Biofouling

The Journal of Bioadhesion and Biofilm Research

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A self-assembled clavanin A-coated amniotic

membrane scaffold for the prevention of biofilm
formation by ocular surface fungal pathogens

Santi M. Mandal, Jahangir Khan, Denial Mahata, Suman Saha, Jayangshu

Sengupta, Osmar N. Silva, Subhayan Das, Mahitosh Mandal & Octavio L.
Franco

To cite this article: Santi M. Mandal, Jahangir Khan, Denial Mahata, Suman Saha, Jayangshu
Sengupta, Osmar N. Silva, Subhayan Das, Mahitosh Mandal & Octavio L. Franco (2017)
A self-assembled clavanin A-coated amniotic membrane scaffold for the prevention of
biofilm formation by ocular surface fungal pathogens, Biofouling, 33:10, 881-891, DOI:
10.1080/08927014.2017.1383400

To link to this article: https://doi.org/10.1080/08927014.2017.1383400

View supplementary material Published online: 19 Oct 2017.

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Biofouling, 2017
VOL. 33, NO. 10, 881–891
https://doi.org/10.1080/08927014.2017.1383400

A self-assembled clavanin A-coated amniotic membrane scaffold for the

prevention of biofilm formation by ocular surface fungal pathogens
Santi M. Mandala, Jahangir Khana, Denial Mahatab, Suman Sahac, Jayangshu Senguptac, Osmar N. Silvad,
Subhayan Dase, Mahitosh Mandale and Octavio L. Francod,f
a
Central Research Facility, Indian Institute of Technology Kharagpur, Kharagpur, India; bRubber Technology Centre, Indian Institute of Technology
Kharagpur, Kharagpur, India; cPriyamvada Birla Aravind Eye Hospital, Kolkata, India; dS-Inova Biotech, Pos-Graduação em Biotecnologia,
Universidade Católica Dom Bosco, Campo Grande, Brazil; eSchool of Medical Science and Technology, Indian Institute of Technology, Kharagpur,
India; fPrograma de Pós-Graduação em Ciências Genômicas e Biotecnologia, Centro de Analises Proteômicas e Bioquímicas, Universidade
Católica de Brasília, Brasília, Brazil

ABSTRACT ARTICLE HISTORY

Amniotic membrane (AM) is frequently used in ophthalmologic surgery for rapid ocular surface Received 20 April 2017
reconstruction. Sometimes it may create a major problem with associated infections after biofilm Accepted 15 September 2017
formation over the membrane. To overcome this problem, AM was coated with the antimicrobial KEYWORDS
peptide clavanin A. The antifungal activity of clavanin A in the native and self-assembled form was Amniotic membrane;
determined against the common ocular surface pathogens Candida albicans, Aspergillus fumigatus, antimicrobial peptides;
Alternaria sp. and Fusarium sp. Biofilm formation over the coated surface was significantly reduced anti-biofilm; clavanin A;
in comparison with the uncoated membrane. The coated membrane revealed effectiveness in terms self-assembled
of biocompatibility, cell attachment colonization when tested in non-cancerous 3T3 and human
embryonic kidney (HEK)-293 cell lines. Clavanin A-coated AM also exhibited excellent physical,
morphological and antifungal characteristics, indicating potential applicability for ocular surface
infection control.

Introduction the ability to reduce ocular surface inflammation and scar-

Major complications of surgical procedures can be caused
by serious bacterial or fungal infection after surgery.
Covering the surgical site with appropriate biomaterial
could prevent such infections (Sarabahi 2012). Recently,
several biocompatible polymers from cellulose, collagen
and chitosan have been developed as coating materials
(Croisier and Jérôme 2013). Moreover, several synthetic
polymers such as polyurethane, polysulfone and polyether
have been also used in biomedical application, but these
have limitations for wound or surgical regions due to their
cytotoxicity (Mogoşanu and Grumezescu 2014).
Amniotic membrane (AM), the innermost layer of the
placenta, has been considered as a coating material, and
it has been used in transplantation for the last 100 years
(Mermet et al. 2007). More attention is now being given to
AM in ophthalmologic uses. AM has high biocompatibil-
ity and antimicrobial characteristics, which make it ideal
for use in ocular surface reconstruction. De Roth (1940)
first reported the use of AM in the eye for the reconstruc-
tion of conjunctival defects. Its revival in 1990 was due to
ring, promoting rapid epithelialization due to the pres-
ence of growth factors and antimicrobial properties. Kim
et al. demonstrated reconstruction in rabbits. As far as the
cornea is concerned, the AM acts as biological contact
lens when transplanted over thin or perforated corneas
(Letko et al. 2001). An AM graft can be used as an effective
biomaterial to improve wound healing in corneo-scleral
ulcerations (Abbasi 2012). It has also been found to be
effective in promoting epithelialization and preventing
corneal perforations in acute fungal infection (Chen
et al. 2006).
AM is widely used in various ocular surface diseases
such as corneal ulcers, corneal abrasions, endogenous
endophthalmitis, neurotrophic keratitis and persistent
epithelial defects, band keratopathy, bullous keratopathy,
conjunctival defect after the excision of a conjunctival mass
(Espana et al. 2002), pterygium, acute chemical injury, and
chronic limbal deficiency (Tseng et al. 1998). Fungal kera-
titis is an inflammation of the cornea that results from fun-
gal colonization or epithelial infiltration and/or invasion

CONTACT Santi M. Mandal mandalsm@gmail.com

Supplemental data for this article can be accessed here. https://doi.org/10.1080/08927014.2017.1383400
© 2017 Informa UK Limited, trading as Taylor & Francis Group
882  S. M. MANDAL ET AL.
of the corneal stroma. Ocular fungal keratitis is the most
frequent disease of the eye and is one of the most com-
mon infectious ocular diseases in India (Thomas 2003).
AM transplantation is sometimes useful for the manage-
ment of infectious corneal ulcer from Staphylococcus spp.,
Pseudomonas spp. and fungi (Tseng et al. 1998; Chen et
al. 2006). AM does not interfere in the penetration of
topical antibiotics to corneas and antibiotic-soaked AM
may act as an effective drug-delivery vehicle (Kim et al.
2001). AM can also protect the ocular surface from being
exposed to unwanted proteolytic damage caused by pro-
teases released by bacteria and inflammatory cells (Lass
1997). However, fungal mat formation over AM has also
been observed, which encouraged the authors to make a
coating over AM using some antimicrobial compounds.
In these circumstances, antimicrobial peptides (AMPs)
appear to be excellent candidates as a coating material.
Biomaterials derived from self-assembled nanostructures
of AMP have shown several biomedical applications (Roy
et al. 2013). Clavanins belong to a family of antimicrobial
peptides that are expressed in the hemocytes of the marine
organism Styela clava (Lee, Zhao, et al. 1997). Similar to
many other naturally occurring antimicrobial peptides,
clavanins exert their antimicrobial effect via permeabili-
zation of the target membranes (Thomas 2003). Clavanins
are multipotent, cationic, α-helical, amphipathic peptides
(Figure 1) with a length of 23 amino acid residues (Lee et
al. 1997; Silva et al. 2015). They are exceptionally rich in
glycine, histidine, and phenylalanine which play impor-
tant roles in the antimicrobial actions of clavanin A (Silva,
Figure 1. Structure and function of clavanin A. Clavanin A may have direct effects on pathogens, eg by membrane disruption through
pore formation in neutral pH. In addition to direct effects, clavanin A may permeabilize the membrane, probably by interacting with
proteins involved in proton translocation in mildly acidic conditions (van Kan et al. 2001).
et al. 2016). The amino acid residues of clavanin A are
VFQFLGKIIHHVGNFVHGFSHVF-NH2.
In this study, clavanin A was used as a material for
coating over AM after self-assembled modification. The
self-assembled structure coated over AM was tested
for antifungal activities against Candida albicans and
Aspergillus fumigatus isolated from patients with kera-
titis. Biofilm formation by multi-drug resistant C. albi-
cans and A. fumigatus strains was evaluated over coated
and uncoated membrane. The biocompatibility of coated
membrane was also evaluated against non-cancerous
mouse embryo fibroblast (3T3) and human embryonic
kidney (HEK) 293 cell lines in vitro.
Materials and methods
Preparation of AM
AM was prepared after minor modifications of a standard
protocol (Kim and Tseng, 1995). Placenta was acquired
from Bellevue Hospital, Kolkata, India. The maternal
donor was confirmed negative in serological screening
for human immunodeficiency virus (HIV), hepatitis B and
C, and syphilis. Under a laminar air hood, this placenta
was cleared of blood clots with Earle’s balanced salt solu-
tion (HiMedia, Mumbai, India) containing 40 μg ml−1 of
gentamycin, 100 μg ml−1 of penicillin G and 2.5 μg ml−1 of
amphotericin B. The AM was separated from the chorion
by dissection and the amnion with epithelial/BM (base-
ment membrane) side up was flattened and spread onto

a nitrocellulose paper (Figure S1). Then the nitrocellu-
lose paper along with the AM was cut into the required
dimensions and stored in vials containing Dulbecco’s
modified Eagle’s medium (DMEM) (HiMedia) with anti-
biotics (100 μg ml−1 of penicillin G) and 100 μg ml−1 of
streptomycin).
Peptide synthesis
Clavanin A was synthesized by Peptides 2.0 (Chantilly,
VA, USA) using N-9-fluorenylmethyloxycarbonyl (Fmoc)
solid-phase synthesis, and purified by high-performance
liquid chromatography (HPLC). The sequence and degree
of purity (>95%) was confirmed by MALDI-TOF analyses
(Figure S2).
Preparation of self-assembled clavanin A
Clavanin A was added to chloroform (2 mg ml−1) and
further sonicated for 10 min to form clear dispersion of
solution. The pH of the solution was checked and main-
tained at 7.0. One ml of clavanin solution was taken in a
2 ml centrifuge tube and rotated at 1,000 rpm in a vor-
tex shaker (Tarson 3020 Spinix Vortex Shaker, Tarsons
Products Pvt Ltd, New Delhi, India) for 12 h following
the method described earlier.
Circular dichroism (CD)
The CD spectrum was acquired by a Jasco J-815 spec-
tropolarimeter (Maryland, USA) equipped with a Jasco
PTC-423 S Peltier temperature controller. The scanning
rate was 50 nm min−1 with a 2 s response time and
the data-pitch was 0.5 nm in continuous mode. The
scanning range was 300–190 nm. The baseline was cor-
rected by subtracting the chloroform as blank (Silva et
al. 2012).
Fourier-transform infrared spectroscopy (FTIR)
The KBr (200 mg) disk was pressed, 20 μl of each solution
(peptide and self-assembled peptide) were dropped onto
a disk and air dried. Data were acquired in a Shimadzu
8400 FTIR spectrophotometer. Absorbance spectra were
obtained from 4,000 to 400 cm−1 with a 4 cm−1 resolution.
Background spectra were also collected and subtracted.
Scanning electron microscopy (SEM) analyses
For SEM images of self-assembled clavanin A, 5 μl of stock
solution (10 mg ml−1) were placed on the glass coverslip
and dried under room temperature. Samples were fixed
onto a graphite stub and kept in an auto sputter coater
BIOFOULING  883
(E5200, Bio-Rad, Hadapsar, Pune, India) under low vac-
uum for gold coating up to 120 s. Surface morphology
was analyzed by a scanning electron microscope (JEOL
JSM 5800, GenTech Scientific, NY, US) with an accelerated
voltage between 5 and 20 kV.
Coating on AM
AM was washed with sterile phosphate buffer saline (PBS)
containing 100 μg ml−1 of penicillin G and 2.5 μg ml−1 of
amphotericin B and cut into pieces. The AM was deprived
of amniotic epithelial cells by incubation with 0.02% eth-
ylenediamine tetra acetic acid (EDTA) at 37℃ for 2 h. The
appropriate amount of the self-assembled peptide solution
(10 μl cm−2 of 10 mg ml−1) was applied to the entire surface of
the AM, which was then placed horizontally at room temper-
ature for 30 min (Samanta et al. 2013). The AM with peptide
solution was air dried for 30 min in a laminar hood and
vacuum-packed at room temperature. Finally, γ-radiation
(9 kGy) was used in a Gamma Chamber 5000 (Manufactured
by Department of Atomic Energy, GOI, Mumbai, India) to
sterilize the resultant clavanin A coated AM (CC-AM).
Fungal strains and growth conditions
C. albicans SJ11, A. fumigatus JM3, Alternaria sp. SS1
and Fusarium sp. SS2 were used in this study. They were
provided by the Ocular Microbiology Laboratory of the
Priyamvada Birla Aravind Eye Hospital in Kolkata, India
(Samanta et al. 2013). All the strains were isolated from
patients associated with keratitis (Sengupta et al. 2012).
Inoculum suspensions were prepared from fresh five-day-
old cultures grown on Sabouraud agar plates. The colo-
nies were covered with 5 ml of PBS with 5% Tween 20.
For Aspergillus spp., the inocula were attained by softly
rubbing the colonies with a sterile loop and the isolates
were shaken for 30 s with a vortex. The suspension (5%
Tween 20) was transferred to a sterile tube. The inoculum
count (0.5 × 103 CFU ml−1) was adjusted by counting the
colonies. The adjusted suspensions were further quanti-
fied by plating on Sabouraud agar plates and incubated
at 35°C.
Antifungal susceptibility testing
The minimum inhibitory concentrations (MIC) of both
free and self-assembled clavanin A were determined
against the fungal pathogens following the guidelines of
the Clinical and Laboratory Standards Institute (CLSI). In
brief, 200 μl of the Roswell Park Memorial Institute medium
(RPMI) 1640 medium were added to each well of a 96-well
microplate along with fungal inoculum (106 CFU ml−1).
Both clavanin A and self-assembled clavanin A were used
884  S. M. MANDAL ET AL.
at a concentration range of 1–1.95 μg ml−1 (Chandra et al.
2001). Two wells of the plate served as growth (without
antifungal) and sterility (without inoculum) controls. The
plates were incubated at 35℃ for 72 h. Experiments were
carried out in triplicate on three different sets.
Biofilm formation on AM
To evaluate the biofilm formation, C. albicans, A. fumiga-
tus, Alternaria sp. and Fusarium sp. were collected from
mature solid culture plate. Then the fungal strains were
added over both coated and uncoated membranes which
were cut into small pieces. The membranes were then
cultured separately in six-well plates containing RPMI
medium. The final concentrations of fungal inoculum
over each membrane were 2.5 × 103 CFU ml−1 and they
were cultured for 72 h at 35℃. After 72 h incubation the
membranes were removed and the planktonic cells were
washed gently with PBS(1×) buffer.
Biofilm quantification
Several methods have been employed to quantify biofilm
formation, such as crystal violet (CV) staining, the XTT
(2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazoli-
um-5-carboxanilide) reduction assay and the alamarBlue
assay (Ramage et al. 2001; Jin et al. 2003; Pettit et al. 2005).
Biofilm quantification by CV staining
In brief, after biofilm formation both the coated and
uncoated membranes were washed gently in PBS and
allowed to air dry for 20 min at 35°C. The membranes
were stained with 0.4% aqueous CV solution for 45 min.
Afterwards, the membranes were washed three times with
Milli-Q sterile water and destained in 200 μl of 95% eth-
anol in an Eppendorf tube. After 45 min, 100 μl of dis-
taining solution (95% ethanol) from each membrane were
transferred to a 96-well plate, diluted 1:4 with sterile water
(as samples exhibiting a very deep blue color give ‘offscale’
absorbance values) before being measured with a plate
reader (Multiskan Spectrum-1500 spectrophotometer,
Thermo Fisher Scientific India Pvt Ltd, Powai, Mumbai,
India) at 595 nm. The absorbance values of the negative con-
trols (membrane without any fungal cells) were subtracted
from the values of the test wells to minimize background
interference (Ramage et al. 2001). Data are presented as the
means of absorbance values of three replicate tests.
XTT-reduction assay for biofilm quantification
The quantitative measurement of biofilm formation was esti-
mated using an XTT-reduction assay (Nett et al. 2011). In
brief, XTT (2,3-bis(2-methoxy-4-nitro-5-sulfo-phenyl)-2H-
tetrazolium-5-carboxanilide; Sigma, Kolkata, West Bengal,
India) was prepared at 0.5 g l−1 in Ringer’s lactate solution.
An aliquot of stock XTT was thawed, and menadione
(Sigma; 10 mM prepared in acetone) was added to a final
concentration of 1 mM. The samples were then incubated
for 3 h at 37°C. The XTT reduction was quantified using
a Multiskan Spectrum-1500 spectrophotometer (Thermo
Fisher Scientific India Pvt Ltd, Powai, Mumbai, India) by
OD490. Data are presented as the means of three replicates.
AlamarBlue assay for biofilm quantification
AlamarBlue (HiMedia; code-TC235) solution was pre-
pared at a concentration of 20 μg ml−1 in methanol pro-
viding a clear blue-colored solution which is reduced by
metabolically active cells to the pink colored resorufin.
The membranes were placed in culture medium for 72 h
at 35℃ as described in previous section on biofilm for-
mation on AM. The membranes grown without inocula
(negative control) and biofilm forming membranes (both
coated and uncoated) were washed gently with PBS buffer.
Then the membranes were sunk into a 96-well plate con-
taining 200 μl of alamarBlue solution, shaken gently and
incubated for 1 h at 37°C. Membranes (both negative and
positive control) were removed and measured at 570 and
600 nm in a microplate reader. The percentage alamar-
Blue reduction was calculated according to the following
equation:
[(O2 × A1) − (O1 × A2)∕(R1 × N2) − (R2 × N1)] × 100
where O1 = the molar extinction coefficient of oxidized
alamarBlue (Blue) at 570 nm, ie 80,586; O2 = the molar
extinction coefficient of oxidized alamarBlue at 600 nm, ie
117,216; R1 = the molar extinction coefficient of reduced
alamarBlue (Red) at 570 nm, ie 155,677; R2 = the molar
extinction coefficient of reduced alamarBlue at 600 nm,
ie 14,652 (McBride et al. 2005); A1 = the value measured
from the test wells at 570 nm; A2 = the value measured
from the test wells at 600 nm; N1 = the value measured
from the negative control well at 570 nm; and N2 = the
value measured from the negative control well at 600 nm.
Assays were performed three times, and the average
percentage reduction expressed as the means of triplicates
with standard deviation (SD).
SEM analyses of C. albicans on clavanin A-coated
AM (CC- AM)
AM and clavanin A-coated AM were characterized by SEM
(Hamid et al. 2014). C. albicans cells were taken from the
log phase of growth. The cells were then washed three
times and resuspended in PBS. The cells were treated with

self-assembled clavanin A with its individual MIC concen-
tration for 1 h. Again, cells were repeatedly washed with
PBS and 5–10 μl of the resuspended solution were placed
on the lysine-coated glass coverslip following the drop-cast
method (Samanta et al. 2013). Membranes were gradu-
ally dehydrated by immersing in 70% ethanol for 6 h fol-
lowed by 95% ethanol (two changes at 1 h each). Finally, to
ensure complete dehydration, the membranes were passed
through 100% ethanol solution for 2 h. The membranes
were then fixed in 2.5% glutaraldehyde solution in PBS.
After that the membranes were washed gently in PBS and
allowed to vacuum dry overnight. The dried membranes
were mounted on aluminum stubs and sputter coated with
gold. The surface morphology was analyzed by SEM (JEOL
JSM5800) with an accelerated voltage from 5 to 20 kV.
In vitro biocompatibility of CC-AM
Non-carcinoma mouse embryo fibroblast (3T3) and
human embryonic kidney (HEK) 293 cell lines were
cultured as monolayers in DMEM supplemented with
10% (v v–1) fetal bovine serum (FBS) and antibiotics.
The AM and CC-AM were spread over the cover glass
contained in a 35 mm Petri dish. Each test membrane
was then placed onto 3T3 fibroblast cell cultures with
a seeding density of 25 × 103 cells cm−2. After two days
incubation at 37°C in the presence of 5% CO2, the MTT
[(3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphe-
nyl)-2-(4-sulfophenyl)-2H-tetrazolium)] assay was per-
formed to quantify the cell cytotoxicity level following
the method described earlier by Mandal et al. (2012). The
fibroblast cells were cultured on coated and uncoated AM
with self-assembled clavanin A. Cell growth was moni-
tored after incubation for 48 h in both the membranes.
The cells over the membrane were counted under a light
microscope. The dimensions of the membrane were meas-
ured and the cell density over the membrane was also
determined.
Statistical analysis
Biofilm formation in different membranes (including
positive and negative controls) was tested in three repli-
cates and the SD was determined from each set of data.
Significance level was determined by comparing means
by using the Student t-test and a p-value was considered
significant when < 0.05.
Results and discussion
AM has received a great deal of attention as an effective
material to control several ocular surface diseases. AM is
well documented in transplantation for reconstruction of
BIOFOULING  885
the corneal surface with persistent epithelial defects, bul-
lous keratopathy, keratitis and corneoscleral ulcers (Dogru
et al. 2003; Burman et al. 2004). More recent literature
also supports the extended use of AM as a substratum
for ex vivo cultivation of limbal, corneal and conjunctival
epithelial cells (Sangwan et al. 2007). However, a limita-
tion was pragmatic when fungal growth occurred over
the membrane by biofilm formation. Therefore, a strategy
has been developed for coating the AM with an antifungal
peptide, clavanin A.
Characterization of self-assembled clavanin A
The molecular interaction of self-assembled clavanin was
characterized by CD and FTIR spectroscopic methods. In
the CD spectrum of clavanin, a prominent peak was not
observed between 240 nm and 220 nm for n–π* transition
for before and after self-assembly. It has been observed that
the peak at 200 nm shifted to 190 nm due to π–π stack-
ing interaction (Greenfield 1999). Two different negative
band shifts occurred in self-assembled clavanin A, as 193
to 197 nm and 197 nm to 201 nm may be the conforma-
tional change of β sheet structure (Figure 2(A)). Another
positive peak shift was observed at 190 nm, which shifted
to 192 nm for α-helix secondary structure. The plausible
noncovalent interaction in self-assembled clavanin A was
characterized by FTIR spectroscopy (Figure 2(B)). The
spectrum of the free clavanin A molecule revealed a broad
stretching peak at 3,413 cm−1 for hydroxyl groups and
3,296 cm−1 for amide groups (N–H), which are the char-
acteristic signals of peptides. Moreover, a sharp absorption
band was observed at 1,660 cm−1 for the stretching mode
of the C=O bond in amide groups. Absorption around
2,955 cm−1 is assigned to the symmetric stretch (–C–H) of
methylene and methyl groups of aliphatic chains in amino
acid (Scalarone et al. 2007). In self-assembled clavanin, the
amide C=O and N-H stretching frequency were shifted
at a lower frequency from 1,660 to 1,631 cm−1 and 3,296
to 3,257 cm−1, respectively, indicating that the clavanin
molecules aggregated through strong hydrogen bonding
interaction between amide groups. Moreover, the SEM
image of self-assembled clavanin A exhibits a mesoporous
crosslink network apparently with good structural stabil-
ity (Figure 3).
SEM analysis
The SEM images were obtained from both coated and
uncoated AMs. The AM on its own showed a clear epi-
thelial layer with prominent nucleus, whereas upon treat-
ment with 0.02% EDTA solution the AM was uncovered
(denuded AM) and collagen fibers were visualized.
After coating over collagen fibers, a clear morphological
886  S. M. MANDAL ET AL.

(A) (B)
1000
1000
975

950

Elipticity
800
925

900
Elipcity

600 875
190 195 200 205
Wavelength (nm)

400

200
200 220 240 260 280
Wavenumber (nm)

Figure 2. Characterization of self-assembled clavanin A. CD spectrum (A) and FTIR spectrum (B) of pure clavanin A (in black) and self-
assembled clavanin A (in red).

Figure 3. FE-SEM image of self-assembled clavanin A. Images were obtained from native clavanin A, magnification 7.70 KX (a) and self-
assembled clavanin A, magnification 10.0 KX (b).

Figure 4. SEM images of amniotic membranes (AM). Native amniotic membrane; magnification 100× (a); denuded AM, 5.0 KX (b) and
clavanin A coated AM, 5.0 KX (c).
 BIOFOULING  887

change was observed due to the coating of self-assem-

bled clavanin A. The self-assembled clavanin A forms a
­nanoporous-like structure, coated over the collagen fiber
present in the basement membrane of AM, as shown in
Figure 4. Collagen fiber and self-assembled clavanin A
become tightly packed, because of their hydrophobic outer
surface, which reveals their strong hydrophobic interac-
tion. Moreover, there is a chance of another cross-linking
reaction between the free amine of clavanin A with the
carboxyl groups of collagen, which promotes the forma-
tion of covalent bonds.
These bonds are also helpful, and along with hydropho-
bic interaction they stabilize the biomaterial structure and
contribute to its mechanical strength (van Kan, Ganchev,
et al. 2003).

Antifungal activity and biofilm inhibition analysis

Both clavanin A and self-assembled clavanin A were tested
for antifungal activity against C. albicans, A. fumigatus,
Alternaria sp. and Fusarium sp. The self-assembled pep-
tide showed same antifungal activity (MIC 15.62 μg ml−1)
against A. fumigatus and Fusarium sp. compared with pure
clavanin A, whereas activity was doubled when tested
against C. albicans and Alternaria sp. (MIC 7.81 μg ml−1).
The XTT and alamarBlue reduction assay (Figure 5(B)
and (C)) were done to quantify the metabolic activity of
the biofilm. The results showed significant colonization
of the uncoated membrane, whereas fungal colonization
was significantly (p < 0.05) less in self-assembled coated
AM. CV staining was used to quantify biofilm forma-
tion (Figure 5(A)) by the pathogens over the coated and
uncoated membrane. These data were corroborated by
using SEM image analysis (Figure 6).
It has been reported that clavanin A has strong anti- Figure 5. Measurement of biofilm formation by different
bacterial activity in vitro and in vivo (Silva et al. 2015; methods. Crystal violet was used to quantify all the cells present
in the biofilm (A); XTT (B) and the alamarBlue (C) reduction assay
Silva, Fuente-Núñez, et al. 2016). Here, it is proved that were used to quantify the metabolically active cells in a biofilm
its antifungal activity might be due to the cationic and formed over only amniotic membrane (AM) and clavanin-coated
amphipathic nature of peptides, which are rich in glycines, amniotic membrane (CC-AM). Data are presented as means of
histidines, and phenylalanines. The Gly, His, and Phe res- three replicate tests, with SD. A p-value < 0.05 is considered as
idues play important roles in the antimicrobial actions statistically significant (*).
of clavanin A (van Kan, Demel, et al. 2003). Fungi grow
well in acidic medium, so in these conditions the peptide together, the pH-dependent activity of clavanin A is thus
becomes highly positively charged, due to the protona- comparable to histidine-rich peptides.
tion of four histidines within the clavanin A sequence However, in contrast to histatins, which translocate the
(Rydengård et al. 2008). Previous studies have shown that membranes of C. albicans, and induce cell death by non-
the antimicrobial activity of clavanin A was significantly lytic release of ATP (Koshlukova et al. 1999), clavanin A
increased at low pH as compared to neutral pH (Silva, acts directly on fungal membranes, causing disruption
Alves, et al. 2016). Furthermore, the antimicrobial effect of (Figure 7). This presumably is due to specific interactions
histatin 5 is increased at low pH (MacKay et al., 1984) and of clavanin A with membrane proteins that are involved
variants of magainin and histidine-rich peptides LAH4 in generating trans-membrane ion gradients. It has been
have been shown to increase antibacterial activity at low reported that at low pH, clavanin A showed strong activity
pH compared to neutral pH (Mason et al. 2006). Taken against C. albicans (Malik et al. 2016).
888  S. M. MANDAL ET AL.

Effect of self-assembled clavanin A against
C. albicans
The interactions between fungal cell membranes and
self-assembled clavanin A peptide were observed by SEM
(Figure 7). SEM images clearly indicate a morphologi-
cal change after treatment with the peptide for 1 h. This
indicates that the target should be found on the surfaces
such as lipid bilayer or cell walls. Clavanin A may cause
cell membrane disruption by the rapid dissipation of the
transmembrane potentialSilva et al. 2015). Interaction
of amphipathic antimicrobial peptide with cell mem-
brane occurs due to the balance of hydrophobic and

Figure 6. SEM images obtained from amniotic membrane after fungal biofilm formation. Images taken after fungal growth for 48 h
over uncoated membrane (left panel; a, C. albicans; c, A. fumigatus; e, Alternaria sp.; g, Fusarium sp.) show strong fungal adhesion to the
surface and no biofilm was visualized over coated membrane (right panel; b, C. albicans; d, A. fumigatus; e, Alternaria sp.; g, Fusarium sp.).
Magnifications: a, 1.0 KX; b, 1.0 KX; c, 2.33 KX; d, 30.00 KX; e, 500 ×; f, 500 ×; g, 500 ×; h, 500 ×.

Figure 7. Effect of self-assembled clavanin against C. albicans. SEM images of C. albicans on uncoated surface of amniotic membrane,
magnification 5.00 KX (a); SEM image of C. albicans after treatment with self-assembled clavanin A, magnification 5.00 KX (b).
Figure 8. Cytocompatibility of coated and uncoated AM. The
cytotoxic effects of coated and uncoated AM were determined
following a dose-dependent cytotoxicity assay in which 3T3 cells
were counted using a hemocytometer under a light microscope
after culturing over coated and uncoated AM (a), and the cell
viability assay was monitored with MTT reagent using the HEK
293 cell line where no significant cytotoxicity was observed up to
a concentration of 50 μg ml−1 (b).
BIOFOULING  889
electrostatic interactions. The non-polar amino acids in
the side chain of peptide interacts with the hydrocarbon
core of the cell membrane, whereas electrostatic inter-
actions occur between negatively charged phospholipid
head groups and positively charged cationic antimicro-
bial peptides (Dathe et al. 1997). The self-assembling
structure of peptide may facilitate the disruption of cell
membrane by the formation of a pore like structure
(Matsuzaki et al. 1997).
Biocompatibility
The self-assembled modified AM is intended for appli-
cation in fungal infections of the ocular surface, so the
interactions of biological tissue materials with fibro-
blast and epithelial cells were considered. Therefore,
the compound was tested against HEK-293 and 3T3
cell lines. The comparative results between AM and
coated AM are shown in Figure 8(A). The cell den-
sity was found to be lower in the uncoated membrane
when compared with the coated counterpart. Coated
AM could serve as an ideal scaffold since the 3T3 cell
constructed a colonial structure after incubation for
48 h, which is shown by phase contrast microscopy and
the level of cytotoxicity was significantly (p <0.05) less,
as is also confirmed with the MTT assay against HEK
293 cell lines (Figure 8(B)). Although the surface of the
ocular environment is different than that studied here,
the biocompatibility found herein represents an impor-
tant first step in the study of the immune response and
regenerative potential of a biomaterial with self-assem-
bled clavanin A.
890  S. M. MANDAL ET AL.
Conclusions
In the last decade, AM has brought about major advances
in the reconstructive surgery of the ocular surface, but
there is always a risk of microbial infections after surgery.
The usefulness of AM coated in the antimicrobial pep-
tide, clavanin A, in reducing biofilm formation by ocular
pathogens was demonstrated. In the light of this finding,
the preparation of self-assembly-based biomaterials and
coatings over biomedical materials are an attractive pros-
pect for the near future.
Disclosure statement
No potential conflict of interest was reported by the authors.
Funding
This work was supported by DST-SERB [grant number
EMR/2015/000367], CAPES, CNPq, FUNDECT and FAPDF.
ONS holds a postdoctoral scholarship from the National
Counsel of Technological and Scientific Development (CNPq)
- Brazil [grant number 300583/2016-8].
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