Synthesis, Characterization and Chromatographic Applications of Antimicrobial Cryogels

Full text

Synthesis, Characterization and Chromatographic Applications
of Antimicrobial Cryogels
Antimikrobiyal Kriyojellerin Sentezi, Karakterizasyonu ve
Kromatografik Uygulamaları
Research Article
K. Erol
/ Hacettepe J. Biol. & Chem., 201 7, 45 (2), 187–195
Kadir Erol
Hitit University, Osmancık Ömer Derindere Vocational Higher School, Department of Property Protection and Safety, Corum, Turkey.
ÖZ
Patojenik bakterilerin sebebiyet verdiği hastalıklara karşı antibakteriyel malzemeler son yılllarda olduça önemli
bir ilgi merkezi haline gelmiştir. Bu çalışma kapsamında antimikrobiyal poli(2-hidroksietil metakrilat-glisidil
metakrilat), pol(HEMA-GMA), kriyojeller sentezlenmiş ve yapıya L-Arjinin aminoasidi üzerinden Ag(I) iyonları
immobilize edilmiştir. Yapının karakterizasyonu için; şişme testi, Frouer dönüşümlü infrared (FT-IR) spektroskopisi,
taramalı elektron mikroskobu (SEM), yüzey alanı (BET), elementel ve ICP-OES analizleri yapılmıştır. L-Arjinin amino
asidinden Ag(I) şelatlayıcı ligand olarak yararlanılmış ve kriyojellerin melittin proteini için adsorpsiyon kapasitesi
173.9 mg protein/g kriyojel olarak tespit edilmiştir.
Anahtar Kelimeler
Antimikrobiyal, kriyojel, L-Arjinin, Ag(I).
ABSTRACT
Antibacterial materials, in the last years, have become an important center of attention against diseases be-
cause of pathogenic bacteria. Within the scope of this study, antimicrobial poly(2-hydroxyethyl methacrylate-
glycidyl methacrylate), poly(HEMA-GMA), cryogels were synthesized and Ag(I) ions were immobilized to the struc-
ture through the amino acid L-Arginine. For characterization of the structure; swelling test, Fourier transform
infrared (FT-IR) spectroscopy, scanning electron microscopy (SEM), surface area (BET), elemental analysis and
ICP-OES methods were performed. The L-Arginine amino acid was used as an Ag (I) chelating ligand and the melit-
tin protein adsorption capacity of cryogels was determined as 173.9 mg/g cryogel.
Key Words
Antimicrobial, cryogel, L-Arginine, Ag(I).
Article History: Re ceived: Sep 9, 2016 ; Revised: Oct 2, 2 016; Accepted: Jan 20, 2017; Availab le Online: Apr 1 , 2017.
DOI: 10.15671/HJBC. 2017.151
Correspondence to: K. Erol, Hitit University, Osmancık Ömer Derindere Voc. High. Sc., Dep. of Property Protection and Safety, Çorum, Turkey.
Tel: +90 364 611 5030 Fax : +90 364 611 5133 E-Mail : kadirerol86@gmail.com

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/ Hacettepe J. Biol. & Chem., 2017, 45 (2), 187–195
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INTRODUCTION
Studies on “Turkey’s Important Plant Areas”
have been dated back to the early 1990s.
The results ofInfectious diseases caused by
pathogenic bacteria are a threat to human health
substantially. Silver metal, ion and compounds
has been used as antibacterial material for the
treatment of ulcers, wound healing and for the
protection the food and water for centuries [1].
As promising alternatives, silver salts and pile
metals, silver nanoparticles and the coordination
complexes containing silver metal are increasingly
being used as a new antibacterial agents [2-7].
Metal-organic coordination complexes, in
recent years, because of their potential functional
applications in areas such as separation,
magnetism, catalysis, luminescence, and
membrane gain a lot of attention alongside the
interesting topology [8-13]. One of the materials
used as membrane in the literature is cryogel.
Cryogel membranes, in the past decade,
are polymeric structures been mainly used for
adsorption processes. The cryogels, hydrogel
derivatives, with three-dimensional mega pores
are synthesized in frozen medium, by using
water-soluble monomer/polymer solution in the
presence of an initiator and an activator via free
radical polymerization [14-16].
The frozen ice crystals work as porogen
during polymerization, and the porous structure
formed as a result of melting of frozen ice crystals
is becoming quite helpful and useful material for
chromatographic processes [17].
Among the other parameters making these
polymers attractive, possessing larger pore sizes
(up to 100 microns), high mechanical properties,
high biocompatibility and reusability performance
and the presence of the flow channel linked with
each other can be mentioned [18-20].
In addition, the existence of macropores
in cryogel structure allow the mass transfer of
macromolecular compounds and the migration
of the cells efficiently. Due to these properties,
cryogel are used in tissue engineering [21-24],
as the separation matrix in chromatographic
processes [25-32] and as bioreactor [33,34].
The polymers with different characteristics
can be obtained using the support material and
surface modifications. The molecules selected
according to the structure of the target molecule
cryogel is coupled to the cryogel structure and
the separation and purification process of target
molecule is performed in accordance with affinity
base. The cryogels, under favor of extremely to be
open to modification, can have desired properties
simply and quickly. Starting with this idea, to put
the cryogel interaction with silver ions having
antibacterial feature to have the cryogel with
antibacterial properties is among the options that
come to mind first.
Primarily, poly(2-hydroxyethyl methacrylate-
glycidyl methacrylate), poly(HEMA-GMA), cryogels
were synthesized within the scope of this study.
The cryogel was modified with the immobilization
of L-arginine amino acid and finally reacted with
the silver ion to obtain cryogels with antibacterial
feature. The Ag(I) ion binding feature of the amino
acid L-Arginine, was played a significant role in the
selection as ligand [35]. The cryogels synthesized
were characterized via swelling test, scanning
electron microscope (SEM), Fourier-transform
infrared spectroscopy (FT-IR), elemental analysis
and surface area (BET) methods. In the last stage,
the antibacterial and antifungal properties of
cryogel has been examined.
MATERIALS and METHODS
Material
2-Hydroxyethyl methacrylate (HEMA),
glycidyl methacrylate (GMA), ethylene glycol
dimethacrylate (EGDMA), silver nitrate, ammonium
persulfate (APS), sodium dodecyl sulphate (SDS),
L-arginine, N,N,N‘,N’ -tetramethylethylenediamine
(TEMED) and melittin (from honey bee venom)
was supplied from the company Sigma (St. Louis,
USA). All other chemicals are of analytical purity
and ultra pure (18 MΩ.cm) water in all studies
were used.

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The Synthesis of Poly(HEMA-GMA) Cryogels
GMA (500 µL), HEMA (5000 µL) and distilled
water (6500 µL) were mixed to obtain monomer
phase. The disperse phase was obtained by mixing
sodium dodecyl sulfate (SDS, 1 g) distilled water
(25.60 mL) and EGDMA (2.4 mL). Then the two
phases were mixed with each other and cooled in
an ice bath for 15 minutes before the addition of
APS (20 mg) and TEMED (100 µL). The mixture
obtained was remained at -20°C for 24 hours.
The resulting cryogels were cut in the shape of
membrane (disc). Until the sodium dodecyl sulfate
and other monomer residues were removed from
the medium and the washing water were clear,
the cryogels were washed with distilled water in
a rotator (Multi Bio RS-24 Biosan, Latvia) at the
stirring rate of 10 rpm with the change of washing
water in every 15 minutes.
The Immobilization of Silver onto Poly(HEMA-
GMA) Cryogels
The cryogel membranes (20 unit) was stirred
firstly in the solution of NaOH (10 mL, 1 M) for 2
hours and then in the solution of arginine (10 mL,
5 mg/mL, pH 5.0 acetate buffer) for 24 hours
after washed with distilled water several times,
and finally in the solution of AgNO3 (10 mL, 5 mg/
mL) for 6 hours. The membranes on which silver
was immobilized were washed several times with
distilled water and ethanol (Figure 1).
Characterization Studies
Swelling Test
The water retention capacity of poly(HEMA-GMA)-
Arg@Ag(I) was determined using distilled water.
For this, dry cryogel membranes were carefully
weighed, then thrown into the distilled water in
the isothermal water bath and remained at 25°C
for 30 minutes. Then membranes were placed into
a filter paper to wipe quickly the water retained
on the surface and weighed again to calculate the
water-retention capacity.
The following equation was used to determine
the capacity of water retention.
Water Retention Capacity (%)= [(Ws-Wo)/Wo]x100 (1)
In this equation, Wo and Ws are the weights (g) of
dry and water-retained membrane, respectively.
SEM Analysis
The surface morphology of cryogel membranes
were examined using scanning electron
microscopy (SEM, FEI/Quanta 450 FEG, USA). The
membrane dried by lyophilisation was tailored for
SEM analysis and was put on the double-sided
tape on the SEM holder. The sample was then
coated with a thin gold layer under vacuum. The
sample obtained was, then, inserted into the SEM
device and imaged at different magnification.
FT-IR Analysis
In the determination of the characteristic
functional groups of poly(HEMA-GMA) cryogels,
the Fourier transform infrared spectrometer
(Thermo Scientific Nicolet 6700 FT-IR
Spectrometer, USA) was used. Cryogels were
dried and pulverized primarily (about 2 mg) and
made into pellets homogeneously with anhydrous
potassium bromide powder (KBr) (98 mg, IR
grade, Merck, Germany) and the FT-IR spectrum
was obtained in the wave number range of 400-
4000 cm-1.
Elemental Analysis
About 2-3 mg of sample was dried totally put
into the sample holder of the elemental analysis
(Elementar vario PYRO cube, Germany) device.
The analysis was performed at 1120oC for
combustion tube and at 850oC for reduction tube
to obtain the N% in the sample.
The Determined of Silver Amount Immobilized
onto the Cryogels
The ICP-OES (Spectro Arcos, Germany) device was
used to determine the silver amount immobilized
onto the poly(HEMA-GMA)-Arg cryogels. In
this technique, the sample is excited by
electromagnetic induction up to 10000 K by argon
plasma and the quantity of elements excited are
determined by the specific wavelength emitted.
Plasma is obtained by the excitation of argon
gas electromagnetically via a radio frequency
(RF) generator in induction coils. This happens
by ionization of the incoming gas via hot plasma
continuously.

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Figure 1. The reaction mechanism of poly(HEMA-GMA)-Arg@Ag(I) cryogel formation.

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Surface Area Analysis
The specific surface area of the membranes
was determined with the device Brunauer-
Emmett-Teller (BET; Quantachrome Autosorb®
iQ-Chemi, USA). The cryogel samples dried with
lyophilisation were incubated at 35°C at 100 mbar
for 6 hours under vacuum to eliminate oxygen
and moisture in the pores. Then, the cryogel
samples were treated with nitrogen gas at room
temperature.
Antimicrobial Studies
The micro broth dilution method was used to
test the antimicrobial activities of cryogels. The
microdilution testing protocol was selected to
obtain the MIC [36]. Bacterial and C. globrata
suspensions that were grown overnight in Mueller-
Hinton broth and were standardized in double-
strength Mueller-Hinton broth to 108 CFU/mL
using McFarland No: 0.5 standard solution. The
dimethylsulfoxide (0.002 g/mL, 2 mL) solution
was used to prepare the stock solutions. 100 µL
suspension of each microorganism and compound
tested were added into the wells, respectively. As
a positive growth control, the sterile distilled water
and the medium were used. The first well, in which
there is no turbidity, was identified as the minimal
inhibitory concentration (MIC) at the end of the
incubation at 37oC for 24 h. Chloramphenicol
and ketoconazole were used as the standard
antibacterial agent and whereas as antifungal
agent, respectively.
The Application of Adsorption/Desorption
Using Melittin Protein
The melittin protein, which is a small polypeptide
and have antimicrobial feature, was selected
to determine the adsorption/ desorption
performance of poly(HEMA-GMA)-Arg@Ag (I)
membranes against biomolecules [37]. The
acetate buffer solution of 4 mL (pH: 5) was mixed
with melittin solution of 1 mL (100 mg/L) and
stirred in a rotator for 15 minutes. Then, in order
to determine the protein concentration prior to
adsorption, sample of 200 µL was separated and
the cryogel membrane was placed into the tube
and stirred at 20 rpm for 30 minutes. At the end
of this period, the membrane was taken from the
adsorption medium and to determine the protein
concentration after adsorption a sample of 2 mL
was taken from the adsorption environment. The
adsorbed amount of melittin was determined
via UV-VIS spectrophotometer (Double Beam
PC 8 Auto Cell Scanning UVD-3200 Labomed,
INC., USA) at 280 nm wavelength. The melittin
adsorption capacity of cryogels was determined
according to the following formula:
q = [(Ci-Cf) x V]/m (2)
Here, q is the amount of adsorption (mg / g), Ci
is the concentration of melittin solution (mg/L)
before adsorption, Cf is the concentration of
melittin solution (mg/L) after adsorption, V is the
volume of the adsorption medium (L) and m is the
mass of dry adsorbent (g).
The NaCI solution (10 mL) of 1 M was used for
desorption studies. To determine the reusability
of cryogels, NaOH solution (10 mL) of 50 mM
was used to regenerate cryogels exposed to
desorption in between successive adsorption-
desorption cycles.
RESULTS and DISCUSSION
Characterization Studies
At the end of the swelling test, the swelling rate of
the cryogel was determined as 301.3%. Accordingly,
the dry cryogel of 1 g has about water retention
of 3 g. According to the SEM image of cryogels
examined, macro-pores and interconnected flow
channels were drawn attention (Figure 2). When
the FT-IR spectrum of poly(HEMA-GMA)-Arg
cryogel was examined, the peaks were observed
at 3442 cm-1 (alcohol, -OH), at 2951 cm-1 (alkane,
C-H) and 1731 cm-1 (carboxylic acid, C = O) (Figure
3-a). The peaks for poly(HEMA-GMA)-Arg@Ag (I)
at 3442 cm-1 (alcohol, -OH), at 2951 cm-1 (alkane,
C-H), 1731 cm-1 (carboxylic acid, C=O) and also at
461 cm-1 (N-Ag (I) bond) are clearly visible (Figure
3-b). These results confirm the inclusion of silver
into the polymeric structure [38].
According to the elemental analysis results,
the amount of L-arginine incorporated into the
cryogel structure was estimated as 389 µmol/g
polymer. The amount of Ag(I) immobilized onto
the cryogels with the surface area of 6.289 m2/g
was found as 140.5 µmol/g polymer.

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Antimicrobial Studies
The microorganisms (Candida utilis, Escherichia
coli, Enterobacter aerogenes, Pseudomanas
aeruginosa, Bacillus subtilis, Staphylococcus
aureus) were used to antimicrobial activity
analysis of cryogel dispersion solution (1 mg/mL)
prepared in DMSO-distilled water (1:1) mixture. At
the end of the studies performed, the cryogel
solution have shown antimicrobial activity and
the minimum inhibitory concentration (MIC) was
determined as 0.25 mg/mL.
Melittin Adsorption/Desorption
The adsorption capacity of cryogels for melittin
protein was estimated as 173.9 mg/g at the end
of the adsorption-desorption experiments. This
result indicates that the poly(HEMA-GMA)-
Arg@Ag(I) cryogels have effective adsorption
performance and may be used in chromatographic
processes. It has also been found to have 99%
of melittin desorbed from the melittin-adsorbed
cryogels (Figure 4). Thus, poly(HEMA-GMA)-Arg@
Ag(I) cryogels have reusability feature in the
adsorption studies.
CONCLUSION
It can be reported at the end of the studies
performed that poly(HEMA-GMA) -Arg @ Ag (I)
cryogels have antimicrobial activity. In light of the
data obtained, the materials under consideration
is the promising materials to be used in medical
applications. In addition, the high adsorption
capacity of these cryogels is also advantageous in
order to increase the diversity in chromatographic
applications.
ACKNOWLEDGEMENT
Because of the contribution to the work, I would like to
express my heartfelt thanks of Prof. Dr. Nevzat Şahin (19
Mayıs University, Faculty of Arts and Sciences, Department
of Biology) and Asst. Assoc. Dr. Demet Tatar (Hittite
University, Osmancik Ömer Derindere Vocational Higher
School, Medical Services and Technical Division).
Figure 2. The SEM image of poly(HEMA-GMA)-Arg@Ag(I) cryogel.

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Figure 3. FT-IR spectra of a) Poly(HEMA-GMA)-Arg b) Poly(HEMA-GMA)-Arg@Ag(I) cryogels.

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