Fabrication of calixarene based protein scaffold by electrospin coating for tissue engineering

Article abstract of DOI:10.1166/jnn.2018.15381

In this study, calixarene was synthesized by using different functional groups as p-tert-butyl- Calix[4]arene ester and amides. Calixarene nanofibers were produced by electrospin coating. Protein immobilization onto the calixarene nanofibers was carried out with human serum albumin (HSA). The maximum amount of binding on produced three different calixarene nanofibers (DE, 2-AMP and 3-AMP) was compared by using a fluorescence technique for protein analysis. Result showed that maximum binding amount was found to be as 177.85 mg cm-2 for 3-AMP surface. The protein binding was also characterized by using SEM, TEM, AFM and FT-IR. From obtained results, calixarene-albumin nanofiber was also fabricated by spin coating using 3-AMP which has ability max binding of protein.

Full text of DOI:10.1166/jnn.2018.15381

Article
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Nanoscience and Nanotechnology
Vol. 18, 5292–5298, 2018
Copyright © 2018 American Scientific Publishers
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Printed in the United States of America
Fabrication of Calixarene Based Protein Scaffold by
Electrospin Coating for Tissue Engineering
Esra Maltas Cagil1ꢀ2ꢀ∗, Fatih Ozcan3ꢀ4, and Seref Ertul3
1Faculty of Pharmacy, Selcuk University, 42075, Konya, Turkey
2International Centre of Excellence in Pharmacy, Selcuk University, 42075, Konya, Turkey
3Department of Chemistry, Selcuk University, 42075, Konya, Turkey
4Advanced Technology Research and Application Center, 42075, Konya, Turkey
In this study, calixarene was synthesized by using different functional groups as p-tert-butyl-
Calix[4]arene ester and amides. Calixarene nanofibers were produced by electrospin coating. Pro-
tein immobilization onto the calixarene nanofibers was carried out with human serum albumin (HSA).
The maximum amount of binding on produced three different calixarene nanofibers (DE, 2-AMP
and 3-AMP) was compared by using a fluorescence technique for protein analysis. Result showed
that maximum binding amount was found to be as 177.85 mg cm−2 for 3-AMP surface. The pro-
tein binding was also characterized by using SEM, TEM, AFM and FT-IR. From obtained results,
calixarene-albumin nanofiber was also fabricated by spin coating using 3-AMP which has ability
max binding of protein.
IP: 62.197.220.193 On: Fri, 15 Jun 2018 22:01:48
Keywords: Nanofiber, Albumin, Protein, Calixarene, Electrospin Coating, Fluorescence,
Copyright: American Scientific Publishers
Nanotechnology, Biomedical, Scaffold, Tissue Engineering, Pharmaceuticals.
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1. INTRODUCTION
Recently, nanoparticles (NPs) systems based on pro-
teins including hemoglobin, globulin, lysozyme, gelatin,
collagen, casein, soy and whey protein have been stud-
ied for encapsulation duet to their enhanced properties of
bioavailability, absorbability and low toxicity.18 Protein-
nanoparticles systems have been extensively investigated
in vitro and in vivo drug delivery and encapsulation of
bioactive compounds because of its capability to form
self-assembled NPs and more importantly, its capability
for sustained drug release.19ꢀ20 Due to the binding affin-
ity to neutral polymers and proteins via hydrogen bonding
and hydrophobic interactions, metal ions for immobilized
metal affinity, protein nanoparticles systems were utilized
to form robust nanocarrier film or platform.21ꢀ25 Some lim-
itation of the proteins such as low stability and high cost
synthesis have limited widespread applications of the pro-
teins. Therefore, immobilization protein onto ideal sub-
strate materials with desirable properties such as available
particles size, surface area and functionalization with dif-
ferent group, is necessary. There are different strategies
for protein immobilization, such as cross-linking, covalent
attachment, adsorption on solid supports or entrapment in
silica/polymer matrices.26
Nanotechnology is a growing science that is extensively
used for synthesis of inorganic and organic materials, have
unique physical, chemical and biological functions.1ꢀ2 The
materials produced at the nanoscale level have attracted
increasing attention in the fields such as drug delivery,
protein affinity, diagnostic, biomedical imaging and engi-
neering by development of nanotechnology.3–8 A large
amount of the nanomaterials based on polymers, chitosan,
graphene, gold and magnetic nanoparticles had been inves-
tigated due to their unique properties such as good biocom-
patibility, a large specific surface area and pore volume,
controllable particle size. Another advantage of nanopar-
ticles which is pore size ranging from 2 nm to 50 nm
are excellent candidates for biological applications.9–12
Nanoparticles based nanoscience and biotechnology have
shown great promise especially in biomedical applica-
tions such as drug/gene delivery, biosensing, bioimag-
ing and photothermal therapy.13–15 Nanocarriers have been
designed as platforms to integrate therapy and diagnostics,
which are emerging directions in biomedical.16ꢀ17
∗Author to whom correspondence should be addressed.
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doi:10.1166/jnn.2018.15381

Cagil et al.
Fabrication of Calixarene Based Protein Scaffold by Electrospin Coating for Tissue Engineering
4
H
O
1) NaOH, HCHO
2) Difenilether
i
HO
OH OH
O
HO
K2CO3
Acetone
ii
1
Br
O
4
Ethyl bromoacetate
4
O
O
iii
OH
O
HO
O
CH2Cl2/CH3OH
O
OH
O
O
HO
O
O
O
NH2
HN
NH
(DE)
2
N
2-aminomethyl-pyridine
N
N
3
(2-AMP)
iv
CH2Cl2/CH3OH
NH2
N
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3-aminomethyl-pyridine
Copyright: American Scientific Publishers
4
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O
O
OH
O
HO
O
HN
NH
N
N
4
(3-AMP)
Scheme 1. Schematic illustration of the synthesis of p-tert-butyl-calixarene derivatives. (i) Formaldehyde, NaOH, diphenyl ether, (ii) bromoethylac-
etate, Acetone, reflux, 48 h, (iii) 2-(aminomethyl)pyridine, CH2Cl2/methanol (1:1) at room temperature, (iv) 3-(omethyl)pyridine.
Calixarenes are widely used in supramolecular chem-
istry. Due to having a cyclic structure, the calixarenes
which can be functionalized easily with polar and apo-
lar groups are good carrier for cations, anions and neutral
molecules. The calixarenes can be used in many biolog-
ical applications because of the easy-functionalized and
having large surface area.27–29 In recent years, the usage
of the nanofibers fabricated by electrospun method is
getting increased.30 In literature, polymers having large
molecular weight are available to produce as nanofibers.31
The calixarene which is also one of the key members
of chemistry has recently begun to combine with poly-
mers in fabrication of the nanofibers. In the report of
Chen et al., polyacrylonitrile (PAN) was prepared as
670 nm dia fiber via electrospun method by using differ-
ent derivatives of p-tert-butyl-calix[8]arene. These p-tert-
butyl-calix[8]arene derivatives were immobilized on PAN.
Then, resulted fibers were used in absorption of congo
red.31 A study of polyacrylonitrile polymers (PAN) by
pulling the nanofibers was on electrospinning method. In
another study, C-Methylcalix[4] resorcinarene was immo-
bilized onto the PAN nanofibers with 10–40 nm of
diameter by using dipping method.32 Derivatives of differ-
ent p-tert-butyl-calix[4]arene has also been functionalized
by using PAN additive nanofibers for removal of toxic
anions.33
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Fabrication of Calixarene Based Protein Scaffold by Electrospin Coating for Tissue Engineering
Cagil et al.
Figure 1. Fluorescence spectra of human serum albumin at 280 nm of excitation wavelength.
In this paper, it is the first time that calixarene-albumin
nanofibers have been prepared. The new nanofiber systems
were developed by electrospin coating with three differ-
ent calixarene to evaluate protein binding. Human serum
albumin (HSA) was used as a model protein to examine
the binding characteristics of calixarene nanofibers for tis-
sue engineering.
2. EXPERIMENTAL DETAILS
All of the reagents used in this study were obtained
from Merck (Germany) or Sigma Aldrich (USA) and used
without further purification. CH2Cl2 was distilled from
CaCl2/MeOH over Mg and stored over molecular sieves.
Anions were used as their sodium salts. Thin layer chro-
matography (TLC) was performed using silica gel on
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glass TLC plates (silica gel H, type 60, Merck). Sol-
Copyright: American Scientific Publishers
vents were dried by storing them over molecular sieves
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(Aldrich; 4_A, 8e12 mesh). All aqueous solutions were
prepared with deionized water that was passed through a
Millipore Milli-Q Plus water purification system. Melting
points of the molecules were determined on a Gallenkamp
DE
2-AMP
3-AMP
1
apparatus. H and 13C NMR spectra were obtained using
4000
3500
3000
2500
2000
1500
1000
500
a Varian 400 MHz spectrometer operating at 400 MHz.
The prepared nanofiber mats were characterized by using
a Bruker Vartex 70 ATR-FTIR, instrument in the range
4000–400 cm−1. A Zeiss EVO LS10 model scanning elec-
tron microscope was used to take SEM images. EDX anal-
ysis was done with 123 eV Bruker detector on Zeiss EVO
LS10 electron microscope. Inovenso basic system electro-
spinner was used for nanofiber production. Fluorescence
measurement was done with using Perkin Elmer LS-55
Fluorescence spectrophotometer.
Wavenumber (cm–1
)
The calixarenes such as 5,11,17,23-Tetra-tert-butyl-
25,26,27,28-tetrahydroxycalix[4]arene 1,5,11,17,23-Tetra-
tert-butyl-25,27-bis methoxycarbonylmethoxy) 26,28
dihydroxycalix[4]arene 2(DE), 5,11,17,23-Tetra-tert-butyl-
25,27-bis(2 aminomethylpyridineamido)-26,28 dihydroxy
calix[4]arene 3(2-AMP) 5,11,17,23-Tetra-tert-butyl-25,27-
bis(3-aminomethylpyridineamido)-26,28 dihydroxycalix[4]
arene 4(3-AMP), (Scheme 1) were synthesized according
to the reported literatures Scheme 1.34–36
HSA
DE-HSA
2-AMP-HSA
3-AMP-HSA
4000
3500
3000
2500
2000
1500
)
1000
500
Wavenumber (cm–1
Figure 2. FTIR spectra of nanofibers and protein immobilized nano-
fibers DE, 2-AMP and 3-AMP; HSA, DE-HSA, 2-AMP-HSA and
3-AMP-HSA.
In this study, calixester DE and amides 2-AMP and
3-AMP nanofibers were produced by electrospinning of
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J. Nanosci. Nanotechnol. 18, 5292–5298, 2018

Cagil et al.
Fabrication of Calixarene Based Protein Scaffold by Electrospin Coating for Tissue Engineering
Figure 3. SEM images of nanofibers (a) DE (b) 2-AMP, (c) 3-AMP at 1 ꢁm and 200 ꢁm.
100–120% (w/v) solution in DMF. The electrospinning
process was conducted under a fixed electrical voltage of
22–26 kV, while the distance between the needle tip and
the collector was kept as 16 cm. Flow rate was set at
5.5–8.5 mL/h, and the process was performed continu-
ously. The materials were collected on an aluminum foil-
coated square plate collector as nanofiber mats.
1 mg mL−1 of human serum albumin in 20 mM× Tris,
pH 7.4 was mixed for 2 h at 4 ꢀC at a surface area
of cm−2 of all calixarene nanofibers.23 Solution includ-
ing unbounded protein were separated by pipet from the
piece of nanofiber. The pieces of the nanofiber were
washed three times with 20 mM× Tris, pH 7.4. The
nanofibers were washed with the Tris buffer for chemical
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Figure 4. AFM images of nanofibers (A) DE (B) 2-AMP, (C) 3-AMP and TEM images of nanofibers (D) DE (E) 2-AMP, (F) 3-AMP.
J. Nanosci. Nanotechnol. 18, 5292–5298, 2018
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Fabrication of Calixarene Based Protein Scaffold by Electrospin Coating for Tissue Engineering
Cagil et al.
Table I. Atom weights of elements in the nanofibers related to the EDX
results.
characterization. Solution was used to determine the
amount of bounded protein via calibration curve drawn at
280 and 342 nm of excitation and emission wavelengths
by fluorescence spectroscopy. The regression equation of
calibration curve of albumin in 20 mM× Tris, pH 7.4 was
found to be as y = 154ꢂ4x +27ꢂ513, r = 0ꢂ99ꢂ23
Nanofiber
Carbon (%) Nitrogen (%) Oxygen (%) Sulfur (%)
DE
DE-HSA
2-AMP
2-AMP-HSA
3-AMP
3-AMP-HSA
48.1
58.2
45.0
49.6
45.4
55.1
28.5
22.8
30.2
26.3
24.6
16.5
23.3
17.4
24.8
22.9
29.9
21.7
–
1ꢂ4
1ꢂ1
3. RESULTS AND DISCUSSION
10ꢂ8
In this study, three different types of calixarene nano-
fibers abbreviated as DE, 2-AMP and 3-AMP were
synthesized as shown Scheme 1. The calixarenes were
synthesized with different functional groups as p-tert-
butyl-Calix[4]arene ester and amides. Calixarene nano-
fibers were produced by electrospin coating. Then, the
effect of functional group was studied for evaluation of
protein binding.
Figure 2 shows that the ATR FT-IR spectra of the parti-
cles DE, 2-AMP and 2-AMP. The aliphatic C–H stretching
of CH, CH2 and CH3 groups from DE were observed at
2956–2867 cm−1. The bands at 3429 cm−1 and 1750 cm−1
belong to stretching vibrations of –OH groups and ester,
respectively. The bands of aliphatic C–H stretching of
CH, CH2 and CH3 groups observed at 2956–2867 cm−1
and the bands belonged to the amide bond at 1676 cm−1
were overlapped in the spectra of DE, 2-AMP and 3-AMP
nanofibers.
Figure 2 also shows the ATR FT-IR spectra of the par-
ticles HSA, DE-HSA, 2-AMP-HSA and 2-AMP-HSA. In
order to characterize binding of albumin on calixarene
nanofibers, starting materials were compared with albu-
min bounded ones. New peaks at around 2250 cm−1 and
580 cm−1 related to amide bond were observed in the spec-
tra of the three nanofibers. Also, the new band at around
1800 cm−1 indicated that albumin bounded to 2-AMP and
3-AMP. However, a large amount of peaks shifted and
increased in transmittance which is indicated the binding
of protein to all starting materials.39ꢀ40
Transmission Electron Microscopy (TEM) and Atomic
Force Microscopy (AFM) verified the structure of pro-
duced calixarene nanofibers (Figs. 3 and 4(A–F)). Accord-
ing to the data, nanofiber diameters of DE, 2-AMP and
3-AMP were measured as 75–100 nm, 250–350 nm and
180–250 nm, respectively. Scanning Electron Microscopy
(SEM) allowed the verification of the morphological dif-
ferences between nanofibers which were DE, 2-AMP
and 3-AMP and nanofibers-protein. As viewed from
Figures 5(A)–(C), after protein immobilization on the
nanofibers, the morphology greatly changed. Protein bind-
ing results in big differences on the view of each
The surface characteristics such as physical and chem-
ical properties would be effective on binding of biolog-
ical molecules like albumin. For this aim, albumin from
human serum was immobilized on these three calixarene
based nanofibers. The binding amount of albumin was
measured by using Fluorescence Spectroscopy. Albumin
has an emission spectra at 280 nm and 342 nm of an
excitation and emission wavelengths (Fig. 1). After pro-
tein was bounded onto the nanofibers, the remaining solu-
tion was analyzed at these wavelengths. According to the
IP: 62.197.220.193 On: Fri, 15 Jun 2018 22:01:48
results, binding amount of albumin was found to be as
Copyright: American Scientific Publishers
91.45 mg cm−2 for DE, 148.55 mg cm−2 surface for
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2-AMP and 177.85 mg cm−2 for 3-AMP. This showed that
amide bond is more effective for protein binding when
compared with DE and other two calixarene nanofibers
including amide bonds. In addition to amide bond, pyri-
dine ring also contributes to the interaction of protein with
nanofibers. But, when 2-AMP and 3-AMP nanofibers were
compared, 3-AMP exhibited higher affinity than 2-AMP
due to nitrogen in the ring more closer to surface, led to
increase in amount of protein binding to the nanofiber sur-
face. This interaction may also be non-covalent interaction
including hydrogen bond, Van der Waals and hydrophobic
interaction.24ꢀ37ꢀ38
Expected binding mechanism of calixarene nanofibers
to albumin was given at Scheme 1.
A
B
C
Figure 5. SEM images of nanofibers (A) DE-HSA (B) 2-AMP-HSA, (C) 3-AMP-HSA at 1 ꢁm.
J. Nanosci. Nanotechnol. 18, 5292–5298, 2018
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Cagil et al.
Fabrication of Calixarene Based Protein Scaffold by Electrospin Coating for Tissue Engineering
Figure 6. SEM graphs of albumin-nanofibers with 3-AMP.
calixarene nanofibers when compared with bare ones.
Reticulation structures on linear calixarene nanofibers
appeared on the surface due to binding of the proteins
(Figs. 5(A–C)).23ꢀ24
EDX analysis was obtained from SEM images of the
nanofibers. EDX results related to the atom weights (%)
of the each elements in the nanofibers were shown at
Table I. According to the results, protein bounded cal-
ixarene nanofibers have sulfur elements which introduced
to the nanofiber structure due to sulfur elements in the
structure of cysteine and methionine residues in the pro-
tein. In addition, the differences in ratio of the carbon, oxy-
gen, nitrogen and sulfur indicated that the binding amount
nanofiber system with smooth shape and surface to evalu-
ate in biomedical applications such as tissue engineering.
Acknowledgment: We would like to thank The
Research Foundation of Selcuk University (BAP) for
financial support of this work.
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Received: 30 August 2017. Accepted: 23 October 2017.
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J. Nanosci. Nanotechnol. 18, 5292–5298, 2018