooth concentration profile vertically from the target surface towards the top of the cavitation bubble. The mass abundance of NPs varied according to the bubble's shape, which indicated the absence of steep particle density gradient once the bubble reached to its maximum height [22]. Fojtik and Henglein [34] synthesized colloidal NPs using PLAL and demonstrated the scalability [19] and versatility [33] of the method. In the past, Ag, Au, Pt, TiO2 and ZnO nanomaterials with diverse nanomorphologies (quantum dots, quantum wells, nanowires, and

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nanorods) were synthesized for sundry biomedical applications. Lately, renewed interests are generated on the broad range of biomedicine applications (especially as antibacterial activities) adopted with aromatic plants including, root, bark, extracts, and stick [35,36]. Compared to metal NPs, NPs of natural herbs are preferred due to their biocompatibility, low cultivation cost, effortlessness, safe handling, presence of rich active agents and easy stabilization [37,38]. Cinnamon cassia being a genus in the family Lauraceous has been used worldwide as a healthy food ingredient and traditional herbal medicine over several centuries [39]. It possesses notable attributes including non-toxicity, safe, bioactivity, biocompatibility, anti-bacterial efficacy, anti-inflammatory action and antioxidant activity [39–41]. Despite all these known advantages, the mechanism of bioactivity of cinnamon components (stick or power) in the form of NPs (CNPs) against bacteria are not widely explored [42]. Earlier, cinnamon doped metal NPs were synthesized as a stabilizing agent for antibacterial and other biomedical applications [41,42]. Yang et al. [38] prepared palladium NPs doped with the broth of Cinnamomum camphora leaf for antioxidant uses. The polyols and the heterocyclic components were believed to be responsible for the reduction of palladium ions and subsequent stabilization of palladium NPs [38]. Fatima et al. [43] synthesized Ag NPs with cinnamon cassia using chemical method and evaluated their bioactivity against highly pathogenic avian influenza virus subtype H7N3. Gauthami et al. [44] and Sathishkumar et al. [42] produced Ag NPs utilizing the bark extract of Cinnamomum zeylanicum for medicinal application. Zhang et al. [17] and Ma et al. [45] reported the bioactivity of cinnamon oil nanoemulsions/microemulsions against some pathogenic microorganisms. Using wet-milling technique, Bhawana et al. [46] prepared curcumin NPs and determined their antimicrobial properties. So far, majority of the studies focused on the antibacterial activity of cinnamon doped metal NPs. Nevertheless, development of systematic preparation technique for pure CNPs with controlled size distribution and desired antibacterial activities remains challenging. In this work we synthesized CNPs using PLAL strategy and characterized them to determine the influence of varying ablation energy on various properties of CNPs. These NPs were prepared inside methanol medium. The antibacterial activity of as-prepared CNPs were assessed on Gram-negative and Gram-positive bacterial strains such Escherichia coli (EC), Pseudomonas aeruginosa (PA), Bacillus subtilis (BS) and Staphylococcus aureus (SA). Antibacterial activity was tested using agar well diffusion assay and optical density (OD600) measurements. Results are presented, analyzed, and compared.

2. Experimental 2.1. Materials used for CNPs preparation Commercially available cinnamon sticks (Cinnamon Cassia, China) of dimension 80 mm × 20 mm × 2 mm were purchased from the local supermarket (Aeon, Kuala Lumpur, Malaysia). Analytical grade methanol (CH3OH, 96% purity from Sigma Aldrich) was used as liquid media to grow the CNPs. The nutrient agar (Merck) media was used for all bacterial cultures. Mueller Hinton agar (Merck) and penicillinstreptomycin (Sigma Aldrich) were utilized for the assessment of the antimicrobial activity of CNPs. Representative microorganisms of gram-positive and gram-negative bacteria strain including Escherichia coli ATCC 11775, Pseudomonas aeruginosa ATCC 27853, Bacillus subtilis ATCC 21332 and Staphylococcus aureus ATCC 25923 were acquired from the Microbiology Research Laboratory from Faculty of Bioscience and Medical Engineering (Universiti Tecknologi Malaysia). Cinnamon sticks (used as target material) were chopped at a dimension of 20 mm × 10 mm × 2 mm and washed thoroughly using acetone in an ultrasonic bath for 1 h before being rinsed with distilled water to remove the presence of any organic contaminants.

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2.2. Synthesis of CNPs by PLAL method PLAL technique was used to prepare CNPs with six different laser ablation energies. A Q-switched Nd:YAG laser with spot size of 2 mm, pulse duration of 10 ns and repetition rate of 1 Hz at wavelength of 1064 nm under varying ablation energy of 30, 60, 90, 120, 150 and 180 mJ corresponding to the laser fluences of 1.91, 3.82, 5.73, 7.64, 9.55 and 11.46 J/cm2 respectively, were used. First, the cinnamon stick (target) was immersed at the bottom of cubic pyrex container of volume 27 cm3 filled with 5 mL of liquid methanol as growth medium. Fig. 1 displays the schematic diagram for the experimental setup. The cinnamon target was kept at a constant height of 10 mm from the bottom level inside methanol. Then, the laser beam was focused on the surface of cinnamon target through a lens of focal length 80 mm for the duration of 16.30 min and the signal was recorded at the rate of 1000 pulse/s. The separation between the lens and target surface was kept fixed at 17 mm to compensate the refraction of light [47]. The solution is rotated at 12 rpm using a magnetic stirrer (Newport®-UTR80) to achieve homogenous mixture devoid of craters on the surface structure. 2.3. Characterizations of CNPs Optical absorption spectra in the wavelength range of 200–600 nm were recorded using a UV-Vis spectrophotometer (PerkinElmer Lambda 25 Spectrometer). A quartz cuvette having path-length of 0.5 cm was utilized for the absorption measurements. FTIR spectra of the CNPs in the wavenumber range of 500–4000 cm− 1 was recorded on a PerkinElmer Frontier™ Spectrometer using KBr pellets techniques. Crystalline structures of CNPs were examined on a Rigaku SmartLab X-ray Diffractometer which used Cu Kα radiation (wavelength of 0.15406 nm). The average crystallite diameter was estimated from the major diffraction peak using Scherrer equation given by: D¼

Kλ β cos θ

ð1Þ

where λ is the X-ray wavelength, D is the diameter of CNPs, β is the full width at half-maximum (FWHM) of the selected diffraction peak positioned at Bragg diffraction angle of θ and K is the Scherrer constant. The morphology and selected-area electron diffraction (SAED) pattern of CNPs was obtained using a biological transmission electron microscope (BIO-TEM from Hitachi HT7700). A high-resolution transmission electron microscope (HRTEM from JEOL ARM 200F) was

used to image the CNPs. Energy dispersive X-ray (EDX) spectra were recorded for CNPs elemental analysis where the EDX spectrometer was attached to a field emission scanning electron microscope (FESEM, HITACHI SU8020). The particle size distribution and mean particle diameter of the prepared CNPs were determined (averaged with 3 scans) using dynamic light scattering (DLS) on a Zetasizer (Nano-ZS90 Malvern Instruments). All the characterizations were made at room temperature. 2.4. Chemical degradation tests Liquid chromatography-mass spectrometry (LC-MS) was used to evaluate the chemical composition of prepared CNPs suspended in methanol medium. The evaluation was performed using an Agilent Ion Mobility 6560 Q-TOF system coupled with the Agilent 1290 Infinity II UHPLC. Chromatographic separation was performed on a reversephase C18 column (Poroshell 120 EC-C18, 4.6 × 100 mm, 2.7 μm) at 35 °C. The eluents were 0.1% (v/v) formic acid in water and 0.1% (v/v) formic acid in methanol. A CNPs solution of 10 μL was injected to the LC system at the flow rate of 1.0 mL/min. The instrument was operated in positive ion mode to perform full-scan analysis over an m/z range of 100–1000. The sheath gas (12 L/min) at 250 °C, nebulizer (45 psig) and dry gas (10 L/min) at 400 °C was used. Mass accuracy was calibrated online using purine (121.0509 m/z) and HP-0921 (922.0098 m/z). The UV signal was acquired at 254 nm. The chromatograms were used to quantify the CNPs and to evaluate their chemical degradation (chromatographic purity) in the medium. The relative intensities (the area under the curve) of the impurity peaks compared to the CNPs peak were used to estimate the chemical degradation percentages in the laser treated samples. The mass spectrometer that operated in positive electrospray mode with a dual spray configuration allowed an accurate determination of degradation products. 2.5. Antibacterial activity assessment of CNPs The antibacterial activity of CNPs against gram-positive and gramnegative bacterial strains was investigated utilizing agar well diffusion assay [48] in terms of bacterial growth inhibition. The Mueller–Hinton agar was poured into the sterilized petriplates and allowed to solidify at room temperature. After solidification, fresh bacterial culture (EC, PA, BS and SA) was swabbed uniformly over the Mueller–Hinton agar plates using sterile L-shape. Four wells each of diameter 6 mm were made in the agar medium using sterile cork borer. CNPs solutions of

Fig. 1. Schematic diagram of the experimental setup to grow CNPs.


20 μL that were prepared by varying the laser ablation energy from 30 to 180 mJ then poured into the corresponding well. For the control and standard antibacterial agent, the stock solution without CNPs and penicillin-streptomycin were used. The loaded plates were incubated at 37 °C for 24 h. After the incubation, the extent of the formed inhibition zone around the well was monitored and measured (in mm) which authenticated the antibacterial activity of CNPs. Meanwhile, the optical density (OD) at 600 nm was measured using a UV-Vis spectrophotometer (PG Instruments, T60) to examine the bacterial growth after each 5 h of interval over a total duration of 45 h. Freshly grown bacterial culture of volume 180 μL (104 cells/mL) of EC, PA, BS and SA were incubated. Finally, to observe the bacterial cell growth at 37 °C these CNPs solutions were loaded into each flask filled with 20 mL of Muller Hinton broth. 3. Results and discussion 3.1. Mechanism of laser energy ablation assisted growth of CNPs Fig. 2 illustrates the UV-Vis absorption spectra of all synthesized samples as a function of laser ablation energy. The effect of varying laser fluences (1.91, 3.82, 5.73, 7.64, 9.55 and 11.46 J/cm2) on the growth of CNPs was studied. The changes in the solution color (containing CNPs), the intensities, and the absorption peak positions clearly indicated the variation in the CNPs morphology (size and shapes) and crystallinity. This observation agreed well with the findings of Wiley Benjamin et al. [49]. The inset (1) in the figure shows the laser ablation energy dependent size variation of CNPs which gradually turned from colorless into dark brown in short time intervals, which was attributed to the coherent oscillation of electrons at the surface of NPs [50]. Meanwhile, the liquid medium (methanol) exhibited an enhancement in the percentage of optical absorption intensity with increasing ablation energy. This suggested that the NPs number density and size was indeed influenced by the laser ablation energy. It is worth noting that this mechanism of CNPs formation is quite different than the vacuum pulse laser deposition, where the number density and sizes of formed NPs are decided by the condensation of laser ablated plasma plume over the substrate surface [22,24]. At lower energy ablation (1.91, 3.82, and 5.73 J/cm2) the cinnamon target was heated. However, due to the strong confinement of the liquid (methanol) at the surface, the vaporization was inhibited and no plasma plume was formed. In the absence of any vapor plume, the hot target remained in contact with the methanol and promoted the oxidation of NPs

Fig. 2. Laser ablation energy dependent optical absorption spectra of CNPs synthesized in methanol liquid medium. Inset (1): particle size dependent color change, inset (2): peak shift (weak) and inset (3): peak shift.

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compounds [18]. The reaction was initiated with the oxidation of the molten target surface by oxygen splitting of water molecules at the hot target so that the hydroxide NPs could exist on the target surface. The produced cinnamon hydroxide material on the hot target surface was then desorbed and diffused into the methanol. The negative charge of these ultrafine hydroxide particulates restricted their selfaggregation and led to the formation of cinnamon nanocrystallites. Moreover, at higher laser fluences (7.64, 9.55 and 11.46 J/cm2) the liquid absorbed the energy at faster rate than the cinnamon target and resulted the removal of material by reactive sputtering rather than direct laser ablation [24]. When the amount laser energy reaching to the target appeared zero, the produced plasma in the methanol created a cavitation bubble which was eventually expanded and then collapsed, driving highly energetic species into target [22,27]. Irrespective of the laser ablation energy, two characteristic bands were evidenced centered at 261 nm and 321 nm. The broadening of bands verified the size dispersion of nanoparticles grown in the methanol medium [22]. The intense absorption band was allocated to the phenolic acids and their derivatives (flavanols, cinnamaldehyde, phenylpropenes and eugenol) as reported elsewhere [43,35]. The broadening of the weak absorption peak accompanied by a tiny red shift was assigned to the presence of ring of benzoyl and cinnamoyl system in the nucleated CNPs [51]. Inset (2) shows the laser ablation energy dependent shift of the weak peak which was attributed to the presence of aromatic amino acids of proteins structure. Furthermore, it is known that the absorption band in the range of 261–262 nm arises due to the excitation in tryptophan and tyrosine residues in the protein [52]. The intensity of the prominent absorption band in the range of 321–325 nm was steadily increased as well as red shifted up to laser ablation energy of 120 mJ and blue shifted thereafter as depicted in inset (3). This observation was ascribed to the quantum size effects of CNPs as report earlier [24,53]. Furthermore, the recovery of NPs at higher energy was lesser than at lower energy because of the filtering out of the suspension [25]. The liquid suspension containing the CNPs were very stable even after 2 months of incubation, but the optical properties of CNPs was changed due to the structural alteration of terpenoids, phychimcal cinnamaldehyde, eugenol ethyl cinnamate, caryophyllene, carboxyl and hydroxyl groups present in the CNPs suspension which contributed to the distinct aroma and biological activity of CNPs [22]. The FTIR spectra of all samples (Fig. 3(a)) display the presence of various functional groups in CNPs. The FTIR band positions displayed a minor shift accompanied by a change in the intensities. The appearance of broad and strong absorption band of CNPs around 3360 cm−1 was assigned to the hydroxyl group (O\\H) stretching vibration of phenols and alcohols [43,54]. The band centered at 2974 and 2880 cm−1 was allocated to the carbon hydroxyl (C\\H) stretching vibration of alkane. The band at 2502 cm−1 and 1912 cm−1 revealed the presence of\\C≡C\\ stretch of alkynes and aldehydes [54]. The gradual decrease in the absorption intensity of the band around 1912 cm−1 as exemplified in Fig. 3(b) was due to the methanol oxidation and subsequent formation of CNPs [55]. The band at 1658 cm−1 showed a nonlinear increase in the intensity which was associated with the stretching vibration of an aldehyde carbony1 C_O groups of alkenes. This confirmed the growth of CNPs and supported the observed broadening and shift in UV-Vis the peak (Fig. 2). The dominant peak as marked in Fig. 3(b) corresponded to the presence of high levels of cinnamaldehyde and aldehydes in the cinnamon as reported earlier [38,43–45]. The band at 1417 cm−1 (Fig. 3(c)) was allocated to the vibrational bending absorption of C-OH alkane and aromatic amines. Due to the influence of conjugation and aromatic ring, this peak was wider than the normal aldehyde compound [54]. The band at 1031 cm−1 revealed the presence of C\\O stretching of aliphatic amines which signified the consumption of methanol due to the oxidation. The band at 864 cm−1 (Fig. 3(a, c)) was shifted and the intensity was enhanced with increasing ablation energy. The occurrence of this band was assigned to the C\\H bending vibration absorption of alkynes and alkyl halides suggesting

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Fig. 3. Ablation energy dependent FTIR spectra of CNPs in the wavenumber range of: (a) 500–4000 cm−1, (b) 1600–2000 cm−1 and (c) 800–1500 cm−1.

their involvement in the CNPs formation. The presence of these peaks as shown in Fig. 3(c) confirmed that the CNPs were covered by secondary metabolites such as terpenoids, flavonoids, glycosides, phenols, tannins, with different functional groups (ketone, aldehyde, carboxylic acid, etc.) [56]. Fig. 4 displays the XRD pattern of the synthesized CNPs in methanol at optimal laser fluence of 5.73 J/cm2. Five prominent peaks were observed at diffraction angle (2θ) of 15°, 24.44°, 30.21°, 38.24° and 52.61° corresponding to the growth orientation along (001), (220), (002), (311) and (440) lattice planes, respectively. These XRD peaks verified the presence of high crystallinity of the face center cubic (FCC) structured CNPs (DB Card Number 01-074-9D14). The (001) peak was used to calculate the crystallite size (Eq. 1). The average size of cinnamon nanocrystallite was 8.47 nm. Fig. 5(a)–(c) illustrates the TEM and HRTEM images, size distribution, crystalline phase, and EDX spectra of the optimum CNPs sample grown in methanol at laser fluence of 5.73 J/cm2. The TEM image in Fig. 5(a) exhibits the morphology of the CNPs which was found to be predominantly elliptical in shape and uniform. The appearance of such unique morphology of CNPs was attributed to the newly generated

Fig. 4. XRD pattern of the CNPs grown in methanol at 5.73 J/cm2 fluence.

atoms that were trapped by the existing nuclei during the diffusioncontrolled growth process. Inset (a1) in Fig. 5(a) depicts the magnified HRTEM image of a single elliptical CNP shape, where the particle edge appears brighter than the center. This indicates the presence of proteins structure adhered to the surface as supported by the FTIR data. Inset (a2) shows the CNPs size distribution which varied in the range of 2– 41 nm. Fig. 5(b) presents the EDX spectra of the optimum CNPs sample. The presence of appropriate elements such as C, O, Cu, B, Ca, K, Ni, Al, Fe, and Zn clearly verified the nucleation of CNPs. The occurrence of B and K peaks were assigned to the protein capping over CNPs whereas the Cu peak was appeared from the copper grid used to attach the sample. These observations were supported strongly by the FTIR analysis (Fig. 3). Inset (table) of Fig. 5(b) depicts the percentage of the atomic elements present in the CNPs suspension. Fig. 5(c) illustrates magnified TEM image of a selected region (square box). Inset (c1) reveals the SAED pattern and Inset (c2) lattice scale fringes of a single CNP. The occurrences of concentric rings and bright circular spots in the SAED pattern confirmed the growth of nanocrystalline cinnamon particles along different lattice plane with preferred orientation. The magnified view of lattice fringe image exhibited the regular lattice spacing of 0.216 nm (Inset (c3)) corresponding to the (001) lattice plane (Inset (c4)). The effect of laser fluence on the particle size distribution and mean particle diameter of the prepared CNPs are measured (Table 1) using DLS and the chemical degradation percentage estimated by liquid chromatography. With increasing laser fluence, the mean particle size was decreased at the cost of higher degradation [25–27]. The nature of the degradation products induced during laser ablation was investigated with mass spectrometry (Table 2). The empirical molecular formulas for the degradation products (particles) suggested the occurrence of oxidation through the addition of oxygen or elimination of hydrogen in the CNPs sample [14]. After laser fluence, the appearance of beige tint in methanol clearly indicated the suspension of insoluble CNPs which could be isolated. This higher degradation may be attributed to the two-photon absorption (at 257 nm) in the midst of CNPs absorption peak wavelength (200–320 nm). Fig. 6 displays the laser fluence dependent average size of CNPs together with their standard deviation [25,26]. With increasing laser fluence, the particles became homogenous, smaller, and elliptical in


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Fig. 5. CNPs prepared with laser fluence of 5.73 J/cm2 in methanol: (a) TEM image of the optimum sample (Inset: HRTEM of single elliptical CNP and size distribution corresponding to (a) of CNPs), (b) EDX spectra (Inset: At.% and Wt% of detected elements). (c) Magnified TEM image.

shape. The concentration of CNPs appeared highest at 5.73 J/cm2 and the average diameters were reduced beyond this fluence due to the fast diffusion of particles [22]. The photon energy of single laser pulse was readily converted to the internal modes of the NPs which in turn raised the temperature and caused the fragmentation of large NPs [19]. After the diffusion of a single laser pulse into the solution, the NPs temperature returned to the room temperature before the next one was arrived. This repeated heating and cooling of NPs occurred between every laser pulse interval and imparted the NPs stability as reported in the literature [24,17]. The presence of various chemical compounds in cinnamon was identified by LC/MS analysis [35,17]. Fig. 7(a) and (b) show the LC chromatogram of methanol containing cinnamon before and after laser ablation, respectively. Emergence of several new peaks in Fig. 7(b) clearly reveals the existence of CNPs in methanol obtained with laser ablation at optimum fluence of 5.73 J/cm2. Table 2 enlists various cinnamaldehyde and polyphenolic derivatives in CNPs (identified from LC/MS tests) those are responsible for antibacterial activities.

Table 1 Laser fluence/ablation energy dependent CNPs mean size (±standard deviation, SD), percentage chemical degradation (Deg) and sample code. CNPs sample code

Ablation energy (mJ)

Fluence (J/cm2)

Mean size ± SD (nm)

Deg (%)

S2-30

30

1.91

35.79 ± 2.91

S2-60 S2-90 S2-120 S2-150 S2-180

60 90 120 150 180

3.82 5.73 7.64 9.55 11.46

32.45 ± 3.21 28.79 ± 1.02 26.34 ± 0.95 24.37 ± 0.72 21.45 ± 0.45

0.1 ± 0.08 0.3 ± 0.2 0.5 ± 0.3 0.8 ± 0.5 1.2 ± 0.7 2.2 ± 0.8

3.2. Antibacterial activity of CNPs Presently, various metallic and nonmetallic NPs are realized as a viable alternative to antibiotics owing to their high potential towards bacterial multidrug resistance. In this regard, CNPs can physically interact with the cell surface of various bacteria which is particularly important in the case of gram-negative bacteria. Numerous studies have revealed the adhesion and accumulation of different NPs to the bacterial surface [57]. Researches revealed that NPs can damage the bacterial cell membranes leading to structural changes and make the bacteria more permeable [58]. It was established that the NPs size, shape, distribution, and concentration [59,60,61] play a decisive role towards antibacterial actions. Earlier, some studies confirmed that the NPs accumulation on the membrane cell could create gaps in the integrity of the bilayer which predisposes it to a permeability increase and finally bacterial

Table 2 Chemical composition of CNPs at optimum laser fluence of 5.73 J/cm2. Peak no.

Name of identified compound Retention time (min)

Mass (m/z)

1 2

2-Methyl Gramine 3-Methylbutyraldehyde oxime Longifolonine Benzyl beta primeveroside Epicatechin Carfecillin 2-Trimethyl-docosatetraenoyl amine 6-Deoxotyphasterol

0.544 0.999

188.1317 C12H16N2 101.0841 C5H11NO

1.999 2.692 3.503 4.506 37.763

297.1002 402.1521 864.1864 454.1221 419.3561

37.907

434.3735 C28H50O3

3 4 5 6 7 8

Chemical formula

C17H15NO4 C16H20N6O8 C45H36O18 C23H22N2O6 C27H46FNO

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Fig. 6. Laser fluence dependent variation in the average size of CNPs together with standard deviation.

cell death [58,62]. In short, it was acknowledged that NPs overall activity is strongly size dependent [63,64]. Certainly, the size distribution of the NPs is a fundamental parameter for controlling the antimicrobial activity. The bactericidal activity of tiny CNPs (mean size below 30 nm) was found to be optimal against EC, PA, BS and SA [65]. The penetration capacity of smaller NPs into the bacteria is better and their interactions with the membranes, resultant damage, and subsequent cell death are certainly more than larger size CNPs. Electrostatic forces are develop when NPs with a positive size distribution encounter bacteria having negative surface charge. This promotes a closer attraction and interaction between the two entities and possible penetration in the bacterial membranes. NPs with positive charge and reduced size distribution are effective for antibacterial purposes. The surface/volume ratio for NPs being higher than the corresponding bulk material the modalities and amount of the interactions with the bacterial surfaces are facilitated with higher antibacterial activity. Fig. 8 schematically displays the possible toxicity mechanisms of CNPs on bacterial cell. The molecules in CNPs bind to the negatively charged protein and nucleic acid, causing structural changes and deformations of the bacterial cell wall, membranes and nucleic acids. The CNPs also damage the membranes and induce the release of reactive oxygen species, forming free radicals with a powerful antibacterial action.

Fig. 8. Schematic presentation showing the activity of CNPs against bacteria strain.

Furthermore, small CNPs can easily enter the microbial body, causing the damage of its intracellular structures. Consequently, the ribosomes get denatured with the inhibition of protein synthesis, where the translation and transcription are blocked through the binding of bacterial cell related genetic material [66,17]. Protein synthesis has been shown to alter by the treatment of CNPs. The proteomic data have revealed an accumulation of immature precursors of membrane proteins resulting in destabilization of the composition of the outer membrane. 3.3. Antibacterial tests of CNPs using diffusion assays Using agar well diffusion assay, the inhibition zone test was carried out to qualitatively investigate the antibacterial property of the freshly prepared CNPs in methanol medium. All the methanol suspension containing varying diameters of CNPs (1–30 nm) obtained at different laser ablation energy exhibited high inhibitory effect against gram-positive and gram-negative bacterial strains EC, PA, BS and SA as shown in Fig. 9(A), (B), (C), (D), respectively. However, the appearance of the widest diameter of the inhibition zone (in mm) obtained with optimum sample S2–90 (prepared at ablation energy of 90 mJ) confirmed its strongest antibacterial activity. Conversely, the antibacterial activity revealed by the blank/control sample S2-0 (without laser ablation i.e. 0 mJ) was almost negligible. The ablation energy dependent inhibition zone for all

Fig. 7. LC chromatogram of: (a) methanol with cinnamon before laser irradiation, (b) CNPs in methanol after laser ablation at optimum fluence of 5.73 J/cm2.


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Fig. 9. Inhibition zones revealing the antibacterial activity of CNPs in methanol medium (at 37 °C) prepared with different ablation energy when implemented on bacterial strains of: (A, a) E. coli, (B, b) P. aeruginosa, (C, c) B. subtilis, and (D, d) S. aureus.

the bacterial strains EC, PA, BS and SA were measured and compared with the antibiotic as displayed in Fig. 9(a), (b), (c), (d), respectively. The possible mechanism of antibacterial activity of CNPs indicated efficient inhibition of the bacterial surface protein sortase and subsequent prevention of cell adhesion to fibronectin. It was asserted that CNPs could anchor to the bacterial cell walls, disrupt the membranes structure, and then penetrate inside the cells to break down the structure of cell organelles as mentioned in Section 3.2. Fig. 10 (a)–(d) shows the optical density at 600 nm (OD600 in LB media) displaying the antibacterial activity of CNPs prepared at different ablation energy when tested on the bacterial strains of Escherichia coli, Pseudomonas aeruginosa, Bacillus subtilis, and Staphylococcus aureus, respectively. The methanol suspension containing CNPs was treated after every 5 h interval to the total duration up to 45 h to examine the impact of different ablation energy on the antibacterial activity of CNPs. This method was widely used previously to determine the growth of bacteria for the assessment of antibacterial abilities. Generally, the lower OD600 of bacteria suspension after cultivation for a definite time signifies the better antibacterial ability of reagent. The OD600 of all the pure bacterial strains (without adoption with CNPs) and blank/control sample S2-0 was higher with identical absorption intensity and the inhibition zone values (Fig. 9). Moreover, after 5 h of cultivation, all the bacterial strains implemented with CNPs divulged lower OD600, which

was attributed to the reduction in the conglomeration of CNPs. Besides, the suspension containing CNPs (S2-90) prepared at optimal ablation energy of 90 mJ displayed the lowest OD600 especially after 15 h of cultivation as compared to other samples. This suggested that the CNP with lowest average diameter of 28.79 nm in S2-90 sample (large surface area to volume ratio) have significant influence against the bacterial strains growth. Thus, the excellent capacity of CNPs to resist the bacterial growth was consistent with the standard inhibition zone results. 4. Conclusion For the first time we prepared highly crystalline, natural, dispersed, homogeneous, and elliptical CNPs using a simple, inexpensive and yet accurate method called PLAL. These NPs were grown in liquid methanol media using a Q-switched Nd:YAG laser with varying ablation energy in the range of 30–180 mJ. The influence of laser ablation energy on the growth, structure, morphology, and optical properties of CNPs were determined. The antibacterial activity of these CNPs was evaluated. Synthesized CNPs were characterized using UV-Vis, PL, FTIR, XRD, TEM, SAED and EDX measurements. It was shown that by varying the ablation energy, it is possible to control the CNPs morphology in a desired way which is useful for nanomedicinal applications. This procedure revealed high yields and easy operations in the absence of any toxic reagents or

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Fig. 10. Optical density (in LB media) showing the inhibition activity of various CNPs when tested on bacterial strains of: (a) Escherichia coli, (b) Pseudomonas aeruginosa, (c) Bacillus subtilis, and (d) Staphylococcus aureus.

surfactant template. UV-Vis spectra revealed two characteristic absorption bands in range of 261 and 321 nm. FTIR spectra verified the presence of various functional groups of the bioactive component and the bonding structures of the CNPs. The XRD diffraction patterns revealed the high crystallinity of the FCC CNPs. The laser fluence of 5.73 J/cm2 produced the best CNPs morphology wherein the average particle diameter was found to be 28.79 ± 1.02 nm indicating that the methanol medium was favorable for the growth CNPs. LC/MS analysis revealed chemical degradation of CNPs. The antibacterial activity of CNPs was evaluated against EC, PA, BS and SA bacterial strains using the agar dilution and diffusion test. The optimum CNPs sample (grown at 90 mJ) exhibited highest antibacterial activity against E. coli and P. aeruginosa. The higher antibacterial activity of CNPs was attributed to the size effect of tiny NPs and unique morphology. The optical density of the CNPs implemented bacteria growth over the duration of 5 to 45 h also confirmed their antibacterial efficacy. It was established that natural CNPs with inherent antibacterial activity that were grown in the methanol medium at optimal laser fluence of 5.73 J/cm2 are potential candidate for the development nanomedicine.

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