Accepted Manuscript

Synergistic effects of Woodfordia fruticosa gold nanoparticles in

preventing microbial adhesion and accelerating wound healing in
Wistar albino rats in vivo

Navdeep Raghuwanshi, Poonam Kumari, Amit Kumar

Srivastava, Priya Vashisth, Tara Chand Yadav, Ramasare Prasad,
Vikas Pruthi

PII: S0928-4931(16)32248-2
DOI: doi: 10.1016/j.msec.2017.05.134
Reference: MSC 8116
To appear in: Materials Science & Engineering C
Received date: 23 November 2016
Revised date: 18 May 2017
Accepted date: 22 May 2017

Please cite this article as: Navdeep Raghuwanshi, Poonam Kumari, Amit Kumar
Srivastava, Priya Vashisth, Tara Chand Yadav, Ramasare Prasad, Vikas Pruthi , Synergistic
effects of Woodfordia fruticosa gold nanoparticles in preventing microbial adhesion and
accelerating wound healing in Wistar albino rats in vivo, Materials Science & Engineering
C (2017), doi: 10.1016/j.msec.2017.05.134

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 ACCEPTED MANUSCRIPT

Synergistic effects of Woodfordia fruticosa gold nanoparticles in preventing

microbial adhesion and accelerating wound healing in Wistar albino rats in

vivo

Navdeep Raghuwanshi1, Poonam Kumari2, Amit Kumar Srivastava3, Priya Vashisth1,

Tara Chand Yadav1, Ramasare Prasad2 and Vikas Pruthi1*

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1
Molecular Microbiology Laboratory, Biotechnology Department, Indian Institute of

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Technology, Roorkee 247667, Uttarakhand, India.
2
Molecular Biology and Proteomics Laboratory, Biotechnology Department, Indian Institute
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of Technology, Roorkee 247667, Uttarakhand, India.
3
Indian Institute of Integrative Medicine, IIIM (Council of Scientific & Industrial Research),
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Jammu- 180001, India.

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Corresponding author:-

Prof. Vikas Pruthi

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Department of Biotechnology,

Indian Institute of Technology Roorkee (IIT-R),

Roorkee-247667, India

Ph: 091-1332-285530 (office), 091-1332-285110 (Resi.)

Fax: 091-1332-273560Mob: 09997777613

E.mail: vikasfbs@gmail.com

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Abstract

Therapeutic effectiveness of biogenically synthesized Woodfordia fruticosa nano-gold

particles (WfAuNPs) has been claimed in this study which prevents microbial adhesion and

enhanced wound healing potential on Wistar albino rats. The synthesized WfAuNPs were

characterized using several biophysical techniques such as UV-Visible Spectroscopy (UV-

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vis), X-Ray Diffraction (XRD), Dynamic Light Scattering (DLS), Zeta Potential, Fourier

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Transform Infrared Spectroscopy (FTIR), Field Emission Scanning Electron Microscopy

(FE-SEM), Atomic Force Microscopy (AFM) and High Resolution Transmission Electron

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Microscopy (HR-TEM) analysis. The synthesized WfAuNPs in the size range of 10-20 nm
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were used to develop 1% Carbopol 934 based nano gold formulation (WfAuNPs-Carbopol

934). The WfAuNPs-Carbopol 934 nanoformulation were evaluated using viscosity and
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spreadability measurements. The wound healing potential of WfAuNPs-Carbopol 934

monitored up to 12 days was confirmed by performing wound contraction (%),

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epithelialization, and histopathological studies done in vivo on Wistar albino rats. The
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hydroxyproline content was also measured in the re-epithelized skin for quantification of
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collagen content. The effects of WfAuNPs on microbial adhesion leading to biofilm

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formation were evaluated against Candida albicans and Cryptococcus neoformans fungal

strains. The respective Minimum Inhibitory Concentration (MIC80), Biofilm Inhibitory

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Concentration (BIC80) and Biofilm Eradication Concentration (BEC80) values of C. albicans

was found to be 16, 32, 256 g/ml respectively while for C. neoformans it was recorded to

be 32, 64, 256 g/ml respectively. Data obtained, confirmed the effectiveness in preventing

microbial adhesion and wound healing potential of the WfAuNPs as compared to current

marketed formulations.

Key words: Woodfordia fruticosa; WfAuNPs; XRD; HRTEM; Biofilm; Wound Healing.

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1. Introduction
Chronicity in wound patients is the leading cause of death worldwide due to sepsis it causes

[1]. Wound sepsis is the condition where systemic infection occurs at the site of wound and

increase the chances of patients deaths [2]. Various factors such as formation of biofilm,

microbial pathogens and bioburden at the wound site affects the delay in wound healing so

there is an urgent need to develop fast acting and effective formulations for chronic wounds

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infection [3-5]. Recent studies manifest state of the art techniques to develop such novel

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formulations using the aspects of nanobiotechnology as a therapeutic treatment alternative for

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increasing bacterial infections [6,7]. The first documented report on the preparation of gold

nanoparticles (AuNPs) was published in mid-19th century where they were used for the
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purpose of staining of window glass panes and dichroic glass cups of Roman origin [8].

Biogenic AuNPs encompassing properties such as biostability, biocompatibility and less

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toxicity to human cells can be used as potential therapeutic agent [9,10]. Functionalization of

these biogenic metallic nanoparticles with drug of choice can further used as a targeted drug
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delivery system toward specific biomedical applications [11]. Biogenic AuNPs unravels
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insight of biomedical sciences especially in the field of biosensors [12], bio-labelling [13],

sensing [14], bio-imaging, mapping of neuronal activities in Alzheimers disease

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(Optogenetics) [15], cancer [16], and wound healing [17]. These synthesized nanoparticles

can be used in near future as a prospective drug candidate to curb bacterial infections caused
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by hampering and disrupting the biofilm formed at wound sites. Moreover, these biogenic

AuNPs overcoming the microbial resistance in the comparison to other antibiotics used

frequently. The bacteriostatic and bacteriocidal effect of nanoparticles is due to its

remarkable feature of deep penetrating ability within the biofilm and it also helps to curb

development of resistance against nanoparticle formulation as compared to other antibiotics

used frequently [4,5,18]. Recently, other inorganic metallic nanoparticles such as

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upconversion nanoparticles (UCN) conjugated with the photosensitizers have been also used

as a model for photodynamic therapy (PDT) in cancer [19]. Earlier, investigations have also

reported the green synthesized AuNPs using naturally available biomaterials in plants such as

alkaloids, glycosides, terpenoids, flavonoids, proteins/enzymes, organic acids, amino acids

and carbohydrates [20,21,22]. These inorganic biomaterials encompasses different functional

groups (hydroxyl, carbonyl, and amine) which plays a crucial role in its reaction with metal

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(Au+) ions resulting in reduction in size up to nano range (Au0) leading to increased

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intracellular uptake [11,23]. Furthermore, they are also responsible for capping around the

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synthesized AuNPs [24]. On the other hand, the mechanism of AuNPs formation via biogenic

route is still unknown whereas several hypothesis have been proposed for AuNPs synthesis
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[25]. In this study, Woodfordia fruticosa (Kurz) flowers aqueous extract (WfAe) was utilized

for the biogenic synthesis of gold nanoparticles (WfAuNPs). W. fruticosa is locally

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recognized as Dhataki and it comes under the family Lythraceae. In domestic as well as in

international market, flowers of W. fruticosa are in high demand for preparation of herbal
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medicines and other parts of the plant also encompasses wide variety of medicinal properties
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[26]. In tribal areas of Chhattisgarh and some district of Central India, fresh flowers of plant
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are used indigenously for healing purpose of cuts, wounds and in case of emergency
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bleeding, whereas powder of dried flower help in rapid healing of wounds. The whole plant is

traditionally important due to its usage in ayurvedic formulations [27-29]. Dried flowers of
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this plant are also reported to treat wounds and peptic ulcers with suppressing discharge and

increasing granulation [30].

The important phytoconstituent from dried flower part of this plant isolated earlier includes

quercetin-3-O-oxylopyranoside, myricetin-3-O-O-galloyl-d-galactopyranoside, myricetin-3-

Oarbinopyranoside, ellagic acid [31,32] and woodfordin A, B, C along with five oligomers

woodfordin E, F, G, H and I [33,34]. The biogenic synthesized novel WfAuNPs

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nanoformulation ointment gel (WfAuNPs-Carbopol 934) prepared in this investigation. It

showed accelerated wound healing, which manifested noticeable improvements in wound

repair and reduced the chances of biofilm formation at the affected site.

2. Materials and Methods

2.1. Chemicals

Tetrachloroauric acid III (HAuCl4), Potassium bromide (KBr), Carbopol 934, Formalin, L-

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glutamine, Sodium bicarbonate, Morpholinepropanesulfonic acid, XTT [2,3-bis(2-

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methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium- 5-carboxanilide sodium salt] tetrazolium

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salt, Menadione were procured from Sigma Aldrich (St. Louis, USA). Povidone iodine 5%

w/w ointment (Win Medicare, India), Ketamine hydrochloride (Sun Pharmaceuticals Ltd.
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India), Sabouraud dextrose (SAB) agar and RPMI-1640 medium were obtained from

Himedia Laboratories (Mumbai, India). Glasswares were treated with aqua regia (HCl:
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HNO3 = 3:1) for 30 min than carefully washed several times via Milli-Q water (Milli-Q plus

system, Millipore Co.) with high 18.2 M-cm water resistivity and dried by hot air oven for
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the period of 5 h prior to use.

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2.2. Collection and authentication of plant material

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Woodfordia fruticosa flowers were freshly collected in the month of January locally from
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Bhopal (23o25'N, 77o41'E) Madhya Pradesh, India. Flowers were identified and

authenticated by Dr. Zia Ul Hasan, Head of the Department, Department of Botany, Saifia
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Science College, Bhopal, Madhya Pradesh, India. The voucher specimen number

Bot/saifia/14/439 was deposited in Pharmacology Department, Sapience Bioanalytical

Research Lab for future reference.

2.3. Extract preparation and biogenic synthesis of WfAuNPs

The collected flowers of Woodfordia fruticosa were shaded dried for 15 days, then

converted into powder and sieved (Sieve number 80). Powdered flowers (1 gm) were then

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mixed with 100 ml of distilled water in an Erlenmeyer flask (250 ml) and heated at 50 oC

for 20 min. The obtained solution was filtered using Whatman paper no. 1 and stored at 4
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C for further experimental work. Biogenic synthesis of WfAuNPs was done by mixing 5

ml of WfAe in 95 ml of 1 mM HAuCl4 (reaction mixture) at 25 oC with continuous stirring

(120 rpm) using magnetic stirrer. The color change of the reaction mixture instantly from

brownish to ruby red, indicated the formation of gold nanoparticles (WfAuNPs). Three

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times washing (Milli-Q water) was done for the purification of synthesized WfAuNPs by

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centrifugation of samples at 15000 rpm for 10 min. The purified WfAuNPs were collected

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and redispersed in Milli-Q water for characterization.

2.4. Characterization of WfAuNPs

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2.4.1. Spectroscopic analysis

The bioreduction of gold ions by WfAe extract was monitored up to seven days using UV-
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visible (UV-vis) spectrophotometer (Shimadzu-1700) operated at a resolution of 1 nm and

wavelength scanned at 400-800 nm. Potassium bromide (KBr) pellet method was utilized for
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the identification of probable functional groups responsible for reduction and stabilization of
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WfAuNPs. The FTIR (Perkin Elmer Spectrophotometer) spectrum was recorded in the range
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of 4000 cm-1 to 400 cm-1.

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2.4.2. XRD analysis

XRD (Brucker D-8 Advance) pattern of synthesized WfAuNPs were taken at operated
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voltage of 45 kV supplied with 40 mA with CuK (=0.1542 nm) radiation source over 2

value from 20-100 at 0.04 / min with 2 s time constant.

2.4.3. Hydrodynamic size (DLS) and Zeta potential measurements

The hydrodynamic size (Z-Average), zeta potential (surface charge) and polydispersity index

(PDI) of synthesized WfAuNPs were assessed using dynamic light scattering (DLS)
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measurements (Malvern Zetasizer Nano ZS90) machine at 25 C. During this

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analysis, WfAuNPs diluted samples were placed in a folded capillary cell supported with

platinum electrodes and loaded into the sample holder of the analyser.

2.4.4. Microscopic analysis

Surface topologies of synthesized WfAuNPs were checked using microscopic techniques

such as FESEM, HR-TEM and AFM analysis. FE-SEM (Ultra Plus- Carl Zeiss operated at 5

kV coupled with EDX-detector) samples were prepared by transferring a drop of WfAuNPs

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in glass slides and air dried overnight for morphological analysis. Dried slide was then gold

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coated with sputter coater (Denton) to maintain charges during investigation. EDX spectrum

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was taken for the determination of elemental composition in synthesized WfAuNPs. The

AFM samples of WfAuNPs were diluted (10 times) with Milli-Q water and transferred on
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clean glass slide for drying by applying vacuum for 24 h at 25 oC. Dried AFM samples were

then analysed using AFM (AFM-STM, Ntegra Ts-150, Ireland). HR-TEM (FEI Tecnai G2
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operated at 200 kV) samples were prepared by transferring a drop of WfAuNPs in carbon-

coated copper TEM grid and then air dried for 30 min before analysis. Histogram and
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diameter distribution of 20 particles (WfAuNPs) were calculated using Image J 1.49

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software (NIH, USA).

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2.5. Nano-gold formulation (WfAuNPs-Carbopol 934)

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WfAuNPs formulation was developed as described earlier [1]. Briefly, lyophilized biogenic

WfAuNPs was slowly dispersed in 1% (w/v) Carbopol 934 using overhead stirrer (Remi) at
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1000 rpm for 3 h till the gelling was completely hydrated with water and the pH 6.5 was

adjusted by triethanolamine solutions. Triethanolamine is a neutralizing agent which gives

gel forming and swelling properties to Carbopol 934. Similarly 2 % WfAe-Carbopol 934

formulation was also developed. Carbopol 934 polymer was used as a gelling agent that

provides sufficient uniformity for the ease of topical application. Povidone iodine (5 % w/w)

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was used as standard drug for the comparing the wound healing potential of the developed

nanoformulation.

2.6. Characterization of WfAuNPs-Carbopol 934 nanoformulation

2.6.1. Viscosity measurement

The WfAuNPs-Carbopol 934 gel viscosity was determined using Brookfield viscometer

LVDV-II (U.S.A) at 25 1 oC. The spindle number s-64 was used for viscosity measurement

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studies and weighed amount (50 mg) of WfAuNPs-Carbopol 934 sample was placed in

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sample holder and spindle was lowered. The spindle was rotated at the shear rate of 6 s-1 and

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corresponding viscosity (cP) was recorded.

2.6.2. Spreadability
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The spreadability of WfAuNPs- Carbopol 934 nanoformulation was calculated using

slightly modified protocol of Shah et al. 2007 [35]. Briefly, WfAuNPs- Carbopol 934
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nanoformulation (500 mg) was placed at the centre (1 cm in diameter) of pre-marked glass

plates. Different weight (50, 100, 150, 200, 250, 300 and 350 g) were then applied at the top
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of upper glass plate for the period of 5 min. The increase in diameter from the initial value of
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WfAuNPs-Carbopol 934 nanoformulation was noted.

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2.7. Antibiofilm activity

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2.7.1. Media and Culture Conditions

Two fungal strains Candida albicans (MTCC 227) and Cryptococcus neoformans (NCIM
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3541) were selected for the analysis of antibiofilm potential of synthesized WfAuNPs. The

stock culture cells stored at -80 oC were propagated first by streaking onto Sabouraud

Dextrose (SAB) agar plates and then kept overnight for incubation at 30 C. A loopful culture

of C. albicans and C. neoformans were then transferred on the SAB liquid medium (broth)

and kept on orbital shaker at 180 rpm (30 C) for 14-16 h. The pH of the RPMI 1640 medium

with L-glutamine without sodium bicarbonate was buffered with 0.165

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M morpholinepropanesulfonic acid. Lyophilized WfAuNPs stock solution 5 mg/ml was

prepared and stored at 20C till further usage.

2.7.2. WfAuNPs susceptibility testing

As per the recommendations of Clinical and Laboratory Standards Institute (CLSI), Broth

microdilution method were utilized for the testing of synthesized WfAuNPs potential

against planktonic cells of C. albicans and C. neoformans [36]. Two fold serially diluted

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concentrations of WfAuNPs were made in RPMI-1640 medium and each dilution (100 l)

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was transferred on presterilized 96 well microtiter plates. RPMI-1640 medium devoid of

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WfAuNPs was added in control wells. Planktonic cells were harvested during exponential

phase followed by washing with sterile phosphate-buffered saline (PBS) and resuspended in
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RPMI-1640 medium at a density of 410 3 cells/ml. Cells (100 l) from the suspension

media were then poured into the wells with WfAuNPs and control wells to provide
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2103 cells/ml in working volume (200 l). Subsequently, microtiter plates were incubated

for 48 h at 37 C. The absorbance of the samples was measured at OD600 nm after 48 h of

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incubation period.
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2.7.3. Effect of WfAuNPs on C. albicans and C. neoformans biofilm formation and

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preformed biofilm
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Effect of WfAuNPs on biofilm formation was performed using 96-well microtiter plates as

reported earlier [37-38]. RPMI-1640 medium was used for the preparation of cell
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suspension (2106 cells/ml) which was further transferred (100 l) to each well of the

microtiter plates. To investigate the effect of WfAuNPs on the biofilm formation; serially

diluted concentration of WfAuNPs in RPMI-1640 medium (100 l per well) were added so

that final concentration of cells (1106 cells/ml) be achieved. Similarly 100 l of RPMI-

1640 medium devoid of WfAuNPs was dispensed into control wells and the microtiter

plates were then incubated at 37C for 48 h. To assess the effect of WfAuNPs on pre-

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formed biofilm RPMI-1640 medium was used to prepare the cell suspension maintaining

the cell density to 110 6 cells/ml [37,38]. Cell suspension (100l) was then transferred

into microtiter plates and further incubated at 37C for 48 h. The samples were washed

thrice with sterile PBS solution to remove non-adherent cells. To the prewashed biofilm

diluted WfAuNPs (100l) was dispensed into the wells. Control was prepared by using

RPMI-1640 (100 l) medium devoid of WfAuNPs dispensed into the designated wells and

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the microtiter plates were then further incubated at 37C for 48 h. Colorimetric XTT

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reduction assay method was employed for quantitative determination of metabolic activity

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of biofilm.

2.7.4. XTT Assay

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XTT reduced colorimetric assay of biofilm was performed according to earlier

reported method [37]. Briefly, after 48 h of incubation period the culture medium was
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removed from the wells of microtiter plate. Biofilm formed on the wells were then washed

with PBS to get rid of non-adherent cells. The filter sterilized XTT (0.5 g/L in PBS)
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solution (100 l) containing menadione (1 M) was then added to each well of microtitre
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plate as well as to the control. The plates were then incubated for 1 h at 37 oC and read at
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OD492 nm for analysing the reduced formazan product formed.

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2.7.5. Morphological analysis of C. albicans and C. neoformans biofilm

SEM was used to analyse the morphological changes in C. albicans and C. neoformans
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biofilm treated with WfAuNPs. Initially, biofilm of C. albicans and C. neoformans were

developed on FBS treated catheter disc (1 mm diameter) in 12 well cell culture plate

containing (0-256 g/ml) WfAuNPs in RPMI-1640 medium. The plates were kept at 37C

for 48 h. After incubation the catheter discs with biofilm were washed thrice with PBS and

further transferred to another 12-well plates containing 2.5 % glutaraldehyde for 48 h

incubation at 4 oC. Sample were then dried by employing increasing concentration of ethanol

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(30 %, 50 %, 70 %, and 95 %) for 10 min and finally kept at 20 min in absolute alcohol. The

samples were then dried using desiccator and coated with gold-palladium (Denton

Vacuum sputter coater, USA) to visualize them under scanning electron microscope (LEO

435 VP) at 10 kV.

2.8. Wound healing activity (In vivo)

2.8.1. Experimental animals

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Animals were procured from the animal house facilities of Sapience Bioanalytical Research

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Lab (CPCSEA; 1413/PO/E/S/11/CPCSEA), Bhopal, Madhya Pradesh, India. They were

acclimatized (Seven days before wounding) in the standard laboratory conditions (25 2 oC;

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light and dark cycle of 12:12 h; relative humidity 4456 %) with cross ventilated animal
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house as per rules and regulations of Committee for the Purpose of Control and Supervision

of Experiments on Animals (CPCSEA) guidelines, Government of India and feed with

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standard commercial diet (pellet) with water ad libitum. This study has been approved by

CPCSEA and protocol approval number was SBRL/IAEC/April 2016/07.

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2.8.2. Acute dermal toxicity studies

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Acute dermal toxicity studies of prepared ointment formulations (WfAe-Carbopol 934 and
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WfAuNPs-Carbopol 934) were performed according to Organization for Economic

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Cooperation and Development Guidelines (Number 402) on Wistar albino rat [39,40]. The

rats were divided into two groups, each group contains (n=6) animals. WfAe-Carbopol 934
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and WfAuNPs-Carbopol 934 ointment at a dose levels of 2000 mg/kg and 500 g/kg body

weight respectively were applied topically on shaved area of each animals of both groups and

monitored for 14 days. Signs of redness, erythema, changes in fur, behavioural pattern and

mortality were checked and recorded.

2.8.3. Wound healing activity

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In vivo wound healing activity was performed on excision and incision wound models of

Wistar albino rats. Total 30 healthy Wistar albino rats (200 30 g) were used and divided

equally (n=6) into five groups as follows; Group I; Control considered as untreated, Group II;

Carbopol 934 ointment base treated, Group III; Standard, Povidone iodine 5 % (w/w)

ointment treated, Group IV; 2 % WfAe-Carbopol 934 and Group V; 1 % WfAuNPs-

Carbopol 934 treated.

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2.8.4. Excision wound model

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Excision wound was generated at dorsal side according to our earlier reported protocol [41].

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The animal were shaved (circular area approx. 2 cm diameter) and anaesthetized with

ketamine hydrochloride (50 mg/kg, i.p. body weight). The treatments of all the groups with
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different formulations were repeated daily at every 24 h till wound get completely healed. To

determine the therapeutic efficiency of developed nanoformulation on wounded site, the

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wound size was measured on 0, 3, 6, 9 and 12 days using Vernier caliper (Aerospace 300

mm). The animal were monitored every day and not found any adverse effects after topical
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treatments. Consequently, using this model the (%) contraction on wound was measured
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using following formula:

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The epithelization period was assessed after dead tissue dropping from the healed wound
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without disturbing raw wound [42]. Finally, all the healed experimental rats were

anaesthetized and tissue samples were taken at a margin of 5 mm on the normal skin. Neutral

formalin (10 %) solution were utilized for the tissue biopsies after 2 weeks to carry out

Biochemical estimation and histopathological analysis.

2.8.5. Incision wound model

Incision wound model was performed on Wistar albino rats. Animals were grouped and

treated as per excision wound model [43]. Sharp scalpel was used for making 6 cm

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paravertebral incision with full thickness on either side of vertebral column on shaved area.

After complete haemostasis wound was stitched using surgical thread (000 number) and

curved needle (11 number). After stitching, wound was left undressed and animals were

treated continuously for 12 days. Tensiometer was used for measuring the tensile strength of

cured wound on 10th day [44].

2.8.6. Histopathological analysis

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The histopathological analysis was done using standard protocols [41,45]. Briefly, collected

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tissue samples for histopathological analysis were processed in increasing 25 %, 50 %, 75 %,

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90 % and 100 % alcohol concentrations for the complete drying which were later fixed in

paraffin wax. Paraffin fixed skin tissue (5 m) sections were prepared by microtome (Leica
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Biosystems, RM 2235, USA) and overnight dried in oven for staining (Hematoxylin- Eosin

(H & E) and Massons trichrome staining). The overnight dried stained samples were
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analysed using Evosfl, AMG groups, USA, microscope.

2.8.7. Biochemical estimation

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To measure the content of collagen synthesized at wounded sites biochemical estimation was
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performed. Collagen is a vital component of wound healing process comprises 25 % of total

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protein and 70-80 % (dry weight) of skin. Collagen serve as an essential scaffolds in tissues
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regeneration [46]. Re-epithelized wounded site (hydroxyproline concentration) was

determined as described earlier [41]. Briefly, Pyrex tubes were utilized for 3 h at 130 oC for
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hydrolysed tissue samples with HCL (6 N) then the hydrolysate was neutralized at pH 7.0 and

was exposed to Chloramine-T oxidation for the period of 20 min. Afterward, Ehrlich reagent

(2.5 ml) was transferred into test tubes and were dipped in a water bath at 60 C. The test

tubes samples were subjected to ice bath for a period of 25 min and further 6.6 ml of

isopropyl alcohol was also added to each tube. All the samples were stirred thoroughly and

analysed via spectrophotometer at a wavelength of 557 nm. Calibration curve of

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hydroxyproline was used as standard for the estimation of hydroxyproline in each test

sample.

2.8.8. Statistical analysis

Experiments were performed in thrice (n=3) and mean SEM obtained results were analyzed

by using one way ANOVA followed by as Tukey-Kramer Multiple Comparisons Test

employing statistical software (GraphPad, InStat 3). Variations between groups were

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considered significant at P < 0.05 levels.

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3. Results and discussion

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3.1. Biogenic synthesis and characterization of WfAuNPs

In the present investigation, the color change of the reaction mixture containing 5 ml of
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WfAe extract and 95 ml of 1 mM HAuCl4 from dark brown to ruby red were observed

visually which confirmed the initiation of gold ions (Au+) bioreduction into its
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nanoparticulate form (Au0). The change in color of the reaction mixture was primarily due to

the excitation of WfAuNPs surface plasmon resonance (SPR) phenomenon which was
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recorded at 524 nm (Fig. 1a). No color changed and shifting in SPR peaks were observed
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when the UV-visible spectrum of synthesized WfAuNPs were recorded up to 168 h (7 days).

The results were in good agreement on completion of WfAuNPs synthesis and also indicated
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the smaller size and shape of WfAuNPs [47]. Crystalline nature of lyophilized WfAuNPs

examined using XRD techniques, showed the four characteristic diffraction intense peaks at
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2 = 38.0o (111), 44. 2o (200), 64. 8o (220) and 77. 6 (311) respectively (Fig. 1b). The

obtained XRD diffractograms were analysed using PANalytical XPert High Score Plus

software which showed that the particles were face centred cubic (fcc) lattice when matched

with the Joint Committee on Powder Diffraction standards, (JCPDS no. 04-0784). Similar

XRD diffractograms were reported by other researchers while synthesizing biogenic gold

nanoparticles using microwave irradiation techniques at 2450 MHz [48]. Synthesized

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WfAuNPs exhibited the high negative (-29.9) surface zeta potential value and stability (Fig.

1c). The greater surface (negative) charge value might be responsible for active functional

constituents works as capping agents present on the WfAe. Particle size distribution studies

were performed using DLS measurements. The average diameter (d. nm) of synthesized

WfAuNPs were found to be 65.22 nm at 100 % intensity while polydispersity index (Pdl)

values was 0.029 (Fig. 1d). The synthesized WfAuNPs particle size value obtained in DLS

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measurement studies was higher as compared to size obtained in FESEM and HRTEM due to

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the hydrodynamic radius probed with DLS. Similar interpretation has been reported earlier by

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other researchers [49]. Tomoaia et al. 2015 stated that, during DLS measurements studies, the

DLS was calculated to be of greater size due to organic coating on synthesized AuNPs
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whereas in HRTEM, only the particle size is taken into account [50]. Presence of biomaterials

in WfAe responsible for bioreduction and stabilization of WfAuNPs were analysed by FTIR
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analysis. The FTIR peaks of WfAe present at 3401 cm-1 represents hydroxyl phenolic (O-H)

group while the absorption peaks at 2958 cm-1 indicated aromatic C-H stretching vibrations
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(Fig. 2a). The peaks at 2921 and 2818 cm-1 denoted (C-H) aliphatic stretching vibrations. The
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FTIR peaks observed at 1726 cm-1 indicated (C=O) carbonyl group and peaks observed at
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1622 cm-1 exhibited aromatic (C=C) stretching vibrations. Further, the peaks detected
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between 1463-1378 cm-1 showed coupled vibrations of carbonyl group (C=O). Finally the

absorption peaks at 1256, 1161, 1086 cm-1 indicated (C-O) stretching vibrations and peaks at
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832 cm-1 exhibited aromatic C-H bending. After WfAuNPs synthesis the characteristics peaks

in Fig. 2b were shifted in 3438, 2958, 2832, and 1632 cm-1, respectively which could be

assumed that the presence of phenolic hydroxyl groups and proteins (absence of amide III

peak 1726 cm-1) which plays a vital role as reducing agent to be involved in the synthesis and

stabilization of WfAuNPs. The topology of synthesized WfAuNPs were analysed by using

FE-SEM, HRTEM and AFM. FE-SEM image at 100 KX magnification which showed that

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the synthesized WfAuNPs particles were spherical and polydispersed in nature with slight

agglomeration (Fig 3a). In EDX spectrum strong signals of gold atoms were observed

whereas weak signals of carbon, oxygen, sodium and calcium were due to the

phytoconstituents present on the synthesized WfAuNPs (Fig. 3b). The three dimensional (3D)

and two dimensional (2D) structures of synthesized WfAuNPs were analysed by AFM

studies as shown in Fig. 3c-d. The synthesized WfAuNPs were further characterized by

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HRTEM with SAED analysis at different magnifications (50 nm and 10 nm). Data showed

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that the most of the synthesized particles were spherical in nature with the size range of 10-20

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nm and average particle size of 13 1.2 nm respectively (Fig. 3e-h). A few prism and rod

shaped AuNPs particles were also observed by other researchers in HRTEM images while
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working on Galaxaura elongata [51].

3.2. Evaluation of nanoformulation (WfAuNPs-Carbopol 934)

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The 1 % (w/v) WfAuNPs-Carbopol 934 gel viscosity and spreadability was found to be

28360 428 cP at 10 rpm/min and 7.38 1.23 cm2 respectively while the viscosity and
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spreadability of 2 % (w/v) WfAe-Carbopol 934 gel was found to be 17600 324 cP at 20

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rpm/min and 6.91 1.02 cm2. The obtained values exhibited good consistency for topical
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applications in wound healing. After mixing, triethanolamine solution in Carbopol gel get
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converted into three dimensional linkage which unveils the unwinding of acrylic acid

molecules at basic pH (6.5) in the presence of water. The spreadability of the developed
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formulation is an important factor for the ease of delivery of active molecules to the affected

area [1].

3.3. Antibiofilm activity

3.3.1 Antifungal and antibiofilm activity of WfAuNPs against Candida albicans and
Cryptococcus neoformans

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MIC assay was employed to access the effectiveness of WfAuNPs against planktonic cells

of both C. albicans and C. neoformans tested strains. The MIC80 was defined as the lowest

concentration of WfAuNPs that causes 80 % decrease in cell growth as compared to the

control. MIC80 of C. albicans and C. neoformans were found to be 16 g/ml and 32 g/ml

respectively. Our results were consistently similar to Wani et al. 2013 which showed that

the AuNPs exhibited potential antifungal activity against C. albicans due to H+-ATPase

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inhibition [52]. Data showed that the green-synthesized WfAuNPs effectively reduce the

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pathogenic biofilm formation. The BIC80 was defined as the lowest concentration of

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WfAuNPs that causes 80 % reduction in metabolic activity of biofilm formation as

compared to the control. Data showed that C. neoformans has BIC80 (64g/ml) which was
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two fold higher compared to BIC 80 for C. albicans (Table 1). The effect of WfAuNPs on

biofilm formation demonstrated that at 256 g/ml there was 96.7 % and 92.2 % reduction in
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metabolic activity of C. albicans and C. neoformans respectively (Fig. 4a). This finding

suggested that C. neoformans biofilm was less susceptible to the WfAuNPs as compared to
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C. albicans. The BEC80 was defined as the lowest concentration of WfAuNPs that
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eliminates 80 % of biofilm as compared to the control. It was also observed that preformed
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biofilm were less susceptible than planktonic cells forming biofilm when tested using
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different concentration of WfAuNPs (Fig. 4b). Preformed biofilm of both C. albicans and

C. neoformans were found to be equally susceptible to WfAuNPs (BEC 80 256 g/ml) as

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shown in Table 1. Earlier, researchers working on gold nanoparticles have shown its

inhibitory effects of on pathogenic biofilm formation [53,54]. It has been reported that

surface physicochemical properties of the nanoparticles greatly influence the cell adhesion

process and several efforts have been made to understand and control the mechanism of cell

adhesion including surface properties for biomedical application. It has been demonstrated

that the strong electrostatic interaction between nanoparticles and pathogenic cell wall

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surface interrupt adhesion mediated interactions between cells and substrate representing

for this inhibition [55-57].

3.3.2 Morphological changes in the biofilm cells

The changes in cellular morphology of C. albicans and C. neoformans biofilm in the

presence and absence of WfAuNPs were analysed using SEM. Images of biofilm without

gold nanoparticles showed network of densely packed yeast cells having smooth surface

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surrounded by extracellular matrix (Fig. 5a, 5g). C. albicans biofilm formed at BIC80 of

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WfAuNPs (32g/ml) revealed disrupted biofilm while C. neoformans biofilm cells at

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(64g/ml) were reduced, scattered and distorted (Fig. 5b, 5h). At BEC80 concentration of

WfAuNPs (128g/ml) against C. albicans biofilm, cells were visualized to have damaged
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hyphae and ruptured yeast cells. Similarly wrinkled and ruptured cells were also observed

in C. neoformans biofilm at 256 g/ml concentration of WfAuNPs (Fig. 5c, 5i).

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3.4. Acute dermal toxicity studies

No sign of redness, erythema, changes in fur, behavioural pattern and mortality was observed
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at the tested dose levels.

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3.5. Effects of WfAuNPs-Carbopol 934 on wound closure and epithelization period (In

vivo)
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Size differences in cutaneous excision wounds during the course of treatments (23 days)

wound closures rate (WCR) were measured. The WCR of Group I was found to be 7.27
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0.35 mm2, 21.35 0.23 mm2, 43.02 0.29 mm2 and 67.20 0.26 mm2 and for Group II 8.90

0.26 mm2, 22.47 0.45 mm2, 43.84 0.28 mm2 and 69.18 0.27 mm2 on 3, 6, 9 and 12

day respectively, while in standard Group III revealed (19.04 0.40 mm2, 45.40 0.25 mm2,

76.08 0.31 mm2, 96.01 0.21 mm2) WCR as shown in Table 2.

Further, the initiation of healing effects was observed from day 3 of treatment for Group

IV with WCR (14.24 0.26 mm2) while significant effect was recorded in day 6 (35.67

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0.29 mm2), day 9 (60.98 0.39 mm2), day 12 (81.79 0.22 mm2) respectively. Enhancement

in wound healing activity was also observed in Group V with WCR (16.93 0.32 mm2, 40.94

0.34 mm2, 73.15 0.31 mm2 and 93.80 0.15 mm2) in 3 -12 days period which were

consistently comparable with standard Group III. However, as evident from the results Group

I and Group II manifests slow wound healing potential (Fig. 6). Maximum epithelialization

period (15.50 0.22 days) was observed in Group V which was consistent and comparable

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with the standard Group III (14.83 0.30 days) and Group IV (17.50 0.42 days) signifies

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its contribution of Group V in the enhanced epithelialization as well as consuming minimal

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time in whole epithelialization process (P < 0.01). Further, WCR data unveils slow wound

healing potential and epithelization period of Group I (23.16 0.47 days) and Group II
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(21.83 0.30 days).

3.6. Effects of WfAuNPs-Carbopol 934 on Tissue hydroxyproline content

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The presence of high concentration of hydroxyproline around wound area eventually leads to

higher level of collagen which is confirmed by the increased microcirculation of collagen

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fibrils. Significant increase in hydroxyproline level was observed (P < 0.01) in animals
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treated in a dose-dependent manner as compared to group I and group II (Table 2). The order
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of collagen stability for animals treated with different formulations was found to be in
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following order; standard group III > group V > group IV > group II > control group I.

3.7. Effects of WfAuNPs-Carbopol 934 on tensile strength of the wound

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Data obtained from Group V showed higher tensile strength in concentration dependent

manner compared with Group I and Group II (P < 0.01 and P < 0.001). Ideally a wound

healing agents must promote the formation of collagen fibrils on wounded site which in turn

results in the greater tensile strength of wounds which can be evaluated by using tensiometer

(Table 3).

3.8. Histopathological study

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The therapeutic effectiveness of WfAuNPs-Carbopol 934 (Group V) nanoformulation for

wound healing potential in terms of re-epithelialization and skin rejuvenation was observed

using H&E and Massons Trichrome staining assay. Histopathological examination of

excised tissues obtained from the wound of all animals group (Group I to Group V) was

performed on the 16th day and their histopathological features were analysed. Group I

(control) and Group II (Carbopol 934 ointment base) treated animals shows unorganized

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granulation, blood capillaries, less collagen and reduced hair follicles formation (Fig. 7a, b)

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while in Group IV (WfAe-Carbopol 934) manifest irregular hair follicles, monocytes and

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blood platelets regeneration (Fig. 7d). Group III (5% Povidone iodine) shows complete tissue

rejuvenation with the formation of hair follicles, monocytes, blood vessels and collagen fibers
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(Fig. 7c). Furthermore, Group V (WfAuNPs-Carbopol 934) represented the presence of

well-organized collagen fibres in dermis along with blood vessels (angiogenesis), aggregation
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of monocytes and hair follicles at wound site which serves as potential candidates for wound

healing (Fig. 7e). The presence of high collagen content was observed in histological sections
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prepared for Massons Trichrome staining assay with stained untreated control Group I,
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Group II, Group III, Group IV and Group V, treated wounds were represented in Fig. 8 a-e.
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The well-organized extracellular matrix with notable amount of collagen deposition, and
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newly formed blood vessels were observed in Group III (Fig. 8c) and Group V (Fig. 8e) as

compared to the Group I (Fig. 8a), Group II (Fig. 8b), Group IV (Fig. 8d). The appearance of
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reepithelization (red color) on wounded tissue was clearly observed in Group III and Group V

treated wounds. Earlier, investigation have shown that the wound healing potential in 18

days at 250 mg/kg body weight dose and reepithelization period was 19 days while working

on ethanolic extract of W. fruticosa flowers [58]. However, data recorded in this study

manifests marked improvement in wound healing potential and was found to be enhanced in

12 days with lesser dosage requirement however it showed 15 days reepithelization

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period. Further critical examination of excised wound tissue unravels that WfAuNPs-

Carbopol 934 (Group V) not only accelerate the wound-healing process but it also enhances

the tensile strength of the healed skin tissue akin to normal skin. WfAuNPs-Carbopol 934

nanoformulation showed its ability to regulate the collagen deposition together with proper

matrix and spatial arrangement which helps in quick healing of wounded tissue. Fibroblast

differentiation and collagen production may be the contributing factor for the above

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mentioned characteristic wound healing features [59,60]. However, the molecular pathway

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involved in the regeneration of collagen by using nanoformulation of WfAuNPs-Carbopol

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934 further needs to be investigated.

4. Conclusion
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Topical application of WfAuNPs-Carbopol 934 nanoformulation over wounded and

abrasive tissue results in rapid aggregation of collagen fibrils, granular tissue formation and
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rejuvenation of epithelial lining leading to quick healing and closures of wounds as compared

to standard marketed drug (5 % Povidone iodine) and control. Experimental data

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demonstrated that topical application of WfAuNPs-Carbopol 934 nanoformulation to be a

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reliable and easy method to heal wounds and prevents scar formation when tested in vivo
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using Wistar albino rat model. These investigations further unravels additional insight into
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the characteristic wound healing features of WfAuNPs and opens up new scope for clinical

scientist and physicians in development and designing of novel treatment approaches with the
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help of novel nanoformulations.

5. Acknowledgements

The authors are expressing warm thanks to the Sapience Bioanalytical Research Lab, Bhopal,

Madhya Pradesh, India for providing facilities to conduct (in vivo) animal experiments and

Institute Instrumentation Centre (IIC), Indian Institute of Technology, Roorkee for providing

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HRTEM, FESEM, AFM characterization facilities for this work. Authors are also thanks to

All India Council for Technical Education (AICTE, New Delhi, India) for financial support.

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differential responses in keratinocytes and fibroblasts during skin wound healing. Chem.

Med. Chem. 5 (2010) 468-475.

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[60] A. K. Srivastava, P. Khare, H. K. Nagar, N. Raghuwanshi, R. Srivastava,

Hydroxyproline: A potential biochemical marker and its role in the pathogenesis of different

diseases, Curr. Protein Pept. Sc. 17 (2015) 596-602.

Legends to Figures and Tables

Fig. 1. (a) UV-vis spectrum of WfAuNPs at 524 nm for the period of 168 h (b) XRD

diffractogram of WfAuNPs (c) Zeta potential and (d) Hydrodynamic size (DLS)

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measurements of WfAuNPs.

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Fig. 2. FTIR spectrum of (a) WfAe and (b) WfAuNPs.

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Fig. 3. Morphology of synthesized WfAuNPs (a) FESEM (b) EDX (c) AFM 2D (d) AFM 3D

images and micrographs of HR-TEM analysis at (e) 50 nm (f) 10 nm resolution (g)

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Histogram of particle size distribution (h) SAED pattern of synthesized WfAuNPs.

Fig. 4. Effect of WfAuNPs on C. albicans and C. neoformans (a) biofilm formation and (b)
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preformed biofilms.

Fig. 5. SEM images of C. albicans (a-f) and C. neoformans (g-l) revealed effects of
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WfAuNPs at different concentration.

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Fig. 6. Wound healing assessment of full thickness excision wounds in Wistar albino rats.
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Macroscopic appearance of wound closure (Group I to Group V) at 0, 3, 6, 9 and 12th days

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after treatment with different formulations.

Fig. 7. Micrographs of H&E stained wounded tissues for (a) control, (b) Carbopol 934
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ointment base treated, (c) Standard, Povidone iodine 5% (w/w) ointment treated, (d) WfAe-

Carbopol 934 treated and (e) WfAuNPs- Carbopol 934 nanoformulation treated after 15

days of treatment. Re-synthesized blood capillaries (BC), hair follicles (HF), Monocytes

(MC), Blood platelets (BP) and Erythrocytes (EC) are indicated by arrows.

Fig. 8. Micrographs of Massons trichrome stained wounded tissue after 15 days (a) control

(b) Carbopol 934 ointment base treated, (c) Standard, Povidone iodine 5% (w/w) ointment

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treated, (d) WfAe-Carbopol 934 gel treated and (e) WfAuNPs-Carbopol 934

nanoformulation treated. Abbreviations: Re-synthesized collagen fibres, muscles,

erythrocytes and hair follicles, are denoted by arrows.

Table 1. WfAuNPs susceptibility study on C. albicans and C. neoformans and their

respective MIC80, BIC80 and BEC80 values.

Table 2. Effect of WfAuNPs-Carbopol 934 nanoformulation on Wound contraction %,

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Epithelization period and Hydroxyproline content of wounds in excision wound model.

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Table 3. E ect of WfAuNPs-Carbopol 934 nanoformulation on tensile strength of wounds

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in incision wound model.
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Fig. 1.

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Fig. 2.

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Fig. 3.

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Fig. 4.

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Fig. 5.

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Fig. 6.

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Fig. 7.

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Fig. 8.

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Table 1.

Organism and strain MIC80 (g/ml) BIC80 (g/ml) BEC80 (g/ml)

Candida albicans 16 32 256

Cryptococcus neoformans 32 64 256

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Table 2.

Epithelization period Hydroxyproline

% wound contraction
Group (Days) content (g/100 mg
3rd day 6th day 9th day 12th day tissue)
I 7.270.35 21.350.23 43.020.29 67.200.26
23.160.47
P T 32.490.55

II 8.900.26 a* 22.470.45 43.840.28

69.180.27
a***
21.830.30
R I 34.661.02

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19.040.40 45.400.25 76.080.31 96.010.21 14.830.30 44.650.75a***, b***
III a***, b ***
a***, b***
14.240.26
a***, b***
35.670.29
a***, b***
60.980.39
a***, b***
81.790.22
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IV a***, b***,
c***
16.930.32
a***, b***,
c***
40.940.34
a***, b***,
c***
73.150.31
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a***, b***,
c***
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a***,b***, c***

15.500.22 41.630.75a***,
V a***, b***,
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a***, b***,
c*** c***
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a***, b***, a***, b***,
c***
a***, b*** b***, c*

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All values are represented as mean SEM, n=6 animals in each group, Data were analyzed by one-way ANOVA, followed by Tukey-Kramer

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Multiple Comparisons Test, a-significant difference as compared to untreated group (group-I), b-significant difference as compared to ointment

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base treated group (group-II) and c-significant difference as compared to standard group (group-III) and *P<0.05, **P<0.01, ***P<0.001,

ANOVA (Analysis of variance), SEM=Standard error of mean.

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Table 3.

Group Wound breaking strength (g)

I 176.50 2.69
II 179.33 1.38
III 242.86 4.73 a***, b***
IV 217.32 4.08 a***, b***, c***

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V 234.91 1.95 a***, b***

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All values are represented as mean SEM, n=6 animals in each group, Data were analyzed

by one-way ANOVA, followed by Tukey-Kramer Multiple Comparisons Test, a-significant

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difference as compared to untreated group (group-I), b-significant difference as compared to

ointment base treated group (group-II) and c-significant difference as compared to standard
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group (group-III) and *P<0.05, **P<0.01, ***P<0.001, ANOVA (Analysis of variance),

SEM=Standard error of mean.

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Graphical abstract

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Highlights

First report on biogenically synthesized Woodfordia fruticosa gold nanoparticles.

Woodfordia fruticosa (WfAuNPs) gold nanoparticles significantly reduced cell adhesion.

WfAuNPs-Carbopol 934 nanoformulation leads to re-epithelialization of wound.

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Rapid formation of collagen fibers observed via Massons Trichrome staining.

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