Green synthesis of chitosan-cinnamaldehyde cross-linked nanoparticles: Characterization and antibacterial activity

Green synthesis of chitosan-cinnamaldehyde cross-linked nanoparticles: Characterization and antibacterial activity

Journal Pre-proof Green Synthesis of chitosan-cinnamaldehyde cross-linked nanoparticles: characterization and antibacterial activity Rahul Rajkumar Ga...

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Journal Pre-proof Green Synthesis of chitosan-cinnamaldehyde cross-linked nanoparticles: characterization and antibacterial activity Rahul Rajkumar Gadkari, Sheetal Suwalka, Mayank Raj Yogi, Wazed Ali, Apurba Das, Ramasamy Alagirusamy

PII:

S0144-8617(19)30965-8

DOI:

https://doi.org/10.1016/j.carbpol.2019.115298

Reference:

CARP 115298

To appear in: Received Date:

5 April 2019

Revised Date:

7 August 2019

Accepted Date:

5 September 2019

Please cite this article as: Gadkari RR, Suwalka S, Yogi MR, Ali W, Das A, Alagirusamy R, Green Synthesis of chitosan-cinnamaldehyde cross-linked nanoparticles: characterization and antibacterial activity, Carbohydrate Polymers (2019), doi: https://doi.org/10.1016/j.carbpol.2019.115298

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Green Synthesis of chitosan-cinnamaldehyde cross-linked nanoparticles: characterization and antibacterial activity Rahul Rajkumar Gadkari, Sheetal Suwalka, Mayank Raj Yogi, Wazed Ali*, Apurba Das, Ramasamy Alagirusamy Department of Textile Technology Indian Institute of Technology Delhi, New Delhi 110016, India *Corresponding author: E-mail: [email protected]; Tel: +91-011-2659 7952

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Graphical abstract

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Highlights

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 Synthesis of chemically cross-linked chitosan and cinnamaldehyde nanoparticles  Prepared nanoparticles are structurally stable and amorphous in character  Improved antibacterial efficacy as compared to bulk chitosan and cinnamaldehyde  Effective against both Gram-positive and Gram-negative bacteria ABSTRACT

Traditional method of chitosan (naturally available abundant biopolymer) nanoparticles

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synthesis is the ionic cross-linking between chitosan and say, sodium tri-polyphosphate (TPP). These nanoparticles are structurally less stable and basically it’s a conversion of pure bio-based material, chitosan into a hybrid structure of biopolymer and a synthetic chemical. The present work reports a novel attempt to synthesize antimicrobial chitosan nanoparticles by chemical cross-linking with cinnamaldehyde, another eco-friendly bactericidal agent. The synthesized nanoparticles (size range, 80-150nm) were analysed for their surface morphology. X-ray diffraction pattern denoted the amorphous characteristics of the formed nanoparticles. The 1

FTIR analysis revealed formulation of chitosan nanoparticles to be based on Schiff reaction between amino group of chitosan and aldehyde group of cinnamaldehyde. Moreover, NMR analysis also confirms the formulation of cinnamaldehyde cross-linked chitosan nanoparticles. TGA and DSC were performed to analyse thermal characteristics and stability of prepared nanoparticles. Subsequently, the study successfully indicated that the synthesized nanoparticles exhibit synergistic antibacterial activity (98%) against Staphylococcus aureus (Gram-positive) and (96%) Escherichia coli (Gram-negative) bacteria. The MIC and MBC values were found to be 5 mg/mL and 10 mg/mL, respectively for both types of bacteria.

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Keywords: chitosan; cinnamaldehyde; cross-linked nanoparticles; imines; Schiff reaction; antibacterial activity

1. Introduction

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Polysaccharides are widely distributed and easily accessible in nature. These materials are important in different fields owing to their typical structures and characteristics that are

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different from those of conventionally synthesized polymers. Among the range of polysaccharides, cellulose and chitin are the most important biomass resources. Cellulose is synthesized by plants, whereas chitin is obtained from lower animals (Honarkar & Barikani,

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2009). Chitosan, the most important derivative of chitin, can be obtained by deacetylation of chitin under alkaline conditions (Dutta, Ravikumar, & Dutta, 2002; Tsigos, Martinou, Kafetzopoulos, & Bouriotis, 2000). It is a useful biopolymer with unique structural features:

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β-1-4 linked units of poly(d-glucosamine) (80%) and poly(N-acetyl-d-glucosamine) (20%) (S. A. Agnihotri, Mallikarjuna, & Aminabhavi, 2004; Gadkari, Ali, Das, & Alagirusamy, 2017).

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The presence of NH2 groups in chitosan is the reason it exhibits much greater potential than chitin for use in different applications. It has good properties like biodegradability,

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biocompatibility, and antibacterial activity, which make it an interesting polymer for enormous application. Some of the potential applications of chitosan are in the areas of medicine, drug delivery, water treatment, membranes, hydrogels, adhesives, antioxidants, biosensors, functional textiles and food packaging (Berscht, Nies, Liebendörfer, & Kreuter, 1994; Kumar Dutta, Dutta, & Tripathi, 2004; Paul & Sharma C, 2000; Thanou, Verhoef, & Junginger, 2001). Moreover, research community is trying to diversify the applications of chitosan by using its different nano-structures like nanoparticles, nanofibres, scaffolds and nanocomposites

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(Jayakumar, Menon, Manzoor, Nair, & Tamura, 2010; Jayakumar, Prabaharan, Nair, & Tamura, 2010). Chitosan possesses free amino and hydroxyl groups that enable its cross-linking (Anitha, Rejinold, Bumgardner, Nair, & Jayakumar, 2012; Wang, Li, Peng, Huang, & Kong, 2011). Many researchers have used formaldehyde, tripolyphosphate, glutaraldehyde, and polyaspartic acid sodium salt as cross-linkers for the preparation of micro- or nano- sized particles of chitosan (Ali, Joshi, & Rajendran, 2010; Ali, Rajendran, & Joshi, 2011; Gan, Wang, Cochrane, & McCarron, 2005; Gupta & Ravi Kumar, 2000; Gupta & Jabrail, 2006). However, some of these cross-linkers are toxic in nature. Moreover, the particles prepared by using these cross-

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linker showed poor mechanical strength and non-biodegradable property (Anitha et al., 2012; Gan et al., 2005) because the other reactant used was not of natural resource. Thus, it is essential to find a non-hazardous, biodegradable and effective cross-linker for the preparation of chitosan nanoparticles with better mechanical properties.

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Cinnamon (Cinnamomum zeylanicum) of Lauraceae family is a popular spice used widely over the world. It is obtained from the inner bark of several tree species from the genus

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Cinnamomum, and primarily contains essential oils and other derivatives, such as cinnamaldehyde, cinnamic acid, and cinnamate. The most important constituent of cinnamon is cinnamaldehyde (C6H5CH=CHCHO) which is present in its essential oil (Ashakirin S. N.,

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Tripathy M, Patil U. K., & Abdul Majeed A. B., 2017; Rao & Gan, 2014). Owing to its nontoxicity, fungicidal and antibacterial effects, cinnamaldehyde has numerous applications

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(Chang, Chen, & Chang, 2001; Jakhetia V et al., 2010; Subash Babu, Prabuseenivasan, & Ignacimuthu, 2007). Gill and Holley (Gill & Holley, 2004) and Ooi et al. (Ooi et al., 2006) reported that cinnamaldehyde inhibits the growth of various bacteria like Gram-positive, Gram-

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negative and fungi. Since it has an aromatic conjugation and an aldehyde group (Fig. 1), it was envisaged that cinnamaldehyde may be used as a cross-linker for the preparation of chitosan

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particles as well. Furthermore, being an antimicrobial agent, it may also enhance the stability and antimicrobial property of chitosan particles. Several studies have been reported wherein chitosan and cinnamaldehyde have been used

in combination. Rieger and Schiffman (Rieger & Schiffman, 2014) incorporated cinnamaldehyde oil into chitosan/poly(ethylene oxide) solutions and formed cinnamaldehyde impregnated chitosan nanofibrous mat by using electrospun technique that was subsequently cross-linked using toxic glutaradehyde vapours. The mat demonstrated good antibacterial activity against E. coli and P. aeruginosa bacteria. Higueras et al. (Higueras, López-Carballo, 3

Gavara, & Hernández-Muñoz, 2014) reversibly anchored cinnamaldehyde to chitosan films via imino-covalent bonding for designing antimicrobial food packaging material. Lei et al. (Lei et al., 2015) synthesized chitosan emulgels via cinnamaldehyde cross-linking and studied the effect of system parameters on the properties of emulgels. In an another study, Babu et al. (Babu & Kannan, 2012) synthesized chitosan nanoparticles loaded with an anticancer compound baicalein, for their potential use in drug-delivery. However, the preparation of pure chitosan nanoparticles targeting antimicrobial efficacy by chemical cross-linking with cinnamaldehyde has not been explored much in detail. Also, there is dearth of literature citing the detailed characterization and evaluation of antibacterial

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properties of such nanoparticles against microorganisms.

Considering the need for an environmental friendly method for the synthesis of chitosan nanoparticles, and the (favourable) structure of biopolymeric cinnamaldehyde indicative of

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enabling cross-linking, the objective of this research work is to investigate the possibility of synthesising pure chitosan nanoparticles using cinnamaldehyde which targets solely on

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antimicrobial effect. Since both chitosan and cinnamaldehyde are antibacterial in nature, an attempt has also been made to explore their antibacterial activities in individual and combined

2.1. Materials

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2. Materials and methods

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effect in nanoparticulate form which is a unique study in this present article.

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Chitosan with molecular weight 50,000-190,000 Da and deacetylation degree ≥90% (CAS No. 9012-76-4), was purchased from Sigma-Aldrich Chemical Co. Ltd. Cinnamaldehyde (CAS No. 104-55-2) was purchased from Central Drug House Private Ltd., India. Acetic acid, liquid

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paraffin, span-80 and tween-80 were obtained from Merck Life Science Private Ltd., India. Magnesium stearate was procured from Sigma-Aldrich Chemical Co. Ltd., India. Nutrient broth (Merck), Nutrient Agar (Merck) and Agar-Agar (Merck) were used to carry out the antimicrobial testing. The deionized water was obtained from Millipore Milli-Q water purification system.

2.2 Chitosan Nanoparticle (Imine) Synthesis Method

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Chitosan solution was prepared by dissolving 0.1 g purified chitosan (1% w/v) in acetic acid solution (10 mL, 1% v/v) by sonicating in Elma S50H Elmasonic until the solution became transparent and finally the pH was set at 6.0. This solution was used as water phase in the next step. Liquid paraffin (50 mL) containing tween-80 (0.507 mL), span-80 (0.467 mL) and magnesium stearate (0.5 g) was prepared to be used as oil phase. Chitosan solution was added drop wise into the prepared oil phase to form water-oil (W/O) emulsion by using VELP Scientifica OV5 Homogeniser at 20,000 rpm for 1 hour. This process was carried out in a cold water bath in order to neutralise the heat evolved due to high speed homogenisation. Cinnamaldehyde (2% w/v, i.e. 0.02 g cinnamaldehyde in 10 mL of acetone) was dissolved

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in acetone, and 1 mL of this solution was dropped slowly into the prepared emulsion under mechanical agitation (12000 rpm) at room temperature (30±2°C). The cinnamaldehyde worked as cross-linking agent to hold the nano-sized chitosan particles. Thereafter, chitosan nanoparticles were collected by centrifugation at 9000 rpm for 15 minutes at room temperature

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(30±2°C) using Thermo Scientific SORVALL ST8 Centrifuge machine, and subsequently washed with petroleum ether and isopropanol for the removal of oil phase attached to chitosan

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nanoparticles. Finally, the nanoparticles were washed with DI water and freeze-dried at −700C for 24 hours in ilShinBioBase Freeze Dryer. Dry nanoparticles were then collected and characterized. The weight yield obtained was ~60%. In this synthesis process to form chitosan

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nanoparticles, pH of chitosan solution was also varied from 5.0 to 6.5 (5.0, 5.5, 6.0, and 6.5) to observe its effect on the size of particles and its resultant influence on antibacterial activity

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which is discussed in supplementary document (S1).

2.2.1 Reaction scheme for the synthesis of cinnamaldehyde cross-linked chitosan

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nanoparticles.

In the first step of reaction (Scheme 1), H+ (in acid medium) attacks on the lone pair of O of

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aldehyde. Following this, acid-catalyzed addition of a nucleophile i.e. amino group from chitosan, to the carbonyl carbon of an aldehyde takes place. Here, N gets a ‘+’ charge, to compensate which it takes e- from H atom. Next, due to the presence of H+ in acid medium, it attacks on OH group of intermediate, causing removal of H2O. Correspondingly, N gives its lone pair to compensate charge on C atom. Now, N develops electron deficiency, and hence attracts e- from H atom. Finally, N of chitosan forms a double bond with C of cinnamaldehyde, forming an imine based compound.

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2.3 Particle Characterization 2.3.1. Morphology analysis The surface appearance of the particles was examined under a Transmission Electron Microscope (TEM) machine (Phillips CM 12). One drop of the diluted particles suspension (as produced) was placed on a copper grid with the help of a syringe and air-dried without causing any disturbance to it, and then viewed under the TEM. Scanning Electron Microscope (SEM) images were also taken by using ZEISS EVO 50 machine. One drop of the particles suspension was placed on an aluminum plate sample holder and maintained for 12 hours at room temperature in a desiccator for complete drying. The dried sample was coated with a thin layer

pressure, 25 mA current and thereafter observed under SEM.

2.3.2 X-ray diffraction (XRD) analysis

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of gold using Emitech K550X large sample coater (Emitech, Kent, UK) set at 9×10−4 Mbar

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X-ray diffraction patterns of bulk chitosan and chitosan nanoparticles were obtained to analyse their crystalline and amorphous content. The X-ray source used was Cu Kα radiation

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of 80 mA at 40 kV. The samples were scanned at a rate of 4° min−1 on Phillips X-PERT PRO X-ray machine.

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2.3.3 Fourier transform infrared (FTIR) analysis

Infrared spectroscopy (IR) of chitosan, cinnamaldehyde and chitosan was carried out to investigate the cross-linking mechanism. Test pellets were made by mixing 7.0-9.0 mg of

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sample and 45 mg KBr at 400 kg/cm2 pressure for 10 min and tested on Nicolet iS50 FTIR spectrometer at a scan speed of 64 with a resolution of 4 cm-1. All the data were taken in

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transmittance mode.

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2.3.4 Nuclear magnetic resonance (NMR) analysis Solid-state 13C CP/MAS NMR analysis was carried out on JNM-ECA Series (Delta V4.3)

spectrometer at a resonance frequency of 400 MHz. A zirconium oxide rotor spinning at the magic angle of 5 kHz was loaded with 200 mg chitosan nanoparticles (powder form). 3000 scans were taken at 5 s relaxation delay and for 50 ms acquisition time (Rieger & Schiffman, 2014).

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2.3.5 Thermal Analysis Differential scanning calorimetry (DSC) scans were recorded on Perkin Elmer (Model DSC 7). 2 mg each of the samples were accurately weighed in aluminum pans without seals and heated from 50°C to 200°C at a rate of 20°C/min under a nitrogen flow pressure of 40 ppsi. Thermogravimetric analysis (TGA) was performed on Perkin Elmer TAC7/DX TG analyser. The carrier gas was nitrogen with a sample purging pressure of 2.6 kg cm−2. The samples were heated from 50°C to 600°C to record the TG curves.

2.3.6 Antibacterial activity assessment

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The antibacterial activity of the bulk chitosan, cinnamaldehyde and chitosan nanoparticles was tested quantitatively using modified colony counting method (AATCC-100) against Grampositive Staphylococcus aureus (ATCC 25923) and Gram-negative Escherichia coli (ATCC 35218) bacteria. All the required glassware and other handling materials were sterilized before use at standard conditions i.e. 120°C, 15 psi and for 30 minutes.

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In this method, 0.05 g each of bulk chitosan, cinnamaldehyde and chitosan nanoparticles were placed in different test tubes (containing 10mL sterilized deionised water), and a test tube

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without any sample was considered as control. Luria broth (LB) solution with 6.8×107 CFU/mL of S. aureus bacteria and 6.4×108 CFU/mL of E. coli bacteria was inoculated, and 100µL each

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of it was added to all test tubes, which were then incubated at 37°C for 24 hours in a laboratory shaker at 100 rpm. 1 mL of bacterial solutions was taken from all the test tubes and its serial dilutions were made using sterilized deionised water. Dilutions of concentrations 10−3, 10−4,

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10−5 and 10−6 were used for colony counting. Then, 100 μL suspension of the last dilution was spread on to an agar plate and all such plates were incubated at 37°C for 24 hours. Subsequently, the numbers of colony forming units were counted using a colony counter. The

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percentage reduction in number of colonies, representative of the antibacterial activity of samples made from bulk chitosan, cinnamaldehyde and chitosan nanoparticles was calculated

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using equation (i).

Antibacterial activity (%) =

A- B ´100 A

(i)

Where, A is the number of bacteria colonies (CFU/mL) of control sample and B is the number of bacteria colonies of the (corresponding) samples. To examine the minimum inhibitory concentration (MIC), and minimum bactericidal concentration (MBC), the procedure specified by Agnihotri et al. (S. Agnihotri, Mukherji, & 7

Mukherji, 2014) was followed. Optical density (OD) method was used to determine the MIC and MBC values of chitosan nanoparticles against bacteria. The batch assays were subjected to control, 0.01, 0.05 and 0.1 g nanoparticles in LB culture medium (10 mL) containing Grampositive (S aureus, 8.0×107 CFU/mL) and Gram-negative (E. coli, 8.64×107 CFU/mL) bacteria, separately. The batch assays were subjected to continuous shaking at 70 rpm and 37°C. Aliquots of the samples were withdrawn at specific time intervals and the value of OD at a wavelength of 600 nm was measured on a UV spectrophotometer (Shimadzu UV-2450). Bacterial regrowth curves were created by plotting OD values versus time. For assessing MIC or MBC values, all studies were done in triplicates.

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3. Results and discussion 3.1. Size, Morphology of chitosan nanoparticles

Fig. 2 shows the TEM and SEM micrographs of chitosan nanoparticles. TEM was used to characterize the morphology of the particles. Several spherical nanoparticles of size varying

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between 80 to 150 nm can be seen in the TEM image. In the SEM image also chitosan

to be smooth in both the images.

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nanoparticles of the same size range can be observed. The surface of the nanoparticles appears

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3.2. Crystallographic assay

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(b)

(a)

The X-ray diffraction (XRD) analysis is a useful tool in determining the structure and crystallization of any polymer. Crystallographic structures of bulk chitosan and chitosan

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nanoparticles were determined by their XRD patterns shown in Fig. 3 There is a strong peak in the diffractogram of bulk chitosan at 2θ at 21.8°, indicating high degree of crystalline

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morphology (Ali et al., 2010; Kumar, Dutta, & Dutta, 2009). However, no strong peak is found in the diffractogram of chitosan nanoparticles, which is characteristic of an amorphous structure. Chitosan nanoparticles are comprised of a dense network structure of interpenetrating polymer chains cross-linked to each other by cinnamaldehyde conclude the strong interaction occurred between chitosan and aldehyde (Sashikala & Syed Shafi, 2014).

3.3 Fourier transform infrared (FTIR) analysis

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FTIR studies of chitosan, cinnamaldehyde and chitosan nanoparticles were performed to determine their chemical structure. As shown in Fig. 4, FTIR spectrum of bulk chitosan shows peak at around 3358 cm-1 representing N-H stretching vibration of the amide linkage present in its structure, and a broad range referring to the O-H bond vibration. Peak observed at 2863 cm-1 is attributed with the C-H stretching vibration sp2 carbon. Peak obtained around 1661 cm-1 shows presence of amide linkage in chitosan and peak at 1580 cm-1 is assigned to heterocyclic hexane ring. FTIR spectrum of cinnamaldehyde shows peak at 1670 cm-1 which belongs to C=O due to the presence of aldehyde group and another peak at 1616 cm-1 because of aromatic benzene ring in conjugation with alkene. The formulation of chitosan nanoparticles is based on

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Schiff reaction between the amino group of chitosan and aldehyde group of cinnamaldehyde. Hence similar to chitosan, its nanoparticles have broad range of peak with increased intensity which describes O-H bond vibration. Peaks observed at 2910 cm-1and 2863 cm-1 are due to the presence of sp3 and sp2 carbon in structure. Formation of nanoparticles leads to the formation of a new bond C=N in the structure, indicated by the peak at 1634 cm-1. Also, a peak similar to

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that of chitosan is observed at 1580 cm-1 which is attributed to heterocyclic hexane ring. Peak at around 1025 cm-1 is observed in both chitosan and its nanoparticles corresponding to the

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ether groups present in cyclic structure of chitosan.

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3.4 Nuclear magnetic resonance (NMR) analysis

The 13C NMR of the chitosan nanoparticle (Fig. 5) confirms the imine peak at around 154 ppm. The peak at 131.79 ppm clearly indicates the presence of CH-CHO in cinnamaldehyde.

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The carbons of monomer N-acetylglucosamine are observed between 50 and 110 ppm. The small peak at 174.19 ppm can be attributed to the carbonyl group (C=O) of the chitosan (Rieger & Schiffman, 2014; Silva D. J. B, Zuluaga F., 2015). Thus the findings reaffirm that chitosan

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nanoparticles are formed through imine bond where both the cinnamaldehyde and chitosan are

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getting reacted as depicted in the mechanism part of the reaction scheme. 3.5 Thermal properties Thermal properties of chitosan and chitosan nanoparticles were studied by DSC and TGA.

In DSC curves (shown in Fig. 6(a)), the first endothermic peak of chitosan polymer at 66– 130°C is attributed to the evaporation of absorbed and bound water. The similar trend is followed in case of chitosan nanoparticles as well. The peak maxima in case of chitosan nanoparticles is seen to be shifted towards a lower temperature due to easy moisture (entrapped inside the structure) evaporation from the more amorphous structure, as well as the availability 9

of less polar –NH2 group in nanoparticles after chemical cross-linking which could also have had the potential to hold enhanced moisture. TGA of bulk chitosan and developed nanoparticles are shown in Fig. 6(b). From 0°C to 300°C, about 6% weight loss is observed in bulk chitosan. This minimal reduction in weight may be attributed to the loss of adsorbed and bound water. At around 350°C, there is sudden loss of 50% weight of bulk chitosan sample, beyond which, a linear decrease in weight is observed. This is due to breaking of molecular structure of test sample during continuous rise in temperature. On the contrary, in case of chitosan nanoparticles, there is zero or very little weight loss up to 250°C. After this, significant degradation of test sample continues to occur

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with increase in temperature till 600°C. The residual weight in the case of chitosan nanoparticles was higher than in bulk form, which implies that former is more stable state than latter.

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3.6 Antimicrobial activity assessment

The antibacterial efficacy of bulk chitosan, cinnamaldehyde and prepared nanoparticles

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against S. aureus is observed to be 65%, 51% and 98%, respectively, and 62%, 54% and 96%, respectively, against E. coli bacteria (Fig. 7). Chitosan shows better inhibitory activity towards both Gram-positive and Gram-negative bacteria, synergistically in nanoparticles form than in

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bulk form owing to enhanced surface area with higher number of unreacted available free amino groups to interact with the bacterial cell. The adhesion of chitosan nanoparticles to the

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surface of a bacteria is also significantly higher in case of nanoparticulate form which in turns alters its membrane properties and ultimately causes the death (Gadkari et al., 2017; Li et al., 2008). Moreover, size of chitosan nanoparticles prepared by Schiff reaction has immense effect

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on the antibacterial efficacy due to different chemistry of the formed nanoparticles which are obtained by varying the process conditions as mentioned in supplementary document (S1,

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Table S1).

The value of MIC represents the lowest concentration of chitosan nanoparticles required to

inhibit bacterial growth; and MBC value represents the lowest concentration of chitosan nanoparticles that kills 99.9% of the bacteria. The value of MBC was determined from the batch culture studies done for assessing MIC values. The growth rate of S. aureus and E. coli bacteria treated with various concentrations of chitosan nanoparticles is shown in Fig. 8.

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The results show that for all the bacterial stains, introduction of chitosan nanoparticles affected the growth kinetics unlike the control samples (culture grown in absence of chitosan nanoparticles). Moreover, bacterial growth reduces with increase in concentration of nanoparticles. In case of the S. aureus, introduction of 1 mg mL−1 causes ~47% reduction in bacterial density after 24 hours, as compared to control sample. On further increasing the concentration of chitosan nanoparticles to 5 and 10 mg mL−1, absence of bacterial growth is observed as these concentrations correspond to MIC and MBC values, respectively. Similarly, Gram-negative E. coli caused ~46%, ~94% and 100% reduction in cell density after 24 hours, for chitosan nanoparticles having concentration of 1, 5 and 10 mg mL−1, respectively. The

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reduction of bacterial load (percentage antibacterial activity) of bulk chitosan, cinnamaldehyde and chitosan nanoparticles along with MIC and MBC values of the nanoparticles are compared in a tabular form in supplementary document (S1, Table S2). 4. Conclusion

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A unique green method to produce antibacterial nanoparticles has been successfully demonstrated where two biomolecules have been chemically cross-linked to engineer stable

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structure. Herein, chitosan nanoparticles with size ranging between 80-150 nm have been synthesized using cinnamaldehyde as a cross-linker. The formation of these nanoparticles was

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based on the Schiff reaction. Chemical cross-linking of chitosan with cinnamaldehyde was confirmed by FTIR and NMR. Amorphous characteristic of chitosan nanoparticles as revealed by X-ray diffraction pattern also indirectly reflects the cross-linking reaction of both the

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molecules. The antimicrobial activity of chitosan was found to considerably enhance in its nanoparticle form because of enhanced surface area with higher number of available free amino groups to interact with the bacterial cell surface due to nanosize. Thus, this method of synthesis

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of nanoparticles is a total green solution and thereby holds enormous applications in the areas of life sciences and biomedicine. For example, the chitosan nanoparticles, thus synthesized,

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may be coated onto textile substrates to be used as wound dressing materials and other various applications under the category of medical textiles. They may also be used for the development of sustainable antimicrobial fabrics for applications like home textiles, sports wears, filtration and automotive sector, etc. Acknowledgements The authors are grateful to the Department of Science and Technology (DST), Govt. of India for funding this research work under ‘Water Research Initiative’ Scheme (Sanction letter 11

number: DST/TM/WTI/2K15/01G). The authors are also thankful to Central Research Facilities (CRF) and Nanoscale Research Facility of IIT Delhi for providing help to execute various characterizations (SEM, XRD, and FTIR). References

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Fig. 1. Structure of cinnamaldehyde

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Fig. 2. (a) TEM and (b) SEM images of chitosan nanoparticles

Fig. 3. XRD patterns of bulk chitosan and chitosan nanoparticles

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Fig. 4. FTIR spectra of chitosan, cinnamaldehyde and chitosan nanoparticles

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Fig. 5. Solid-state 13C CP/MAS NMR spectrum of chitosan nanoparticles

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Fig. 6. (a) DSC and (b) TGA thermograms of bulk chitosan and chitosan nanoparticles

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Inhibition %

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Bulk Chitosan

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Fig. 7. Antibacterial activity of chitosan, cinnamaldehyde and chitosan nanoparticles

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Fig.8 OD regrowth curves of (a) S. aureus and (b) E. coli, in LB broth

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