- Email: [email protected]
Anti quorum sensing and anti biofilm efficacy of cinnamaldehyde encapsulated chitosan nanoparticles against Pseudomonas aeruginosa PAO1
Accepted Manuscript Anti quorum sensing and anti biofilm efficacy of cinnamaldehyde encapsulated chitosan nanoparticles against Pseudomonas aeruginosa PAO1 Pattnaik Subhaswaraj, Subhashree Barik, Chandrasekhar Macha, Potu Venkata Chiranjeevi, Busi Siddhardha PII:
S0023-6438(18)30663-7
DOI:
10.1016/j.lwt.2018.08.011
Reference:
YFSTL 7323
To appear in:
LWT - Food Science and Technology
Received Date: 11 March 2018 Revised Date:
26 June 2018
Accepted Date: 3 August 2018
Please cite this article as: Subhaswaraj, P., Barik, S., Macha, C., Chiranjeevi, P.V., Siddhardha, B., Anti quorum sensing and anti biofilm efficacy of cinnamaldehyde encapsulated chitosan nanoparticles against Pseudomonas aeruginosa PAO1, LWT - Food Science and Technology (2018), doi: 10.1016/ j.lwt.2018.08.011. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Anti quorum sensing and anti biofilm efficacy of cinnamaldehyde encapsulated chitosan
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nanoparticles against Pseudomonas aeruginosa PAO1
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Pattnaik Subhaswaraj1, Subhashree Barik1, Chandrasekhar Macha2, Potu Venkata Chiranjeevi2
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and Busi Siddhardha1*
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014, India
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Telangana, India
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Department of Microbiology, School of Life Sciences, Pondicherry University, Puducherry-605
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Centre for Biotechnology, Jawaharlal Nehru Technological University, Hyderabad-500 085,
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*Corresponding author
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Dr. Busi Siddhardha,
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Department of Microbiology
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School of Life Sciences
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Pondicherry University
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Puducherry – 605014, India.
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E-mail: [email protected]
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Contact no: +91 9597761788
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ABSTRACT
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The encapsulation of cinnamaldehyde into biodegradable polymeric systems represents a
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possible strategy to overcome the bioavailability issues and enhance the sustained release of the
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drug at the target sites. In the present study, the efficacy of cinnamaldehyde encapsulated
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chitosan nanoparticles (CANPs) in attenuating the quorum sensing (QS) regulated virulence of
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P. aeruginosa PAO1 was investigated. CANPs were synthesized by ionic gelation method,
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characterized by dynamic light scattering (DLS), and transmission electron microscopic (TEM)
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analysis. The DLS and TEM analysis confirmed the synthesis of CANPs with a mean diameter
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of 208.12 nm. The encapsulation efficiency was observed to be 65.04±3.14% and in vitro release
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study confirmed the slow and sustained release of cinnamaldehyde. CANPs showed significant
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anti quorum sensing activity by down regulating the QS regulated virulence factors and
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associated biofilm formation as evidenced from microscopic observation. CANPs also
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significantly altered the swimming and swarming motility of P. aeruginosa PAO1. The present
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results suggested the greater prospective of the application of CANPs as potential anti quorum
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sensing agents as compared to native cinnamaldehyde and suggested new avenues for
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development of novel anti-infective agents in the post antibiotic era.
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Keywords: Cinnamaldehyde, Biofilm, Nanoparticles, Quorum sensing, Rhamnolipids
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1. Introduction In majority of pathogenic microorganisms, production of several bacterial phenotypes such
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as virulence factors, toxins and biofilms are generally controlled by a cascade of cell-to-cell
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communication termed as quorum sensing (QS). Pseudomonas aeruginosa is an opportunistic
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pathogen causing majority of the hospital-acquired infections in immunocompromised patients
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through well defined QS hierarchy of Las, Rhl and PQS system. All the three QS systems
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correlate with each other and have profound impact on the expression of virulence genes, biofilm
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formation and resistance to conventional antibiotics (Chang et al., 2014). In this context, the
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paradigm of developing novel antimicrobials has been shifted towards alternative approaches
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targeting bacterial virulence rather than bacterial killing (Maisuria, de Los Santos, Tufenkji, &
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Deziel, 2016). Though, the development of anti-infectives sounds interesting; use of synthetic
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and semi-synthetic drugs targeting bacterial virulence is still limited to laboratory and pre-
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clinical studies. In this context, natural bioactive compounds with immense therapeutic
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properties serve as the potential alternatives for the development of novel anti-infective agents
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(Alasil, Omar, Ismail, & Yusof, 2015; Bacha et al., 2016).
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Cinnamon is an important dietary phytoconstituent with significant antimicrobial
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properties and showed immense therapeutic properties (Kalia et al., 2015). Cinnamaldehyde (3-
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phenyl-2-propenal) is one of the primary phytoconstituents of cinnamon giving a characteristic
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flavour and odour to cinnamon. Cinnamaldehyde has been used as food flavouring agent and
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food additives in food processing industries (Camacho et al., 2015). Additionally, therapeutic
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potential of cinnamaldehyde as antimicrobial, antibiofilm, antioxidant and anti-hyperglycemic
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agent has also been reported (Gomes, Moreira, & Castell-Perez, 2011; Nostro et al., 2012; Rieger
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& Schiffman, 2014; Camacho et al., 2015). However, high volatility and poor water solubility
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limit its widespread biomedical applications. In this regard, considerable attentions have been
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made towards development of novel drug delivery systems for sustained delivery of
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cinnamdehyde (Tian, Lei, Zhang, & Li, 2015). In the quest for proper drug delivery system, nanotechnology provides an impregnable
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and multidimensional platform for antimicrobial therapy. Due to the potential applications in
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agriculture and biomedicine; the use of nanoparticles are gaining considerable interest for
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combating bacterial infections and associated biofilm formation (Mu et al., 2016). Recently, the
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anti-biofilm potential of gold nanoparticles surface conjugated with cinnamaldehyde was
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investigated against P. aeruginosa and Escherichia coli O157:H7, Staphylococcus aureus and
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methicillin resistant S. aureus (MRSA) (Ramasamy, Lee, & Lee, 2017). The slow and sustained
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release of cinnamaldehyde from Poly (DL-lactide-co-glycolide) (PLGA) nanoparticles efficiently
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altered the inhibition of Salmonella spp. and Listeria spp. suggesting its antibacterial efficacy
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(Gomes et al., 2011). Besides, the antimicrobial efficacy of cinnamaldehyde encapsulated β-
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cyclodextrin and polylactic acid based composite against E. coli and S. aureus was also
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successfully established (Liu, Liang, Zhang, Lan, & Qin, 2017). Among the nanomaterials used
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for targeted drug delivery, the biomedical potential of chitosan based nanocomposites has several
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advantages such as greater efficiency, operational simplicity, ecological feasibility, cost
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effectiveness, increased absorptibility, and most importantly slow and sustained release of the
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embedded drugs at the target sites (Ilk, Saglam, Ozgen, & Korkusuz, 2017).
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In the present study, for the first time the efficacy of cinnamaldehyde encapsulated
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chitosan nanoparticles in down regulating the quorum sensing associated virulence and biofilm
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formation in P. aeruginosa PAO1 was determined.
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2. Materials and methods
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2.1. Chemicals and reagents Chitosan, low molecular weight (>75% deacetylation) and pentasodium tripolyphosphate
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(TPP) were purchased from Sigma-Aldrich, USA. Trans-cinnamaldehyde (98%) was obtained
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from Alfa-Aesar, England. All the chemicals were of analytical grade and used without any
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modifications.
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2.2. Synthesis of cinnamaldehyde-encapsulated chitosan-TPP nanoparticles (CANPs)
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CANPs were synthesized by ionic gelation method with slight modifications (Rejinold et
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al., 2011). Cinnamaldehyde (0.1 ml, 0.5 v/v %) was dropped gradually into preheated chitosan
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solution (0.25% w/v) under magnetic stirring and to the mixture, TPP solution (0.25% w/v) was
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injected drop wise under magnetic stirring at 500 × g for 30 min at ambient temperature and pH
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to yield CANPs (Reiger & Schiffman, 2014). The suspension of nanoparticles was then
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centrifuged at 12,000 × g for 60 min and washed with deionized water (3 times) to remove the
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unloaded cinnamaldehyde.
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2.3. Physicochemical characterization of synthesized CANPs
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2.4.1. UV-Visible spectroscopic analysis of CANPs
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The UV-Vis absorption spectra of the reaction mixture were recorded in the range of 200-
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800 nm using UV-Vis spectrophotometer (Cary 60 Agilent technology) to monitor the synthesis
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of CANPs (Lungu et al. 2014).
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2.4.2. Size distribution of CANPs
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The DLS was used to evaluate the size of the synthesized CANPs using Particle size
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analyzer (Malvern-Nano Series) at 25 °C with scattering angle of 90°. The particle size was
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described as the mean diameter and Z-average value (Jo et al., 2015).
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2.4.3. TEM analysis 5
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The size and morphology of synthesized CANPs were analyzed using high resolution
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transmission electron microscope (HR-TEM) (Jeol/JEM 2100) at an accelerating voltage of 200
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kV (Xing et al., 2016).
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2.4.4. Fourier transforma Infra red (FT-IR) spectroscopic analysis
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FT-IR spectra of CANPs were recorded on Fourier transformation infra red
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spectrophotometer (Thermo Nicolet Model: 6700) using potassium bromide (KBr). Briefly, the
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samples were pelleted with KBr and subjected for FT-IR analysis. The spectra were recorded at
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the range of 500-4500 cm-1 at a resolution of 1 cm-1 (Rejinold et al., 2011).
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2.5. Determination of encapsulation efficiency (EE)
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The encapsulation efficiency of CANPs was determined to validate the amount of
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cinnmaldehyde loaded into the chitosan nanoparticles using UV-Vis spectrophotometer. The
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supernatant was collected after centrifugation and non-encapsulated cinnamaldehyde was
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quantified spectrophotometrically at 285 nm using cinnamaldehyde standard curve (Panwar,
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Pemmaraju, Sharma, & Pruthi, 2016). The EE (%) was calculated by using the following
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equation:
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EE (%) = (Total cinnamaldehyde loaded – non encapsulated cinnamaldehyde)/Total
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cinnamaldehyde loaded × 100
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2.6. In vitro release studies of CANPs
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The in vitro release of cinnmaldehyde from the encapsulated CANPs was studied to evaluate
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the controlled release profile of cinnamaldehyde. The lyophilized CANPs were suspended in
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PBS (0.15 M, pH 7.4) at a concentration of 1 mg/mL and kept on a rotor incubator at 100 × g and
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37 °C. At predetermined time intervals (4 h) up to 24 h, the sample solution was withdrawn and
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centrifuged at 5000 × g for 10 min to separate released cinnamaldehyde. The supernatant was 6
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analyzed by UV-Vis spectrophotometry at 285 nm to quantify the released cinnamaldehyde
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content (Ilk et al., 2017). The percentage of cinnamaldehyde released was determined from the
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following equation,
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Release (%) = Released cinnamaldehyde from CANPs/total amount of cinnamaldehyde in
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CANPs × 100
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2.7. Anti quorum sensing activity of CANPs against P. aeruginosa PAO1
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2.7.1. Determination of Minimum Inhibitory Concentration (MIC)
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The MIC for the native cinnamaldehyde and CANPs were determined as per the
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guidelines of Clinical and Laboratory Standards Institute (Luo et al., 2016). Briefly, overnight
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grown P. aeruginosa PAO1 was added to LB medium supplemented with different
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concentrations of cinnamaldehyde and CANPs and incubated at 37 °C for 24 h.
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2.7.2. Pyocyanin inhibition assay
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The effect on pyocyanin production by P. aeruginosa PAO1 on treatment with sub-MIC
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concentrations of cinnamaldehyde and CANPs was measured by the method described by Luo et
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al. (2016) with slight modifications. Briefly, overnight P. aeruginosa PAO1 with
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cinnamaldehyde and CANPs were kept for incubation for 16-18 h. After the incubation, the
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culture was pelleted and the cell free supernatant containing the pyocyanin was extracted with
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chloroform. The organic layer was re-extracted with 0.2 M HCl. The absorbance of the
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supernatant was measured using a microplate reader at 520 nm.
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2.7.3. Growth curve analysis
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Overnight culture of P. aeruginosa PAO1 was inoculated into freshly prepared LB broth
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supplemented with 500 µg/mL of cinnamaldehyde and CANPs and incubated at 37 °C under 100
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× g in a rotatory shaker. The cell density was measured in UV–Vis spectrophotometer at every 7
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two hour interval up to 24 h to evaluate the effect of cinnamaldehyde and CANPs on bacterial
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growth (Cady et al., 2012).
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2.7.4. LasA Staphylolytic activity Briefly, overnight S. aureus culture was boiled for 10 min and centrifuged at 8000 × g for
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10 min. The pellet was resuspended in 10mM Na2PO4 (pH 4.5) to an OD600 of 0.5. Overnight P.
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aeruginosa PAO1 on treatment with cinnamaldehyde and CANPs was centrifuged at 8000 × g
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for 10 minutes. A 100 µl aliquot of P. aeruginosa test supernatant was added to 900 µl of the S.
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aureus suspension. Optical density of the mixture (OD600) was measured and the activity was
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determined as the change in OD600 per hour per µg of protein and percentage of inhibition was
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calculated as compared to control (Alasil et al., 2015).
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2.7.5. Anti motility assay
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For swimming and swarming assay, overnight P. aeruginosa PAO1 was point inoculated at
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the centre of the swimming medium (1% tryptone, 0.5% NaCl and 0.3% agar) and swarming
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medium (1% peptone, 0.5% NaCl, 0.5% agar and 0.5% of sterilized D-glucose) with sub-MIC
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concentrations (500 µg/mL) of cinnamaldehyde and CANPs. The plates were incubated at 37 °C
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for 16 h (Packiavathy, Agilandeswari, Musthafa, Pandian, & Ravi, 2012).
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2.8. Anti biofilm efficacy of CANPs against P. aeruginosa PAO1 biofilm
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2.8.1. Biofilm formation assay (microtitre plate method)
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Briefly, cinnamaldehyde and CANPs treated P. aeruginosa PAO1 culture were incubated
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overnight in a microtitre plate. After 24 h incubation, biofilms formed by bacterial culture was
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stained with crystal violet (0.1% w/v) for 20 min. After washing with water, the liquid was
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discarded from the wells and the material that remained fixed to the polystyrene was washed
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three times with PBS. Crystal violet bound biofilm was dissolved with 95% ethanol, incubated at
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37 °C for 30 min and the absorbance was measured using a microtitre plate reader at 595 nm
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(Zhang et al., 2014).
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2.8.2. Exopolysaccharides inhibition assay The role of cinnamaldehyde and CANPs on the production of exopolysaccharides (EPS)
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by P. aeruginosa PAO1 was determined according to the method described by Rasamiravaka et
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al. (2015). Briefly, P. aeruginosa PAO1 was incubated for 16 h on treatment with sub-MIC
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concentration of cinnamaldehyde and CANPs. After incubation, the culture was centrifuged at
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10,000 × g for 15 min. The pellets were resuspended in high salt buffer and recentrifuged. To the
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supernatant, 95% ethanol was added incubated at 4 °C. After incubation, the suspension was
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recentrifuged and the precipitate was resuspended in deionized water. To the suspension, equal
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volume of 5% phenol and H2SO4. The absorbance of the mixture was measured at 490 nm.
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2.8.3. Rhamnolipids inhibition assay
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For rhamnolipids extraction, P. aeruginosa PAO1 was grown at 37 °C with agitation at
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100 × g for 16 h in LB medium supplemented with cinnamaldehyde and CANPs. Bacterial
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culture was centrifuged (10,000 × g, 5 min) and cell-free supernatant was mixed with equal
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volume of ethyl acetate and vigorously vortexed. After phase separation, the upper rhamnolipid-
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containing phase was collected and quantified by a methylene-blue based method described by
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Rasamiravaka et al. (2015).
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2.8.4. Cell surface hydrophobicity assay
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Briefly, P. aeruginosa PAO1 culture on treatment with cinnamaldehyde and CANPs was
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grown in LB medium, centrifuged at 8000 × g for 2 min and the cell pellet was washed and
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resuspended in buffer (2.22 g of potassium phosphate trihydrate, 7.26 g of monobasic potassium
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phosphate, 1.8 g of urea, and 0.2 g of magnesium sulphate heptahydrate/litre (pH 7.1)). From the
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cell suspension, 100 µL were transferred to 96 wellplate to determine the initial cell density in
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the aqueous phase. Four hundred microlitres of hexadecane were added to the 900 µL of
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remaining buffer suspended cells and vortexed for 5 min. The aqueous phase was then
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transferred and the OD600 was measured to determine the cell density that remained in the
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aqueous phase (Garcia-Lara et al., 2015). The percentage of cell surface hydrophobicity was
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determined using the formula, Cell surface hydrophobicity (%) = (Initial cell density-final cell
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density)/initial cell density ×100
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2.8.5. Microscopic analysis of P. aeruginosa PAO1 biofilms
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The effect of cinnamaldehyde and CANPs on the biofilm forming ability of P.
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aeruginosa PAO1 was examined by light microscopic and CLSM analysis. For light microscopic
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analysis, biofilms were grown on glass slides submerged with tryptone soy broth (TSB) in 24
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well microtitre plate and incubated at 37 °C for 24 h. After incubation, the glass slides were
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washed with carbinol and stained with 0.1% crystal violet and observed under microscope at
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40X magnification (Sivaranjani et al., 2016). For CLSM analysis, overnight culture of P.
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aeruginosa PAO1 inoculated into freshly prepared LB broth supplemented with sub-MIC
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concentration of cinnamaldehyde and CANPs into a 12 well microtiter plate and coverslips were
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dipped into it and incubated. After 24 h of incubation, coverslips were transferred onto the glass
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slides and stained with acridine orange (0.1%) and kept for 3-5 minutes in dark and observed at
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20X magnification (Ouyang et al., 2016).
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2.9. Statistical analysis
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All the experiments were conducted in triplicate (n=3). All the results were presented as
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mean ± standard deviation (SD). One-way analysis of variance (ANOVA) followed by the
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Tukey–Kramer multiple comparison test (Q test) was used to determine significant differences at
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varied level of significance as compared to control and native cinnamaldehyde. For statistical
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analysis, P <0.05 was considered significant (Rejinold et al., 2011).
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3. Results and discussion
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3.1. Synthesis of CANPs
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Cinnamaldehyde was encapsulated on chitosan biopolymer where TPP was used as a
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cross linker, in order to synthesize CANPs. The CANPs were successfully formed by the
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electrostatic interaction between deacetylated chitosan (which have positively charged -NH2
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group) and TPP (having negatively charged phosphate group). The particle obtained were
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lyophilized and used for further characterization and biological activities.
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3.2. Physico-chemical characterization of synthesized CANPs
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3.2.1. UV-Visible spectroscopic analysis of CANPs
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UV-Vis spectroscopic analysis showed the formation of CANPs with a characteristic change in
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the SPR pattern as compared to native cinnamaldehyde and chitosan.
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3.2.2. Dynamic light scattering (DLS) analysis
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The DLS analysis confirmed the formation and size distribution of CANPs. The size of
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CANPs ranged from 28.21-342.0 nm with a mean particle size of 208.12 nm analyzed by DLS
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(Fig. 1 a). The CANPs dispersion showed a poly dispersity index (PDI) of 0.277.
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3.2.3. TEM analysis
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The TEM micrograph presented in Fig. 1 (b) showed predominantly anisotropic CANPs
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with approximate size of ~ 28.21 nm and the average size of the CANPs was 208.12 nm. The
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size of the CANPs observed in the present study was in agreement with the previous result
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(Aydin & Pulat, 2012).
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3.2.4. FTIR analysis
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From the FT-IR analysis, the presence of similar kind of functional group in CANPs in
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comparison to native cinnamaldehyde and chitosan signifies that cinnamaldehyde was
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successfully encapsulated into chitosan carrier where TPP acts as crossslinker. The FT-IR
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spectra of chitosan showed the peaks at 1071.4, 1379.5, 1650.0, and 2874.6 which correspond to
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C-N stretch aliphatic amine, C-H bend alkane, primary amine, and C-H stretch alkane
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respectively (Fig. 2). The successful loading of cinnamaldehyde to chitosan carrier was identified
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by the introduction of a new peak i.e. at 1686.3 which corresponds to aldehyde (C=O) stretch
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which was present in both cinnamaldehyde as well as CANPs. In addition, another peak was
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observed at 3026.9 corresponding to the aromatic C-H stretch. Hence, the spectrum provided
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insights about the role of various functional groups of cinnamaldehyde in reduction and
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stabilization of CANPs. Similar shift in the FT-IR spectrum was reported earlier for kaemferol
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loaded chitosan nanoparticles (Ilk et al., 2017).
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3.3. Encapsulation efficiency and in vitro drug release studies
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The encapsulation efficiency was measured with the help of using a standard curve of
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cinnamaldehyde. The EE (%) of cinnamaldehyde to chitosan carrier was observed to be 65.04 ±
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2.14%. From the size analysis study and observance of more than 65% encapsulation efficiency
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confirmed the successful synthesis and encapsulation of cinnamaldehyde into the chitosan-TPP
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carrier. The present results suggested higher efficacy of CANPs in terms of size and 12
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encapsulation efficiency than the earlier report (Loquercio, Castell-Perez, Gomes, & Moreira,
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2015). The in vitro drug release profile showed that 29% drug was released in the time interval
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of 12 h in the initial burst. After that approx. 60% drug was released in the next 12 h (Fig. 3).
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This result suggested a slow and sustained release of cinnamaldehyde from CANPs which was
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comparatively better than the earlier report showing 20–50% release of phytochemical in a span
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of 24 h using PLGA nanoparticles (Silva, Hill, Figueiredo, & Gomes, 2014).
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3.4. Anti quorum sensing activity of CANPs
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3.4.1. Determination of MIC and sub-MIC
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The MIC of cinnamaldehyde and CANPs against P. aeruginosa PAO1 was found to be
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1000 µg/mL where the growth and cell density were significantly inhibited as evident from the
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absence of turbidity. Hence, sub-MIC concentrations of 250 and 500 µg/mL were used for
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further activities.
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3.4.2. Pyocyanin inhibition assay
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Pyocyanin is an important virulence factor produced by P. aeruginosa PAO1 and is
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regulated by Rhl QS system. Pyocyanin plays a critical role in altering the host immune system
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by virtue of generating reactive oxygen species (ROS) and it has profound role in development
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of acute and persistent respiratory infections (Hall et al., 2016). In the present study, pyocyanin
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production was significantly decreased with the increasing sub-MIC concentrations of both
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cinnamaldehyde and CANPs (Fig. 4). In the present study, the production of pyocyanin was
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significantly inhibited by 93.24% on treatment with sub-MIC concentration of CANPs whereas
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in case of native cinnamaldehyde, 72.96% of inhibition in pyocyanin production was observed
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which was significantly higher than the earlier report (Luo et al., 2016).
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3.4.3. Growth curve analysis From the growth curve assay, sub-MIC concentration (500 µg/mL) of cinnamaldehyde
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and CANPs treated P. aeruginosa PAO1 exhibited normal growth like that of the untreated
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control but with slow growth rate as compared to control (Fig. 5).
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3.4.4. LasA Staphylolytic activity
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The QS gene expression in P. aeruginosa has interconnection with other signalling
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regulatory systems which responds to various environmental signals. LasA protease is under the
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control of the lasI-lasR system (Alasil et al., 2015). This protease has inhibitory effect on the
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growth of S. aureus, which was significantly reduced on treatment with sub-MIC concentrations
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of both cinnamaldehyde and CANPs. A significant decrease in LasA Staphylolytic activity was
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observed as compared to that of the control when P. aeruginosa test supernatant was grown on
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treatment with sub-MIC concentrations of cinnamaldehyde and CANPs (Table 1).
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3.4.5. Anti motility assay
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The flagellar mediated swimming and pili mediated swarming motility of P. aeruginosa
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PAO1 play crucial role in biofilm formation, development and maturation (Packiavathy et al.,
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2012). The presence of sub-MIC concentration (500 µg/mL) of cinnamaldehyde and CANPs
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significantly affect the swimming and swarming motility of P. aeruginosa PAO1 suggesting its
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role in biofilm disruption which was in accordance to the earlier report (Fig. 6).
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3.5. Anti biofilm efficacy of CANPs
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3.5.1. Biofilm formation assay (MTP method)
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The sub-MIC concentrations of cinnamaldehyde and CANPs exhibited significant
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reduction in biofilm formation as compared to untreated control in a concentration dependent
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manner (Table 1). From the results, 84.06±4.09 and 71.25±3.81% of reduction in biofilm
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formation was observed on treatment with sub-MIC concentrations of cinnamaldehyde and
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CANPs respectively which was significantly higher than previous report with 60% to 90% of
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reduction (Kalia et al., 2015; Sivaranjani et al., 2016).
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3.5.2. EPS inhibition assay
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The production of EPS plays an important role in formation and development of biofilm.
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In the present study, the production of EPS on treatment with sub-MIC concentrations of
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cinnamaldehyde and CANPs was significantly altered with 65.36±3.51 and 80.28±4.05% of
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reduction in EPS production. Table 1 showed the concentration dependent inhibition of EPS
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production by P. aeruginosa PAO1 on treatment with sub-MIC concentrations of
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cinnamaldehyde and CANPs suggesting the efficacy of CANPs and cinnamaldehyde in
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inhibition of biofilm formation and development (Periasamy et al., 2015).
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3.5.3. Rhamnolipids inhibition assay
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A significant and concentration dependent decrease in the production of rhamnolipids
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was observed when P. aeruginosa PAO1 was treated with sub-MIC concentrations of
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cinnamaldehyde and CANPs (Table 1). The production of rhamnolipids by P. aeruginosa PAO1
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has a greater influence on biofilm formation and development. The result of CANPs was
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significantly higher than the earlier report. The potential role of CANPs in inhibiting the
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production of rhamnolipids could be attributed to its role in governing the QS regulated rhlAB
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operon encoding the enzymes responsible for rhamnolipids biosynthesis (Nickzad & Deziel,
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2015; Prateeksha et al., 2017).
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3.5.4. Cell surface hydrophobicity assay
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Cell surface hydrophobicity determines the ability of microorganisms to form biofilms
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and bypass the antibiotic therapy. In the present study, sub-MIC concentrations of
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cinnamaldehyde and CANPs significantly decreased the surface hydrophobicity of P. aeruginosa
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PAO1 in a dose dependent manner which was in accordance with the earlier report (Fig. 7)
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(Garcia-Lara et al., 2015).
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3.5.5. Microscopic observation of biofilms
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The light microscopic analysis of the biofilm formed by P. aeruginosa PAO1 showed
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visible clumping with complex morphology of the biofilm architecture which was significantly
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altered on treatment with sub-MIC concentration (500 µg/ml) of native cinnamaldehyde and
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CANPs suggesting its efficacy in attenuating the antibiotic resistant biofilm formation and
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development (Sivaranjani et al., 2016). CLSM analysis confirmed a significant reduction in the
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thickness of biofilm in the presence of cinnamaldehyde and CANPs as compared to the untreated
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control (Fig. 8). The results suggested the altered biofilm architecture with significant reduction
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in biofilm formation in the treated sample as compared to the clumped and complex biofilm
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architecture in the untreated control which was in accordance with the previous report (Ouyang
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et al., 2016).
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In the present study, for the first time, we demonstrated the QS inhibitory activity of
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CANPs against the QS dependent phenotypic virulence factors and biofilm formation in P.
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aeruginosa PAO1. From the results, the encapsulation of cinnamaldehyde into polymeric
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chitosan nanoparticles enhanced the anti quorum sensing and anti-biofilm potential as compared
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to its native form suggesting its widespread biomedical and pharmaceutical applications. In
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addition, the present study will also give new avenues to the use of CANPs in food processing
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industries for enhanced food preservation strategies against a variety of food-borne pathogens.
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4. Conclusion
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In the present study, as compared to native cinnamaldehyde, the QS attenuation efficacy
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of CANPs was found to be significantly higher suggesting the role of a biocompatible
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nanocarrier system for effective targeting of bacterial virulence with slow and controlled release
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of administered drugs at specific sites. The slow and sustained release of cinnamaldehyde from
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CANPs suggested its persistence to be used as effective quorum sensing inhibitor against other
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pathogens. Besides the biomedical applications, the biocompatible CANPs will also play critical
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role in the food processing industries in the near future.
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Conflict of interest
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The authors confirm that this article content has no potential conflict of interest.
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Acknowledgement
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The authors like to acknowledge Dr. G. Muralitharan, Department of Microbiology,
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Bharathidasan University, Tiruchi, India for providing CLSM facilities. The authors also duly
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acknowledge the Central Instrumentation Facilities (CIF), Pondicherry University for providing
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DLS and FT-IR analysis. The authors also like to thank Sophisticated Test and Instrumentation
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centre (STIC), Cochin, India for providing TEM analysis.
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Table 1. Effect of sub-MIC concentrations (250 and 500 µg/mL) of cinnamaldehyde and CANPs on LasA Staphylolytic activity and production of rhamnolipids, EPS and biofilm by P. aeruginosa PAO1. Quorum sensing inhibitory activity
Percentage of inhibition (%)
Cinnamadehyde
39.50±2.82
51.83±2.67*
55.55±4.06
61.16±4.20NS
39.41±3.66
53.09±2.95*
54.56±2.51
63.59±2.54*
52.07±1.48
63.46±2.76*
65.36±3.51
80.28±4.05*
56.33±4.40
76.08±5.57*
71.25±3.81
84.06±4.09*
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LasA Staphylolytic assay EPS inhibition assay Rhamnolipids inhibition assay Biofilm formation assay (MTP) LasA Staphylolytic assay EPS inhibition assay Rhamnolipids inhibition assay Biofilm formation assay (MTP)
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Concentration (µg/mL)
All the values were represented as Mean ± standard deviation (SD) of 3 replicates (n = 3). * Values are significantly different from native cinnamaldehyde at p<0.05 ** Values are significantly different from native cinnamaldehyde at p<0.01 *** Values are significantly different from native cinnamaldehyde at p<0.001 NS Values are not significantly different from native cinnamaldehyde
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Fig. 1. Determination of size, morphology and distribution of Cinnamaldehyde encapsulated chitosan-TPP nanoparticles (CANPs). (a) Dynamic light scattering (DLS) analysis of CANPs showing the mean diameter of nanoparticles (208.12 nm); (b) Transmission electron microscopic
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(TEM) image of CANPs showing spherical shape of nanoparticles with a diameter of 208.12 nm.
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Fig. 2. FT-IR spectra of (a) Cinnamaldehyde; (b) Chitosan and (c) Cinnamaldehyde encapsulated chitosan-TPP nanoparticles (CANPs), recorded in the range of 500 cm-1 to 4500 cm-1 at a resolution of 1 cm-1 showing the shift of identical absorption peaks corresponding to change in
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chemical groups during the encapsulation of cinnamaldehyde into chitosan nanoparticles.
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Fig. 3. In vitro release of cinnamaldehyde from CANPs over a period of 24 hours at every 4 hour
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interval showing the slow and sustained release of cinnamaldehyde.
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Fig. 4. The effect of sub-MIC concentrations (250, 500 µg/mL) of cinnamaldehyde and cinnamaldehyde encapsulated chitosan-TPP nanoparticles (CANPs) on pyocyanin production in
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P. aeruginosa PAO1 as compared to untreated control. All the values were represented as Mean ± standard deviation (SD) of 3 replicates (n = 3). The results were highly significant (p<0.05) as compared to native cinnamaldehyde.
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Fig. 5. Effect on the growth curve of P. aeruginosa PAO1 on supplementation of native
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cinnamaldehyde and CANPs (500 µg/mL) as compared to the untreated control.
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Fig. 6. Effect of sub-MIC concentration (500 µg/L of cinnamaldehyde and CANPs on P. aeruginosa PAO1 swarming and swimming motility as compared to untreated control. (a) Untreated control swarming plate; (b) Swarming motility of native cinnamaldehyde treated P.
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aeruginosa PAO1; (c) Swarming motility of CANPs treated P. aeruginosa PAO1; (d) Untreated
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control swimming plate; (e) Swimming motility of native cinnamaldehyde treated P. aeruginosa PAO1; (f) Swimming motility of CANPs treated P. aeruginosa PAO1.
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Fig. 7. The effect of sub-MIC concentrations (250 and 500 µg/mL) of cinnamaldehyde and
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CANPs on the bacterial hydrophobicity over the surface as compared to untreated control.
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Fig. 8. Microscopic analysis of P. aeruginosa PAO1 biofilm on treatment with sub-MIC concentration (500 µg/mL) of cinnamaldehyde and CANPs as compared to untreated control. (a) Light microscopic observation of biofilm of untreated control; (b) Light microscopic observation
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of biofilm treated with cinnamaldehyde; (c) Light microscopic observation of biofilm treated with CANPs; (d) CLSM image of biofilm of untreated control; (e) CLSM image of biofilm
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Highlights Ionic gelation method was used for synthesis of CANPs
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TEM analysis confirmed the synthesis of CANPs
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Anti quorum sensing potential of CANPs against P. aeruginosa PAO1 was evaluated
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Anti biofilm efficacy of CANPs was determined against P. aeruginosa PAO1
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CLSM analysis corroborated the biofilm inhibition potential of CANPs
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S0023-6438(18)30663-7
DOI:
10.1016/j.lwt.2018.08.011
Reference:
YFSTL 7323
To appear in:
LWT - Food Science and Technology
Received Date: 11 March 2018 Revised Date:
26 June 2018
Accepted Date: 3 August 2018
Please cite this article as: Subhaswaraj, P., Barik, S., Macha, C., Chiranjeevi, P.V., Siddhardha, B., Anti quorum sensing and anti biofilm efficacy of cinnamaldehyde encapsulated chitosan nanoparticles against Pseudomonas aeruginosa PAO1, LWT - Food Science and Technology (2018), doi: 10.1016/ j.lwt.2018.08.011. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Anti quorum sensing and anti biofilm efficacy of cinnamaldehyde encapsulated chitosan
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nanoparticles against Pseudomonas aeruginosa PAO1
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Pattnaik Subhaswaraj1, Subhashree Barik1, Chandrasekhar Macha2, Potu Venkata Chiranjeevi2
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and Busi Siddhardha1*
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1
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014, India
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2
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Telangana, India
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Department of Microbiology, School of Life Sciences, Pondicherry University, Puducherry-605
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Centre for Biotechnology, Jawaharlal Nehru Technological University, Hyderabad-500 085,
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*Corresponding author
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Dr. Busi Siddhardha,
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Department of Microbiology
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School of Life Sciences
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Pondicherry University
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Puducherry – 605014, India.
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E-mail: [email protected]
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Contact no: +91 9597761788
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ABSTRACT
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The encapsulation of cinnamaldehyde into biodegradable polymeric systems represents a
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possible strategy to overcome the bioavailability issues and enhance the sustained release of the
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drug at the target sites. In the present study, the efficacy of cinnamaldehyde encapsulated
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chitosan nanoparticles (CANPs) in attenuating the quorum sensing (QS) regulated virulence of
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P. aeruginosa PAO1 was investigated. CANPs were synthesized by ionic gelation method,
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characterized by dynamic light scattering (DLS), and transmission electron microscopic (TEM)
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analysis. The DLS and TEM analysis confirmed the synthesis of CANPs with a mean diameter
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of 208.12 nm. The encapsulation efficiency was observed to be 65.04±3.14% and in vitro release
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study confirmed the slow and sustained release of cinnamaldehyde. CANPs showed significant
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anti quorum sensing activity by down regulating the QS regulated virulence factors and
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associated biofilm formation as evidenced from microscopic observation. CANPs also
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significantly altered the swimming and swarming motility of P. aeruginosa PAO1. The present
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results suggested the greater prospective of the application of CANPs as potential anti quorum
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sensing agents as compared to native cinnamaldehyde and suggested new avenues for
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development of novel anti-infective agents in the post antibiotic era.
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Keywords: Cinnamaldehyde, Biofilm, Nanoparticles, Quorum sensing, Rhamnolipids
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1. Introduction In majority of pathogenic microorganisms, production of several bacterial phenotypes such
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as virulence factors, toxins and biofilms are generally controlled by a cascade of cell-to-cell
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communication termed as quorum sensing (QS). Pseudomonas aeruginosa is an opportunistic
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pathogen causing majority of the hospital-acquired infections in immunocompromised patients
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through well defined QS hierarchy of Las, Rhl and PQS system. All the three QS systems
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correlate with each other and have profound impact on the expression of virulence genes, biofilm
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formation and resistance to conventional antibiotics (Chang et al., 2014). In this context, the
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paradigm of developing novel antimicrobials has been shifted towards alternative approaches
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targeting bacterial virulence rather than bacterial killing (Maisuria, de Los Santos, Tufenkji, &
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Deziel, 2016). Though, the development of anti-infectives sounds interesting; use of synthetic
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and semi-synthetic drugs targeting bacterial virulence is still limited to laboratory and pre-
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clinical studies. In this context, natural bioactive compounds with immense therapeutic
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properties serve as the potential alternatives for the development of novel anti-infective agents
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(Alasil, Omar, Ismail, & Yusof, 2015; Bacha et al., 2016).
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Cinnamon is an important dietary phytoconstituent with significant antimicrobial
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properties and showed immense therapeutic properties (Kalia et al., 2015). Cinnamaldehyde (3-
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phenyl-2-propenal) is one of the primary phytoconstituents of cinnamon giving a characteristic
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flavour and odour to cinnamon. Cinnamaldehyde has been used as food flavouring agent and
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food additives in food processing industries (Camacho et al., 2015). Additionally, therapeutic
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potential of cinnamaldehyde as antimicrobial, antibiofilm, antioxidant and anti-hyperglycemic
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agent has also been reported (Gomes, Moreira, & Castell-Perez, 2011; Nostro et al., 2012; Rieger
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& Schiffman, 2014; Camacho et al., 2015). However, high volatility and poor water solubility
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limit its widespread biomedical applications. In this regard, considerable attentions have been
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made towards development of novel drug delivery systems for sustained delivery of
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cinnamdehyde (Tian, Lei, Zhang, & Li, 2015). In the quest for proper drug delivery system, nanotechnology provides an impregnable
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and multidimensional platform for antimicrobial therapy. Due to the potential applications in
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agriculture and biomedicine; the use of nanoparticles are gaining considerable interest for
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combating bacterial infections and associated biofilm formation (Mu et al., 2016). Recently, the
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anti-biofilm potential of gold nanoparticles surface conjugated with cinnamaldehyde was
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investigated against P. aeruginosa and Escherichia coli O157:H7, Staphylococcus aureus and
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methicillin resistant S. aureus (MRSA) (Ramasamy, Lee, & Lee, 2017). The slow and sustained
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release of cinnamaldehyde from Poly (DL-lactide-co-glycolide) (PLGA) nanoparticles efficiently
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altered the inhibition of Salmonella spp. and Listeria spp. suggesting its antibacterial efficacy
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(Gomes et al., 2011). Besides, the antimicrobial efficacy of cinnamaldehyde encapsulated β-
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cyclodextrin and polylactic acid based composite against E. coli and S. aureus was also
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successfully established (Liu, Liang, Zhang, Lan, & Qin, 2017). Among the nanomaterials used
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for targeted drug delivery, the biomedical potential of chitosan based nanocomposites has several
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advantages such as greater efficiency, operational simplicity, ecological feasibility, cost
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effectiveness, increased absorptibility, and most importantly slow and sustained release of the
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embedded drugs at the target sites (Ilk, Saglam, Ozgen, & Korkusuz, 2017).
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In the present study, for the first time the efficacy of cinnamaldehyde encapsulated
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chitosan nanoparticles in down regulating the quorum sensing associated virulence and biofilm
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formation in P. aeruginosa PAO1 was determined.
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2. Materials and methods
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2.1. Chemicals and reagents Chitosan, low molecular weight (>75% deacetylation) and pentasodium tripolyphosphate
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(TPP) were purchased from Sigma-Aldrich, USA. Trans-cinnamaldehyde (98%) was obtained
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from Alfa-Aesar, England. All the chemicals were of analytical grade and used without any
93
modifications.
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2.2. Synthesis of cinnamaldehyde-encapsulated chitosan-TPP nanoparticles (CANPs)
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CANPs were synthesized by ionic gelation method with slight modifications (Rejinold et
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al., 2011). Cinnamaldehyde (0.1 ml, 0.5 v/v %) was dropped gradually into preheated chitosan
97
solution (0.25% w/v) under magnetic stirring and to the mixture, TPP solution (0.25% w/v) was
98
injected drop wise under magnetic stirring at 500 × g for 30 min at ambient temperature and pH
99
to yield CANPs (Reiger & Schiffman, 2014). The suspension of nanoparticles was then
100
centrifuged at 12,000 × g for 60 min and washed with deionized water (3 times) to remove the
101
unloaded cinnamaldehyde.
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2.3. Physicochemical characterization of synthesized CANPs
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2.4.1. UV-Visible spectroscopic analysis of CANPs
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The UV-Vis absorption spectra of the reaction mixture were recorded in the range of 200-
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800 nm using UV-Vis spectrophotometer (Cary 60 Agilent technology) to monitor the synthesis
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of CANPs (Lungu et al. 2014).
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2.4.2. Size distribution of CANPs
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The DLS was used to evaluate the size of the synthesized CANPs using Particle size
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analyzer (Malvern-Nano Series) at 25 °C with scattering angle of 90°. The particle size was
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described as the mean diameter and Z-average value (Jo et al., 2015).
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2.4.3. TEM analysis 5
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The size and morphology of synthesized CANPs were analyzed using high resolution
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transmission electron microscope (HR-TEM) (Jeol/JEM 2100) at an accelerating voltage of 200
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kV (Xing et al., 2016).
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2.4.4. Fourier transforma Infra red (FT-IR) spectroscopic analysis
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FT-IR spectra of CANPs were recorded on Fourier transformation infra red
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spectrophotometer (Thermo Nicolet Model: 6700) using potassium bromide (KBr). Briefly, the
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samples were pelleted with KBr and subjected for FT-IR analysis. The spectra were recorded at
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the range of 500-4500 cm-1 at a resolution of 1 cm-1 (Rejinold et al., 2011).
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2.5. Determination of encapsulation efficiency (EE)
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The encapsulation efficiency of CANPs was determined to validate the amount of
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cinnmaldehyde loaded into the chitosan nanoparticles using UV-Vis spectrophotometer. The
123
supernatant was collected after centrifugation and non-encapsulated cinnamaldehyde was
124
quantified spectrophotometrically at 285 nm using cinnamaldehyde standard curve (Panwar,
125
Pemmaraju, Sharma, & Pruthi, 2016). The EE (%) was calculated by using the following
126
equation:
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EE (%) = (Total cinnamaldehyde loaded – non encapsulated cinnamaldehyde)/Total
128
cinnamaldehyde loaded × 100
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2.6. In vitro release studies of CANPs
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The in vitro release of cinnmaldehyde from the encapsulated CANPs was studied to evaluate
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the controlled release profile of cinnamaldehyde. The lyophilized CANPs were suspended in
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PBS (0.15 M, pH 7.4) at a concentration of 1 mg/mL and kept on a rotor incubator at 100 × g and
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37 °C. At predetermined time intervals (4 h) up to 24 h, the sample solution was withdrawn and
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centrifuged at 5000 × g for 10 min to separate released cinnamaldehyde. The supernatant was 6
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analyzed by UV-Vis spectrophotometry at 285 nm to quantify the released cinnamaldehyde
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content (Ilk et al., 2017). The percentage of cinnamaldehyde released was determined from the
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following equation,
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Release (%) = Released cinnamaldehyde from CANPs/total amount of cinnamaldehyde in
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CANPs × 100
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2.7. Anti quorum sensing activity of CANPs against P. aeruginosa PAO1
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2.7.1. Determination of Minimum Inhibitory Concentration (MIC)
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The MIC for the native cinnamaldehyde and CANPs were determined as per the
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guidelines of Clinical and Laboratory Standards Institute (Luo et al., 2016). Briefly, overnight
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grown P. aeruginosa PAO1 was added to LB medium supplemented with different
145
concentrations of cinnamaldehyde and CANPs and incubated at 37 °C for 24 h.
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2.7.2. Pyocyanin inhibition assay
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The effect on pyocyanin production by P. aeruginosa PAO1 on treatment with sub-MIC
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concentrations of cinnamaldehyde and CANPs was measured by the method described by Luo et
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al. (2016) with slight modifications. Briefly, overnight P. aeruginosa PAO1 with
150
cinnamaldehyde and CANPs were kept for incubation for 16-18 h. After the incubation, the
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culture was pelleted and the cell free supernatant containing the pyocyanin was extracted with
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chloroform. The organic layer was re-extracted with 0.2 M HCl. The absorbance of the
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supernatant was measured using a microplate reader at 520 nm.
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Overnight culture of P. aeruginosa PAO1 was inoculated into freshly prepared LB broth
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supplemented with 500 µg/mL of cinnamaldehyde and CANPs and incubated at 37 °C under 100
157
× g in a rotatory shaker. The cell density was measured in UV–Vis spectrophotometer at every 7
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two hour interval up to 24 h to evaluate the effect of cinnamaldehyde and CANPs on bacterial
159
growth (Cady et al., 2012).
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2.7.4. LasA Staphylolytic activity Briefly, overnight S. aureus culture was boiled for 10 min and centrifuged at 8000 × g for
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10 min. The pellet was resuspended in 10mM Na2PO4 (pH 4.5) to an OD600 of 0.5. Overnight P.
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aeruginosa PAO1 on treatment with cinnamaldehyde and CANPs was centrifuged at 8000 × g
164
for 10 minutes. A 100 µl aliquot of P. aeruginosa test supernatant was added to 900 µl of the S.
165
aureus suspension. Optical density of the mixture (OD600) was measured and the activity was
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determined as the change in OD600 per hour per µg of protein and percentage of inhibition was
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calculated as compared to control (Alasil et al., 2015).
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2.7.5. Anti motility assay
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For swimming and swarming assay, overnight P. aeruginosa PAO1 was point inoculated at
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the centre of the swimming medium (1% tryptone, 0.5% NaCl and 0.3% agar) and swarming
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medium (1% peptone, 0.5% NaCl, 0.5% agar and 0.5% of sterilized D-glucose) with sub-MIC
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concentrations (500 µg/mL) of cinnamaldehyde and CANPs. The plates were incubated at 37 °C
173
for 16 h (Packiavathy, Agilandeswari, Musthafa, Pandian, & Ravi, 2012).
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2.8. Anti biofilm efficacy of CANPs against P. aeruginosa PAO1 biofilm
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2.8.1. Biofilm formation assay (microtitre plate method)
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Briefly, cinnamaldehyde and CANPs treated P. aeruginosa PAO1 culture were incubated
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overnight in a microtitre plate. After 24 h incubation, biofilms formed by bacterial culture was
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stained with crystal violet (0.1% w/v) for 20 min. After washing with water, the liquid was
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discarded from the wells and the material that remained fixed to the polystyrene was washed
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three times with PBS. Crystal violet bound biofilm was dissolved with 95% ethanol, incubated at
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37 °C for 30 min and the absorbance was measured using a microtitre plate reader at 595 nm
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(Zhang et al., 2014).
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2.8.2. Exopolysaccharides inhibition assay The role of cinnamaldehyde and CANPs on the production of exopolysaccharides (EPS)
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by P. aeruginosa PAO1 was determined according to the method described by Rasamiravaka et
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al. (2015). Briefly, P. aeruginosa PAO1 was incubated for 16 h on treatment with sub-MIC
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concentration of cinnamaldehyde and CANPs. After incubation, the culture was centrifuged at
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10,000 × g for 15 min. The pellets were resuspended in high salt buffer and recentrifuged. To the
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supernatant, 95% ethanol was added incubated at 4 °C. After incubation, the suspension was
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recentrifuged and the precipitate was resuspended in deionized water. To the suspension, equal
191
volume of 5% phenol and H2SO4. The absorbance of the mixture was measured at 490 nm.
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2.8.3. Rhamnolipids inhibition assay
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For rhamnolipids extraction, P. aeruginosa PAO1 was grown at 37 °C with agitation at
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100 × g for 16 h in LB medium supplemented with cinnamaldehyde and CANPs. Bacterial
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culture was centrifuged (10,000 × g, 5 min) and cell-free supernatant was mixed with equal
196
volume of ethyl acetate and vigorously vortexed. After phase separation, the upper rhamnolipid-
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containing phase was collected and quantified by a methylene-blue based method described by
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Rasamiravaka et al. (2015).
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2.8.4. Cell surface hydrophobicity assay
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Briefly, P. aeruginosa PAO1 culture on treatment with cinnamaldehyde and CANPs was
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grown in LB medium, centrifuged at 8000 × g for 2 min and the cell pellet was washed and
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resuspended in buffer (2.22 g of potassium phosphate trihydrate, 7.26 g of monobasic potassium
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phosphate, 1.8 g of urea, and 0.2 g of magnesium sulphate heptahydrate/litre (pH 7.1)). From the
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cell suspension, 100 µL were transferred to 96 wellplate to determine the initial cell density in
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the aqueous phase. Four hundred microlitres of hexadecane were added to the 900 µL of
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remaining buffer suspended cells and vortexed for 5 min. The aqueous phase was then
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transferred and the OD600 was measured to determine the cell density that remained in the
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aqueous phase (Garcia-Lara et al., 2015). The percentage of cell surface hydrophobicity was
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determined using the formula, Cell surface hydrophobicity (%) = (Initial cell density-final cell
210
density)/initial cell density ×100
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2.8.5. Microscopic analysis of P. aeruginosa PAO1 biofilms
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The effect of cinnamaldehyde and CANPs on the biofilm forming ability of P.
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aeruginosa PAO1 was examined by light microscopic and CLSM analysis. For light microscopic
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analysis, biofilms were grown on glass slides submerged with tryptone soy broth (TSB) in 24
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well microtitre plate and incubated at 37 °C for 24 h. After incubation, the glass slides were
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washed with carbinol and stained with 0.1% crystal violet and observed under microscope at
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40X magnification (Sivaranjani et al., 2016). For CLSM analysis, overnight culture of P.
218
aeruginosa PAO1 inoculated into freshly prepared LB broth supplemented with sub-MIC
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concentration of cinnamaldehyde and CANPs into a 12 well microtiter plate and coverslips were
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dipped into it and incubated. After 24 h of incubation, coverslips were transferred onto the glass
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slides and stained with acridine orange (0.1%) and kept for 3-5 minutes in dark and observed at
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20X magnification (Ouyang et al., 2016).
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2.9. Statistical analysis
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All the experiments were conducted in triplicate (n=3). All the results were presented as
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mean ± standard deviation (SD). One-way analysis of variance (ANOVA) followed by the
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Tukey–Kramer multiple comparison test (Q test) was used to determine significant differences at
227
varied level of significance as compared to control and native cinnamaldehyde. For statistical
228
analysis, P <0.05 was considered significant (Rejinold et al., 2011).
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3. Results and discussion
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3.1. Synthesis of CANPs
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Cinnamaldehyde was encapsulated on chitosan biopolymer where TPP was used as a
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cross linker, in order to synthesize CANPs. The CANPs were successfully formed by the
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electrostatic interaction between deacetylated chitosan (which have positively charged -NH2
234
group) and TPP (having negatively charged phosphate group). The particle obtained were
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lyophilized and used for further characterization and biological activities.
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3.2. Physico-chemical characterization of synthesized CANPs
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3.2.1. UV-Visible spectroscopic analysis of CANPs
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UV-Vis spectroscopic analysis showed the formation of CANPs with a characteristic change in
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the SPR pattern as compared to native cinnamaldehyde and chitosan.
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3.2.2. Dynamic light scattering (DLS) analysis
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The DLS analysis confirmed the formation and size distribution of CANPs. The size of
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CANPs ranged from 28.21-342.0 nm with a mean particle size of 208.12 nm analyzed by DLS
243
(Fig. 1 a). The CANPs dispersion showed a poly dispersity index (PDI) of 0.277.
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3.2.3. TEM analysis
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The TEM micrograph presented in Fig. 1 (b) showed predominantly anisotropic CANPs
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with approximate size of ~ 28.21 nm and the average size of the CANPs was 208.12 nm. The
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size of the CANPs observed in the present study was in agreement with the previous result
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(Aydin & Pulat, 2012).
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3.2.4. FTIR analysis
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From the FT-IR analysis, the presence of similar kind of functional group in CANPs in
251
comparison to native cinnamaldehyde and chitosan signifies that cinnamaldehyde was
252
successfully encapsulated into chitosan carrier where TPP acts as crossslinker. The FT-IR
253
spectra of chitosan showed the peaks at 1071.4, 1379.5, 1650.0, and 2874.6 which correspond to
254
C-N stretch aliphatic amine, C-H bend alkane, primary amine, and C-H stretch alkane
255
respectively (Fig. 2). The successful loading of cinnamaldehyde to chitosan carrier was identified
256
by the introduction of a new peak i.e. at 1686.3 which corresponds to aldehyde (C=O) stretch
257
which was present in both cinnamaldehyde as well as CANPs. In addition, another peak was
258
observed at 3026.9 corresponding to the aromatic C-H stretch. Hence, the spectrum provided
259
insights about the role of various functional groups of cinnamaldehyde in reduction and
260
stabilization of CANPs. Similar shift in the FT-IR spectrum was reported earlier for kaemferol
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loaded chitosan nanoparticles (Ilk et al., 2017).
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3.3. Encapsulation efficiency and in vitro drug release studies
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The encapsulation efficiency was measured with the help of using a standard curve of
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cinnamaldehyde. The EE (%) of cinnamaldehyde to chitosan carrier was observed to be 65.04 ±
265
2.14%. From the size analysis study and observance of more than 65% encapsulation efficiency
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confirmed the successful synthesis and encapsulation of cinnamaldehyde into the chitosan-TPP
267
carrier. The present results suggested higher efficacy of CANPs in terms of size and 12
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encapsulation efficiency than the earlier report (Loquercio, Castell-Perez, Gomes, & Moreira,
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2015). The in vitro drug release profile showed that 29% drug was released in the time interval
270
of 12 h in the initial burst. After that approx. 60% drug was released in the next 12 h (Fig. 3).
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This result suggested a slow and sustained release of cinnamaldehyde from CANPs which was
272
comparatively better than the earlier report showing 20–50% release of phytochemical in a span
273
of 24 h using PLGA nanoparticles (Silva, Hill, Figueiredo, & Gomes, 2014).
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3.4. Anti quorum sensing activity of CANPs
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3.4.1. Determination of MIC and sub-MIC
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The MIC of cinnamaldehyde and CANPs against P. aeruginosa PAO1 was found to be
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1000 µg/mL where the growth and cell density were significantly inhibited as evident from the
278
absence of turbidity. Hence, sub-MIC concentrations of 250 and 500 µg/mL were used for
279
further activities.
280
3.4.2. Pyocyanin inhibition assay
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Pyocyanin is an important virulence factor produced by P. aeruginosa PAO1 and is
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regulated by Rhl QS system. Pyocyanin plays a critical role in altering the host immune system
283
by virtue of generating reactive oxygen species (ROS) and it has profound role in development
284
of acute and persistent respiratory infections (Hall et al., 2016). In the present study, pyocyanin
285
production was significantly decreased with the increasing sub-MIC concentrations of both
286
cinnamaldehyde and CANPs (Fig. 4). In the present study, the production of pyocyanin was
287
significantly inhibited by 93.24% on treatment with sub-MIC concentration of CANPs whereas
288
in case of native cinnamaldehyde, 72.96% of inhibition in pyocyanin production was observed
289
which was significantly higher than the earlier report (Luo et al., 2016).
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3.4.3. Growth curve analysis From the growth curve assay, sub-MIC concentration (500 µg/mL) of cinnamaldehyde
292
and CANPs treated P. aeruginosa PAO1 exhibited normal growth like that of the untreated
293
control but with slow growth rate as compared to control (Fig. 5).
294
3.4.4. LasA Staphylolytic activity
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The QS gene expression in P. aeruginosa has interconnection with other signalling
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regulatory systems which responds to various environmental signals. LasA protease is under the
297
control of the lasI-lasR system (Alasil et al., 2015). This protease has inhibitory effect on the
298
growth of S. aureus, which was significantly reduced on treatment with sub-MIC concentrations
299
of both cinnamaldehyde and CANPs. A significant decrease in LasA Staphylolytic activity was
300
observed as compared to that of the control when P. aeruginosa test supernatant was grown on
301
treatment with sub-MIC concentrations of cinnamaldehyde and CANPs (Table 1).
302
3.4.5. Anti motility assay
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The flagellar mediated swimming and pili mediated swarming motility of P. aeruginosa
304
PAO1 play crucial role in biofilm formation, development and maturation (Packiavathy et al.,
305
2012). The presence of sub-MIC concentration (500 µg/mL) of cinnamaldehyde and CANPs
306
significantly affect the swimming and swarming motility of P. aeruginosa PAO1 suggesting its
307
role in biofilm disruption which was in accordance to the earlier report (Fig. 6).
308
3.5. Anti biofilm efficacy of CANPs
309
3.5.1. Biofilm formation assay (MTP method)
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The sub-MIC concentrations of cinnamaldehyde and CANPs exhibited significant
311
reduction in biofilm formation as compared to untreated control in a concentration dependent
312
manner (Table 1). From the results, 84.06±4.09 and 71.25±3.81% of reduction in biofilm
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formation was observed on treatment with sub-MIC concentrations of cinnamaldehyde and
314
CANPs respectively which was significantly higher than previous report with 60% to 90% of
315
reduction (Kalia et al., 2015; Sivaranjani et al., 2016).
316
3.5.2. EPS inhibition assay
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The production of EPS plays an important role in formation and development of biofilm.
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In the present study, the production of EPS on treatment with sub-MIC concentrations of
319
cinnamaldehyde and CANPs was significantly altered with 65.36±3.51 and 80.28±4.05% of
320
reduction in EPS production. Table 1 showed the concentration dependent inhibition of EPS
321
production by P. aeruginosa PAO1 on treatment with sub-MIC concentrations of
322
cinnamaldehyde and CANPs suggesting the efficacy of CANPs and cinnamaldehyde in
323
inhibition of biofilm formation and development (Periasamy et al., 2015).
324
3.5.3. Rhamnolipids inhibition assay
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A significant and concentration dependent decrease in the production of rhamnolipids
326
was observed when P. aeruginosa PAO1 was treated with sub-MIC concentrations of
327
cinnamaldehyde and CANPs (Table 1). The production of rhamnolipids by P. aeruginosa PAO1
328
has a greater influence on biofilm formation and development. The result of CANPs was
329
significantly higher than the earlier report. The potential role of CANPs in inhibiting the
330
production of rhamnolipids could be attributed to its role in governing the QS regulated rhlAB
331
operon encoding the enzymes responsible for rhamnolipids biosynthesis (Nickzad & Deziel,
332
2015; Prateeksha et al., 2017).
333
3.5.4. Cell surface hydrophobicity assay
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Cell surface hydrophobicity determines the ability of microorganisms to form biofilms
335
and bypass the antibiotic therapy. In the present study, sub-MIC concentrations of
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cinnamaldehyde and CANPs significantly decreased the surface hydrophobicity of P. aeruginosa
337
PAO1 in a dose dependent manner which was in accordance with the earlier report (Fig. 7)
338
(Garcia-Lara et al., 2015).
339
3.5.5. Microscopic observation of biofilms
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The light microscopic analysis of the biofilm formed by P. aeruginosa PAO1 showed
341
visible clumping with complex morphology of the biofilm architecture which was significantly
342
altered on treatment with sub-MIC concentration (500 µg/ml) of native cinnamaldehyde and
343
CANPs suggesting its efficacy in attenuating the antibiotic resistant biofilm formation and
344
development (Sivaranjani et al., 2016). CLSM analysis confirmed a significant reduction in the
345
thickness of biofilm in the presence of cinnamaldehyde and CANPs as compared to the untreated
346
control (Fig. 8). The results suggested the altered biofilm architecture with significant reduction
347
in biofilm formation in the treated sample as compared to the clumped and complex biofilm
348
architecture in the untreated control which was in accordance with the previous report (Ouyang
349
et al., 2016).
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In the present study, for the first time, we demonstrated the QS inhibitory activity of
351
CANPs against the QS dependent phenotypic virulence factors and biofilm formation in P.
352
aeruginosa PAO1. From the results, the encapsulation of cinnamaldehyde into polymeric
353
chitosan nanoparticles enhanced the anti quorum sensing and anti-biofilm potential as compared
354
to its native form suggesting its widespread biomedical and pharmaceutical applications. In
355
addition, the present study will also give new avenues to the use of CANPs in food processing
356
industries for enhanced food preservation strategies against a variety of food-borne pathogens.
357
4. Conclusion
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In the present study, as compared to native cinnamaldehyde, the QS attenuation efficacy
359
of CANPs was found to be significantly higher suggesting the role of a biocompatible
360
nanocarrier system for effective targeting of bacterial virulence with slow and controlled release
361
of administered drugs at specific sites. The slow and sustained release of cinnamaldehyde from
362
CANPs suggested its persistence to be used as effective quorum sensing inhibitor against other
363
pathogens. Besides the biomedical applications, the biocompatible CANPs will also play critical
364
role in the food processing industries in the near future.
365
Conflict of interest
366
The authors confirm that this article content has no potential conflict of interest.
367
Acknowledgement
368
The authors like to acknowledge Dr. G. Muralitharan, Department of Microbiology,
369
Bharathidasan University, Tiruchi, India for providing CLSM facilities. The authors also duly
370
acknowledge the Central Instrumentation Facilities (CIF), Pondicherry University for providing
371
DLS and FT-IR analysis. The authors also like to thank Sophisticated Test and Instrumentation
372
centre (STIC), Cochin, India for providing TEM analysis.
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Table 1. Effect of sub-MIC concentrations (250 and 500 µg/mL) of cinnamaldehyde and CANPs on LasA Staphylolytic activity and production of rhamnolipids, EPS and biofilm by P. aeruginosa PAO1. Quorum sensing inhibitory activity
Percentage of inhibition (%)
Cinnamadehyde
39.50±2.82
51.83±2.67*
55.55±4.06
61.16±4.20NS
39.41±3.66
53.09±2.95*
54.56±2.51
63.59±2.54*
52.07±1.48
63.46±2.76*
65.36±3.51
80.28±4.05*
56.33±4.40
76.08±5.57*
71.25±3.81
84.06±4.09*
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LasA Staphylolytic assay EPS inhibition assay Rhamnolipids inhibition assay Biofilm formation assay (MTP) LasA Staphylolytic assay EPS inhibition assay Rhamnolipids inhibition assay Biofilm formation assay (MTP)
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Concentration (µg/mL)
All the values were represented as Mean ± standard deviation (SD) of 3 replicates (n = 3). * Values are significantly different from native cinnamaldehyde at p<0.05 ** Values are significantly different from native cinnamaldehyde at p<0.01 *** Values are significantly different from native cinnamaldehyde at p<0.001 NS Values are not significantly different from native cinnamaldehyde
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Fig. 1. Determination of size, morphology and distribution of Cinnamaldehyde encapsulated chitosan-TPP nanoparticles (CANPs). (a) Dynamic light scattering (DLS) analysis of CANPs showing the mean diameter of nanoparticles (208.12 nm); (b) Transmission electron microscopic
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(TEM) image of CANPs showing spherical shape of nanoparticles with a diameter of 208.12 nm.
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Fig. 2. FT-IR spectra of (a) Cinnamaldehyde; (b) Chitosan and (c) Cinnamaldehyde encapsulated chitosan-TPP nanoparticles (CANPs), recorded in the range of 500 cm-1 to 4500 cm-1 at a resolution of 1 cm-1 showing the shift of identical absorption peaks corresponding to change in
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chemical groups during the encapsulation of cinnamaldehyde into chitosan nanoparticles.
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Fig. 3. In vitro release of cinnamaldehyde from CANPs over a period of 24 hours at every 4 hour
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interval showing the slow and sustained release of cinnamaldehyde.
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Fig. 4. The effect of sub-MIC concentrations (250, 500 µg/mL) of cinnamaldehyde and cinnamaldehyde encapsulated chitosan-TPP nanoparticles (CANPs) on pyocyanin production in
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P. aeruginosa PAO1 as compared to untreated control. All the values were represented as Mean ± standard deviation (SD) of 3 replicates (n = 3). The results were highly significant (p<0.05) as compared to native cinnamaldehyde.
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Fig. 5. Effect on the growth curve of P. aeruginosa PAO1 on supplementation of native
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cinnamaldehyde and CANPs (500 µg/mL) as compared to the untreated control.
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Fig. 6. Effect of sub-MIC concentration (500 µg/L of cinnamaldehyde and CANPs on P. aeruginosa PAO1 swarming and swimming motility as compared to untreated control. (a) Untreated control swarming plate; (b) Swarming motility of native cinnamaldehyde treated P.
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aeruginosa PAO1; (c) Swarming motility of CANPs treated P. aeruginosa PAO1; (d) Untreated
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Fig. 7. The effect of sub-MIC concentrations (250 and 500 µg/mL) of cinnamaldehyde and
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CANPs on the bacterial hydrophobicity over the surface as compared to untreated control.
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Fig. 8. Microscopic analysis of P. aeruginosa PAO1 biofilm on treatment with sub-MIC concentration (500 µg/mL) of cinnamaldehyde and CANPs as compared to untreated control. (a) Light microscopic observation of biofilm of untreated control; (b) Light microscopic observation
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of biofilm treated with cinnamaldehyde; (c) Light microscopic observation of biofilm treated with CANPs; (d) CLSM image of biofilm of untreated control; (e) CLSM image of biofilm
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Highlights Ionic gelation method was used for synthesis of CANPs
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TEM analysis confirmed the synthesis of CANPs
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Anti quorum sensing potential of CANPs against P. aeruginosa PAO1 was evaluated
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Anti biofilm efficacy of CANPs was determined against P. aeruginosa PAO1
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CLSM analysis corroborated the biofilm inhibition potential of CANPs
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