Professional Documents
Culture Documents
PII: S0141-8130(16)32489-8
DOI: http://dx.doi.org/doi:10.1016/j.ijbiomac.2017.03.095
Reference: BIOMAC 7261
Please cite this article as: Iqra Munir, Sadia Ajmal, Muhammad Raza Shah,
Aftab Ahmad, Abdul Hameed, Syed Abid Ali, Protein–Drug Nanoconjugates:
Finding the alternative proteins as drug carrier, International Journal of Biological
Macromoleculeshttp://dx.doi.org/10.1016/j.ijbiomac.2017.03.095
This is a PDF file of an unedited manuscript that has been accepted for publication.
As a service to our customers we are providing this early version of the manuscript.
The manuscript will undergo copyediting, typesetting, and review of the resulting proof
before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that
apply to the journal pertain.
International J. Biological Macromolecules
Research Paper
Iqra Munira, Sadia Ajmala, Muhammad Raza Shaha, Aftab Ahmadb, Abdul Hameeda, Syed Abid
Alia*
a
H.E.J. Research Institute of Chemistry, International Center for Chemical and Biological
*
Corresponding author: HEJ Research Institute of Chemistry, International Center for
Chemical and Biological Sciences (ICCBS), University of Karachi, Karachi-75270, Pakistan (Ali
ABSTRACT
Present study was conducted to establish the interaction of bovine fetuin-A to validate its binding
modalities with doxorubicin (Dox). Fetuin-A was purified to highest purity and monodispersity.
Green synthesis of fetuin-A conjugated gold nanoparticles (F-GNPs) have been performed
giving typical UV-maxima with subtle variation in fourier transform infrared spectroscopy
(FTIR). Atomic force microscopy (AFM) revealed spherical shaped, polydisperse F-GNPs of
varying sizes, complementing the radius of hydration (19.5-62.4 nm) by dynamic light scattering
(DLS). Circular dichroism (CD) analysis of fetuin-A with respect to Dox interaction shows
remarkable reduction in ellipticity with increasing concentrations of Dox (20-120 μM). Fetuin-
A:Dox and F-GNPs:Dox at variable concentrations revealed significantly enhanced absorption
spectra, while a continuous decrease in florescence (560 nm). This effect was more drastic when
Dox interact with fetuin-A as compared to F-GNPs. Some known antimicrobial drugs were also
investigated under similar conditions, giving strong quenching effect in a dose dependent manner
suggesting the significant yet differential interactions. In cytotoxicity assay, fetuin-A:Dox
conjugates revealed less toxicity as compared to F-GNPs:Dox and Dox alone. In-silico studies of
the fetuin-A:Dox complex suggest that the drug binds in the major grove between beta-sheet and
long loop region of D1 domain and stabilized by several hydrogen bonds.
Abbreviations
Bovine serum albumin (BSA), Hydrophobic Interaction Chromatography (HIC), Size Exclusion
Microscopy (AFM), Dynamic Light Scattering (DLS), Circular Dichroism spectroscopy (CD),
Keywords
Nanoparticles
1. INTRODUCTION
The abundance and variations in the level of serum glycoprotein in diverse physiological and
pathological state have made them subject of great concern since many years. Among them, one
such protein ‘Fetuin’, first described by Pedersen in 1944, is always under discussion because of
their functional diversity and involvement in a wide variety of pathological conditions [1]. In
recent years, plasma proteins have grown up significantly as drug transporters in the clinical
settings [2]. Bovine fetuin, belongs to cystatin super family, is the most dominant globular
plasma protein in fetal calf serum, and in particular, a species homologue of the well-known
plasma protein a2-HS-glycoprotein found in humans [3]. The reported molecular mass ranges
from 51 to 67 kDa and variation in mass is mainly related with the difference in glycosylation
level [4]. It shows around 30% carbohydrate contents (holding six carbohydrate moieties), and
possess 8% sialic acid residues at its terminal end [5]. The primary structure reveals 21 aromatic
residues present in the complete sequence of fetuin-A (i.e., signal peptide + mature sequence),
bringing the count to 19 residues in mature sequence. Amongst them 9 residues belong to
phenylalanine (Phe), 8 residues of tyrosine (Tyr), while only a single tryptophan (Trp) residue is
reported in the mature fetuin-A [6]. Moreover, At least two variants of fetuin family (i.e., fetuin-
A and B) have been discovered so far. While, the term ‘‘fetuin-B’’ was introduced to enlighten
another fetuin-like molecule in mammals, which shows similarity in the molecular weight as
well as structure of fetuin-A and thus effortlessly purified from serum or plasma. Further studies
revealed that FETUA and FETUB genes are located contiguous to each other in all identified
A number of studies have suggested fetuin-A as a multifunctional protein [4], ranging from
its role in neurodegenerative and cardiovascular diseases [9-11], bone metabolism [12], vascular
calcification [13, 14], inflammatory and immune functions [15], metabolic disorders such as
insulin resistance [10] and diabetes mellitus [16], atherosclerosis, and tumor cell proliferative
signaling etc. [17]. It has also been reported by various studies that in blood, the precipitation of
calcium phosphate minerals is predominantly inhibited by fetuin protein [18]. In addition, some
lipid carrier functions have also been found to be associated with fetuin-A which may perhaps
influence the inflammation, calcification and/or apoptosis [19]. In contrast, fetuin-B has been
reported with a major role in fertilization [20]. Other functions similar to fetuin-A include tumor
The most frequently used nanomaterial i.e., GNPs has gathered great attention recently due to
their stability and tunable intrinsic optical properties. With their high affinity for sulfhydryl -SH
groups, GNPs can readily be conjugate with antibodies and different other proteins which have
been further incorporated into diagnostic applications [22]. It is the simplicity of GNPs synthesis
(into diverse shape and size) that promotes the investigators to discover the ultimate prospective
of these nanoparticles for different medicinal purposes; in particular, for drug delivery,
liberation, sensors, imaging etc. Nanoparticles in general not only improves the solubility of drug
with enhanced therapeutic progression (i.e., extended circulation time) but also boosts the uptake
of drug by the desired target. Amongst them, the modern application has been confined to the
treatment of cancers [23]. Dox, an effective and current chemotherapeutic drug, is regularly used
for the treatment of wide variety of carcinomas. It acts in cancer cells via two distinct
DNA and cellular membrane by generation of reactive oxygen species [24]. Yet another novel
mechanism for induction of cellular toxicity by doxorubicin has been discovered recently i.e.,
activation of CREB3L1 transcription factor via ceramide synthesis [25]. Being an anthracycline,
it has a basic amino sugar (water soluble) moiety, and a daunosamine glycosidically linked to
aglycone (water insoluble). The linkage is formed at C7 of a four-ringed aglycone (Fig. 1). This
aglycone resides in quinone family with substituted naphthacene, a 4-methoxy, and 9-hydroxyl
plus hydroxyacetyl group [26]. The major side effect related to Dox dosage is the cardiotoxicity
In blood, drug distribution and elimination can be influenced by the binding to a protein
which ultimately affects the pharmacokinetics of drug by altering the intensity as well as
duration within the medium. Binding of drug to a particular protein can be reversible or
irreversible, the later one showing permanent binding, thus forming covalent bonds and making
the drug least available for therapeutic purpose. In contrast, the reversible binding is more
favorable; allowing slow and sustained release of active drug. The initial binding involved here
comprises electrostatic interactions, followed by van der waals forces. In order to get more
stability, the drug-protein complex may sometimes also undergo configurational changes. It is
also interesting to observe that the molecules of proteins are relatively larger (as compared to
drugs), thereby holding more than one binding sites for the similar drug [28]. Besides several
drug delivery systems, peptides and proteins conjugates have now emerged with great potential
of active targeted therapy in many inflammatory and tumor related diseases. In addition to the
to proteins (with a linker ethylene glycol) is also giving promising results, with enhanced
pharmacokinetics and reduced toxicity [29]. The drug, after being linked to proteins (having high
molecular weight) can take over higher circulating characters and gaining more exposure to
tumor cells [30]. Since plasma proteins have prominence in altering the therapeutic and
distribution characters of drug, the protein-drug conjugate strategy, therefore has much
significance in cancer cure; for a reason that speedy growth of the cancer cells becomes possible
The increasing significance of fetuin-A for both diagnosis and therapeutics has come to the
consideration of the pharmaceutical industries [31]. Being a large group of binding proteins, it
mediates the availability and transport of diverse variety of cargo substances in the bloodstream.
Hence, the current study was designed to establish the binding modalities of native fetuin-A to
Dox in order to highlight the distinguished abilities of fetuin-A to work more efficiently as drug
carrier. F-GNPs were also synthesized for the comparative analysis. Studies were conducted
utilizing diverse spectroscopic (UV/Vis, FTIR and fluorescence), structural elucidation (CD and
AFM), DLS and molecular docking methods. Purification of fetuin-A was conceded to validate
the availability of impure fetuin-A fractions in the market. Furthermore, these spectroscopic
techniques were also utilized to established the interaction of fetuin-A and its conjugated GNPs
with Dox and some frequently used antimicrobial drugs to complement the specific yet
2.1. Materials
The purified fetal bovine serum fetuin-A (≥98%) was purchased from Sigma-Aldrich
(cat#F3004) and further subjected to purification prior to use (see proceeding section). Purified
fetuin-A type-IV fraction from other companies such as Merk-Calbiochem (cat#341506) and
Alfa-Aesar (cat#J60437) were also purchased and used for comparative studies. Dox
(cat#D1515) and Borane tert-butylamine complex (cat#180211) were purchased from Sigma-
and ciprofloxacin) were purchased from Oxide (UK). All other chemicals and reagents used were
For purity and comparative analysis, purified fetuin-A type-IV were subjected to Native
PAGE and size exclusion chromatography (SEC) using TSK-2000SW column (7.5 mm x 300
mm; Tosoh Bioscience, Japan) equilibrated and eluted in 50 mM Tris-HCl buffer, pH7.2,
containing 100 mM NaCl and 0.02% NaN3 using fast-protein liquid chromatographic system
(FPLC- ÄKTAbasic, GE Healthcare, UK). The flow rate was maintained at 0.4 ml.min-1 and the
eluate was monitored at 280 nm. Purified bovine serum albumen (BSA, Merck Germany) under
similar conditions was used as a standard. The data was analyzed by automated software
The purified fetuin-A (Sigma cat#F3004) was selected (because of less contamination of
BSA) and subjected to hydrophobic interaction chromatography (HIC) using TSK-gel Phenyl-
5PW column (1000 Å; 75 x 7.5 mm; 10 ; Tosoh Bioscience, Japan) in FPLC systems. The
column was equilibrated in 1 M ammonium sulphate in 0.5 M phosphate buffer (pH7.2) and
eluted with 0.25 M phosphate buffer (pH7.2) containing 25% ethanol. The flow rate was
maintained at 0.4 ml.min-1 and the eluate was monitored at 280 nm. The final purity as well as
monodispersity of the fetuin-A obtained from HIC was achieved by SEC using BioSep-SEC-
s4000 (7.8 x 300 mm, Phenomenex, USA) column under similar aforementioned conditions.
Protein concentration was measured by the modified dye-binding assay [32] using BSA as a
Austria).
The crude fractions as well as purified proteins was analyzed for their purity and molecular
mass under native (Native PAGE) [33] as well as standard dissociating and
PROTEAN-3 Cell (Bio-Rad Lab, UK). The proteins were separated in 6-10% resolving and 5%
stacking gels at 140 V for 1 h and at the end of electrophoresis the gel was stained either with
0.2% Colloidal Commassie Blue G-250 [35, 36] or 0.5% Alcian blue glycoprotein stain [37].
2.2.2. Identification of Fetuin-A by N-Terminal Sequencing
For the identification of fetuin-A, two major bands of the purified protein from 10% SDS-
PAGE were transferred to PVDF membrane using electro-blotting apparatus (Bio-Rad, USA).
After confirming the successful transfer by reversible staining using 0.2% Ponseu S stain,
membrane was washed with Milli-Q water, dried and subjected to Edman degradation analysis
using solid phase protein sequencer (ABI-491 Applied Biosystems, USA) [38].
All glassware was washed with tap water and then rinsed by freshly prepared aqua regia
(HCl–HNO3, 3:1). Later glassware was washed again thoroughly with Milli-Q H2O and oven
dried before use. The F-GNPs were prepared by using method recently described by us [39] with
slight modification. Briefly, 50 μL of purified fetuin-A (0.1 mM) was added drop by drop to 5
mL of tetrachloroauric (III) acid solution HAuCl4.3H2O (0.1 mM) in water at constant shaking.
At 530 nm, the highest absorbance was observed with a ratio of 5:1 (v/v, Au: fetuin-A). Change
in color of reaction mix was observed from pale yellow to deep violet. This mixture was kept on
stirrer for 24 h at room temperature (~26 oC). The F-GNPs were centrifuged (14,000 rpm for 10
min) and precipitate was collected. Prepared F-GNPs were characterized further by using
The protein-loading capacity on F-GNPs was estimated using protocol defined by Mukherjee
et al., [40]. Briefly, protein conjugated nanoparticles in a known quantity were mixed to SLS-
NaOH solution (5%). Mixture was kept overnight at room temperature followed by
centrifugation at 14,000 rpm for 15 min. Protein released in the supernatant was estimated by
Bradford [32] method as mentioned in section 2.2, values were extrapolated from calibration
The purified proteins (i.e., BSA and fetuin-A) as well as F-GNPs were subjected to DLS
studies using Laser Spectroscatter-201 system (RiNA GmbH Berlin, Germany) with a He-Ne
laser providing a 690 nm light source and an output power in the range of 10-50 mW. 50
measurements of autopiloted run at every 20 s, with a delay of 1s, was used at room temperature
(~26 oC). Sample (~20 µL) was introduced in to a quartz SUPRASIL® cell (light path 1.5 mm,
Hȅllma Germany) for measurement. A fixed 90° scattering angle is used to collect the scattered
light. Analysis for autocorrelation function was carried out with the program CONTIN to attain
hydrodynamic radius (RH) distributions. The RH is related to the diffusion coefficient by the
Einstein-Stokes equation. Data analysis was done using PMgr v3.01p17 software provided with
Topological images for F-GNPs were collected by AFM as per protocol described by Shah et
al [42]. Briefly, the desired solution of F-GNPs (~10 µL) was placed on freshly cleaved mica for
few minutes, rinsed with Milli-Q water and followed for drying with nitrogen. Utilizing Agilent-
5500 AFM/SPM microscope (USA), images were acquired instantly in tapping mode using soft
silicon probes (NCL; Mean width = 38 μm, nominal length = 225 μm, nominal resonant
frequency = 190 kHz, force, nominal force constant = 48 N/m). Using random spot surface
selection strategy, the images were collected from each samples solution with as low as eight
2.6. Spectroscopy
The FTIR spectra were recorded using Shimadzu IR-460 (Japan) by mixing a 1:1 mixture of
F-GNPs and KBr. This powder mixture was then pressed in a mechanical compressor to form a
UV–visible spectra were recorded with a Ultrospec 4300-pro UV/Vis spectrophotometer (GE
Healthcare, UK) with a path length of 1 cm and at room temperature (~25 °C). For interaction
studies, a fixed concentration of fetuin-A (0.1 mg) was mixed with increasing concentrations of
Dox (i.e., 20-280 μM) and incubated overnight at room temperature, followed by UV-Vis full
scan spectrum measurements (200-700 nm). Identical procedure was applied for the group of
(Molecular Devices, USA), using 96 well standard fluorescent plates (NuncTM, Germany). Final
concentration of fetuin-A was 0.1 mg.ml-1, to which the required volume of Dox was added
discretely to attain the preferred concentration of the drug (i.e., 20-240 μM). Final volume of
each reaction was maintained as 300 µL. The emission range for intrinsic measurements was
selected as 300-700 nm, with an excitation of protein at wavelength 280 nm. The data was
Circular dichroism (CD) spectra of the similar samples were collected on a Spectro-
polarimeter (Jasco J810, Japan) at 0.5 nm resolution using 1 mm path length sample cell. An
average of four full-wavelengths scans was monitored in the both far-UV amide region (190-250
nm) and near-UV region (250-350 nm) at 25 °C. Variable spectra (i.e., Dox interaction with
fetuin-A) were also followed by CD measurements. The solvent subtracted spectra as such or
converted to mean residue ellipticity [θ] (MRE = deg·cm2·dmol-1) were acquired [38]. CD
spectra were evaluated to estimate the proportions of secondary structure by on-line DichroWeb
server [43].
To check the effect in human blood plasma, heparinized tubes were used to collect blood
sample through venous puncture of healthy volunteer after ethical approval. Blood was
centrifuged for 5 min at 4000 rpm to separate plasma. The plasma added to fetuin-A alone and F-
GNPs solution (without drug) was used as control, while the working solutions were
incorporated with variable concentrations of Dox (40 - 120 μM) to fetuin-A and F-GNPs in
plasma solution. The fluorescence spectra were recorded as mentioned in preceding section (2.6).
The NCI-H460 cell line was purchased from American type culture collection (ATCC,
USA). Cells were grown and maintained using RPMI-1640 (GIBCO, Aukland, NZ)
supplemented with 2 mM L-glutamine, 2 g/L D-glucose, and 1.5 g/L sodium bicarbonate, 10%
heat inactivated-FBS (Hyclone, USA) and 1% antibiotic solution in a humified (95% air:5%
CO2) incubator at 37 oC. The cells were regularly passaged on reaching 80% confluency using
trypsin-EDTA in a t75 flask. MTT assay was performed to evaluate the effects of protein fetuin-
A alone, Dox alone, fetuin-A: Dox conjugates and F-GNPs: Dox on cells’ viability. 10,000 cells
per well were seeded in a 96-well microtiter plate. The next day, cells were treated with various
yl)-2,5-dipheynyltetrazolium bromide) dye (Biobasic, Canada) was added to the wells followed
by 4 h incubation. Later the dye was removed and formazan crystals were solubilized in DMSO
Devices, USA). The cytotoxicity at different concentrations and IC50 of the samples were
calculated.
All results are presented as the mean ± standard deviation of the triplicate experiments. One-
way analysis of variance (ANOVA) was used to compare the treated and control groups, whereas
The amino acid sequence of bovine fetuin-A was obtained from the protein database
(accession#CAA34596.1). Three-dimensional (3D) structure of Dox was obtained from the Zinc
database (accession# 3918087, ZINC 12. Jan 1, 2012. Production Release Oct. 18, 2005) [44,
Homology/analogY Recognition Engine V2.0 (Phyre2). Phyre2 protein fold recognition server
takes an unknown protein sequence as input, builds a profile using PSI-BLASTs, and
proteins (SCOP) and protein data bank (PDB). The predicted model was further evaluated for
Molecular interaction studies were performed with SwissDock, a web based server for
protein (fetuin-A) and small molecule (Dox) fast docking services [46, 47]. SwissDock is based
on the CHARMM force field with EADock DSS docking software [48], whose algorithm
comprises of the following four major steps: 1) several binding modes are generated either in a
box (i.e., local docking) or in the vicinity of all possible target cavities (i.e., blind docking); 2) at
the same time, their CHARMM energies are estimated on a grid; 3) the binding modes with the
most favorable energies are evaluated with FACTS, and clustered; 4) at the end most favorable
clusters can be visualized online and downloaded. UCSF Chimera, a visualization system for
exploratory research and analysis [49] was used for molecular structure viewing and graphical
imaging.
The major limitation in research and medical application of serum carrier proteins (such as
bovine fetuin-A) includes the extensive posttranslational modifications (PTMs), purity because
of the contamination of other abundant serum proteins (like albumin) and monodispersity. Even
highly purified fractions of bovine fetuin-A available in the market contain contamination of
BSA which leads to the artifacts and difficulties in final interpretation. Thus we have
demonstrated here the purification of bovine fetuin-A from the most purified fraction available in
market such as Sigma-Aldrich. Sequencial steps of chromatography was used comprising of HIC
followed by SEC using FPLC system (Fig. 2a, b). Partially purified fetuin-A fractions from other
companies were also tested for comparison and shows BSA contamination (Suppl. Fig.S1). The
SDS PAGE analysis of the purified two proteins under dissociating/denaturing conditions (i.e.,
reduced) resolved two bands for bovine fetuin-A (while single pure band under Native PAGE,
Fig. 2b insert) which was also confirmed by glyco-staining and nicely differentiate the heavily
glycosylated fetuin-A and non-glycosylated BSA (Fig. 3a). For further confirmation, 2 bands of
bovine fetuin-A (Fig. 3b) were electro-transferred onto the PVDF membrane and subjected to
Edman degradation for N-terminal sequencing. Both bands of bovine fetuin-A revealed identical
thus suggesting the degradation under dissociating/denaturing conditions as have also been
DLS is a promising tool that gives significant structural information about biological
radius, monodispersity and the presence of soluble proteins aggregates [52]. Figure 4 provides a
detailed view of the conducted DLS studies on purified fetuin-A and BSA at physiological pH
7.4 (Fig. 4a, b). Bovine fetuin-A shows a highly monodisperse protein signal, with RH 9.4 nm
±0.22 and 7% polydispersity. As very little is known about fetuin-A, it is rather difficult to
extend any further interpretation, whether the protein exist as monomer or a homodimer (likely)
in solution as RH calculated for purified BSA (almost same size) under similar conditions is 4.94
nm ±0.17 which is quite comparable to the previous reports i.e., RH = 3.3-4.1 nm [53]. CD, being
an excellent technique for quickly calculating the secondary structure of proteins, can also be
utilized to study protein interactions and estimating its binding and folding/unfolding properties.
Figure 4c depicts the further complementation of the purity of fetuin-A using CD as a secondary
structure analysis tool. The far-UV CD spectra between 190-250 nm, indicates the nature of its
secondary structure. The characteristic spectrum of purified native fetuin-A shows a clear
negative ellipticity band at 208 nm along with a shoulder at 220 nm (but not 222 nm) which is an
indicative of the protein’s low alpha helical content [54]. The calculated native spectrum for the
composition of secondary structure shows % yields as 7.4% helix, 59% sheets and 30% random
coil, thus indicating a prominent β–sheet protein. In contrast, a clear enrichment in the alpha
helicity pattern has been observed for purified BSA protein, illustrating two minima i.e., at 222
and 208 nm; enlightening that BSA has predominantly α-helical structure (Fig. 4c).
After thorough purification and characterization of bovine fetuin-A from its contaminant
BSA, biosynthesis of F-GNPs has been performed. During the preparation of reaction mixture,
gold ions reduction and F-GNPs formation was confirmed subsequently by mixing fetuin-A with
gold chloride solution. A rapid change in the color from light yellow to violet was observed. The
suspension was checked further by UV/Vis spectroscopy, giving maximum absorption peak at
530 nm (Fig. 5a), hence resembled the typical GNPs suspension maxima between 520-530 nm as
per defined by Buso et al., [55]. When optimizing the different HAuCl4 and protein fetuin-A
ratios, complete gold ions reduction was observed at 5:1 v/v. A sudden decrease however, was
noticed on increasing this ratio (with respect to gold ions) 6:1; indicating decrease in GNPs due
to aggregation [39, 41]. Results for protein loading experiment, estimates the exact quantity of
fetuin-A loaded on GNPs which was found to be 2.6 x 10-4 mM. Similarly, the calculated
concentration of GNP solution (as prepared) was 1.0 x 10-4 mM, underneath the supposition that
all gold ions (Au3+) have converted to Auo. Previous studies indicate that increasing the
homogenizing speed ultimately enhance the loading capacity of GNP formulations. These
finding goes well with the fact that, higher the homogenizing speed, greater is the variation in
configurations of proteins and their changes [57]. Thus F-GNPs formation was also characterized
by means of FTIR, where absorbance peaks for fetuin-A has been observed between 500–3500
cm-1 regions (Fig. 5b). For free protein, absorbance bands including 3396 for –O–H stretching
and 3232 for amide B region have been observed. Similarly, bands at 1635 and 1548 cm-1 for
Amide I and II regions are found (respectively), contributed by C-O stretch and N–H bending in
plane, linked with C-N stretching. Both these peaks particularly the former one, narrate with the
protein’s secondary structure. Among the overall distribution of Amide I region, peaks 1610-
1640 are in general for β–sheet patterns of protein. Moreover, peak at 1294 might appear due to
C–O group of proteins and disappearance of such peaks due to the bioreduction can be due to the
fact that these groups are accountable for the gold [Au III] ions reduction. Cumulatively,
considerable changes were observed in FTIR pattern of fetuin-A when present alone or in
conjugation with GNPs. The F-GNPs were further assessed for the size and morphological
characters using AFM and DLS. AFM analysis reveals spherical shaped, polydisperse F-GNPs of
(Fig. 6a, b). Additional complementation of the size distribution was also established by DLS
which revealed the RH in a range of 19.5 - 62.4 3-13% and 43% polydispersity (Fig. 5c).
In order to check the stability of F-GNPs against the different concentrations of salt, variable
heating and pH, series of experiments was conducted (Fig. 7). F-GNPs were found to be very
stable at room temperature (~25 - 30 °C) for several days, without any noticeable change in their
wavelength and/or absorption profile. Similar results were found on heating F-GNPs suspension
maximum absorption of surface plasmon peak, with no evident effect on peak position and
accompanying precipitation (Fig. 7a). This effect can be explained on the bases of "electronic
scattering rate and increased electron-surface / electron-defect scattering rate can be noticed due
temperature hence excites the electrons to a high-energy level [58]. As the energy state of
electron dictate its velocity, an increased velocity of the electrons brings a large damping
constant and thus a rapid dephasing, thereby resulting in reduced absorption of plasmon band
[39].
Effect of different molar concentrations of salt (i.e., NaCl) on surface plasmon peak of F-
GNPs was also performed. As demonstrated in Fig. 7b, a sequential increase in the NaCl
concentration resulted in consistent decrease in the absorption maximum for F-GNPs. This rapid
phenomenon contributed by chloride (Cl−1) ions [59]. In contrast to a previous report, protein
patuletin coated gold nanoparticles lost the total absorption maxima at even 1 M NaCl [37], thus
F-GNPs show higher stability against salt mainly because of the compact structure of fetuin-A
and heavy PTMs, like glycosylation and phosphorylation [60, 61]. Additionally, F-GNPs were
also analyzed for stability at variable pH which alters the stable structure of protein either by
affecting its solubility or by manipulating the charges. No significant change in the profile of F-
GNPs has been observed, with only a minor decrement in the absorbance maxima which might
Fetuin-A has been associated with various metabolic and pathological processes. Being a
negative acute phase protein, it shows strongest association with adipokines and subclinical
markers in inflammation e.g. leptin, TNF-α, adiponectin, CRP and resistin etc. Functionally, it
also shows positive correlation with diversified atherosclerotic risk factors including metabolic
syndrome, diabetes and obesity [62]. In addition, it has also been reported as a foremost adhesive
protein in serum, to facilitate the growth signaling in breast cancer [4]. Considering all such
binding properties, it has been worthy to investigate fetuin-A as a natural drug carrier protein.
Thus, a comparative drug interaction or drug loading studies of fetuin-A alone and F-GNPs was
also conducted.
CD spectroscopy was used as investigative tool in order to look into the secondary structural
alterations in fetuin-A with respect to Dox interaction. CD spectra were recorded for fetuin-A
both in the absence and presence of Dox, with experimental molar ratios as 1:0, 1:1, 1:3 1:6 for
intense minima 208 nm along with a shoulder at 220 nm and maxima at 198 nm), when
measured in far-UV region on sequential addition of the Dox, a reduction in ellipticity was
noticed (Fig. 8a). This can be associated with the interference of Dox with supportive structural
interactions and bonding forces of protein, resulting in the structural transitions and hence
alteration in the native structure of fetuin-A. It is quite remarkable to observe that high molar
ratio of fetuin-A: Dox brought a drastic deviation in the secondary structural contents which can
region between 250 - 350 nm gives the information about characteristics tertiary structure of a
protein. The signal produced in this region is the cumulative effect of disulfide bonds and
aromatic moieties. Figure 8b shows the near-UV CD spectra of fetuin-A. The results are in good
agreement with the previously reported data on fetuin-A structure [63]. Interestingly, along with
the previously reported data at 256 and 289 nm, two more major maxima at 319 and 324 nm
have been observed for fetuin-A when the wavelength range was increased till 330 nm. This
region was considered in order to justify Dox-protein interaction, as the Dox gives maxima
predominantly in this region. Strong quenching effect has been observed for fetuin-A when
treated with Dox, in conjunction with another maximum at 328 nm related to Dox signal.
Furthermore, original signal for the drug was also varied in the region i.e., 295 and 270 nm when
Fig. 9a, b demonstrates the absorption spectra of fetuin-A: Dox and F-GNPs: Dox at variable
concentrations. Pure fetuin-A alone displays typical absorption maxima at 280 nm, while Dox
alone at ~480 nm but this absorption pattern has been significantly enhanced with respect to the
increasing concentrations of Dox (i.e., 20 - 240 μM). This increment was found to be more
drastic when Dox interact with fetuin-A as compared to F-GNPs suggesting that the interaction
of drug to protein itself is much significant than that of drug to protein-GNPs. More precise
in proteins due to higher sensitivity of its indole group to electronic shifts. Intrinsic fluorescence
is exclusively offered by amino acids; tyrosine, tryptophan, and phenylalanine. The changes in
the spectra obtained by excitation at 295 nm is related to the tryptophan presence, whereas
variations in the protein structure at 280 nm excitation is featured for both tyrosine/ tryptophan
residues [64]. In order to complement Dox induced alteration in the structure of fetuin-A and F-
GNPs, fluorescence properties (maxima at 560 nm) was also measured, with varying
concentration of Dox (20-240 μM). As shown in Fig. 9c, d, a continuous decrease in the
emission of florescence was observed with a slight change in the spectral pattern. It is important
to note that the quenching effect of Dox to fetuin-A was quite drastic with the initial
concentrations (20-60 μM), and dropped further when drug to protein ratio was increased (Fig.
9c). Similarly, a concentration dependent decline in the florescence intensity has also been
observed with a trivial shift in the florescence spectra, but this change in florescence intensity of
F-GNPs: Dox was not as significant as observed in fetuin-A: Dox (P <0.05) interaction (Fig. 10).
Possible reason for this could be the less available binding sites for Dox on fetuin-A, due to its
conjugation to GNPs.
3.5. Quenching effect of Dox on Fluorescence Intensity of Fetuin-A and F-GNPs in Human
Blood Plasma
As dealing with a plasma protein i.e., fetuin-A, it was reasonable to check the quenching
effect of Dox on fluorescence intensity of fetuin-A and F-GNPs when incorporated with human
plasma. Figure 11 depicts that the different components of human plasma do not affect the
dependent quenching effect on the fluorescence intensity (maxima at 560 nm) of F-GNPs was
observed after spiking of Dox in human plasma (Fig. 11a). In contrast, no drastic effect was
observed for fetuin-A: Dox in human plasma (Fig. 11b) suggesting the tight binding of Dox to
fetuin-A alone (as compared to F-GNPs), slow release of drug into the bloodstream, not allow
the drug to interact with other components of plasma, and thus resulting in low quenching effect.
Established results also confirm the fact that more a drug bind to the protein, less efficiently it
Besides Dox, the recognition properties of fetuin-A alone and F-GNPs was also investigated
for some other commonly uses antimicrobial drugs (i.e., gentamycin, vancomycin, amoxicillin,
tetracycline and ciprofloxacin). Along with UV absorbance, the fluorescence emission was also
monitored at excitation wavelength of 280 nm, where a florescence maximum was followed at
560 nm for both drug: fetuin-A and drug: F-GNPs (Suppl. Fig. S2-6). Interestingly, all
concentration dependent manner suggesting the differential interactions with both fetuin-A alone
and F-GNPs (Fig. 10). As can be noticed in figure 10, each drug shows higher binding affinity
towards fetuin-A (i.e., protein alone), as compared to the F-GNPs. The explanation for such a
significant difference with respect to fluorescence quenching could be the availability of more
complementary binding sites of protein fetuin-A for respective drugs, and conjugating the
protein with GNPs hence reduces the available interaction sites. Moreover, different chemical
nature of the tested drugs and as a result varied nature of interactions with protein fetuin-A could
be another possible reason for differential interactions. Due to the very significant and
sensors.
For comparative cytotoxicity profile, purified protein fetuin-A and Dox alone, fetuin-A: Dox
conjugates and F-GNPs: Dox were subjected to MTT assay using non-small lung carcinoma cell
line. Results revealed that Dox alone inhibit the cell growth at the IC50 value of 0.051 µM (SD
0.02), while purified protein fetuin-A does not inhibit growth even at the highest tested
calculated for the fetuin-A: Dox conjugates (i.e., 0.43 µM 0.04) is almost twice the value
established for F-GNPs: Dox (0.21 µM 0.02) thus suggesting the slow and sustain release of
drug when conjugated with protein and/or protein coated GNPs. The results are in good
agreement with the earlier work of Kratz [66], with similar drug Dox and albumin as drug
carrier, suggesting that protein-drug conjugates work more efficiently then free drug.
Although in-solution studies give a much closer correlation to physiological environment, but
it is rather complex to find out the exact binding location of a drug with its corresponding target.
In the present study, findings of spectroscopic experiments were further complemented with the
molecular modeling and docking studies. The established 3D structure of fetuin-A consists of
three domains (i.e., two N-terminal D1 and D2 domains, and a C-terminal D3 domain), amongst
which D1 (residues Ileu1 - Leu110) and D2 (residues Asn138 - Leu232) are well compact, stabilized
by disulfide bonds and cystatin-like domains, while D3 (residues Leu284 - Ileu341) has a proline-
rich sequence thus comprising much of the loop regions (Fig. 12a). Interestingly, the only
tryptophan residue (i.e., Trp51) present in fetuin-A primary structure is found to be located in a
surface loop region of D1 domain which is quite unusual to be exposed thus suggesting that
protein might exist as homodimer so the Trp51 is protected and sandwich at the interface. RH
measured for the native bovine fetuin-A (i.e., RH = 9.4 ±0.22 nm) which is quite high comparing
to the same size protein (e.g. BSA), thus nicely supporting this hypothesis (Fig. 4b).
The favored sites for the binding of Dox to fetuin-A was established with SwissDock, a web
based server for protein and small molecule fast docking services. Upon blind docking of the
ligand to protein, it has been observed that the principal regions of Dox to fetuin-A interaction
are present typically in the loop regions (Fig. 12b-c). The best fit fetuin-A: Dox complex,
however, suggest that the drug binds in the grove between beta-sheet and long loop region of D1
domain. Amino acid residues: Thr41, Leu42, Asn43 from beta-sheet (as main chain) and Asn81 (as
main and side chain) from the accompanying loop region shows direct hydrogen bonding
between fetuin-A and Dox molecule (Fig. 12b and Insert). Such polar interaction usually acts as
an “anchor” and facilitate the other non-polar hydrophobic bonding of protein’s side chain to the
Dox structure, and thereby strongly locating the 3D space position of drug (Dox) in the binding
pocket of protein (fetuin-A). As aforementioned, Trp51 is also present at the distal end of beta-
sheet contributing in Dox binding with a loop region in D1 domain, thus binding of Dox in the
same region may also bring conformation changes in microenvironment of Trp51 as observed
4. Conclusions
The main aim of present work has been to purify bovine fetuin-A and the biosynthesis of F-
GNPs. A methodical exploration for the characterization as well as interaction between purified
fetuin-A and F-GNPs with Dox have also been illustrated using UV/Vis, FTIR, fluorescence
spectroscopy, CD, AFM, DLS and in-silico studies. Dox is a known anticancer drug and
conducting its binding studies with plasma protein fetuin-A has been significantly important as
fetuin-A is a normal carrier protein in blood. Thus, it has an immense capacity to become a drug
delivery vehicle for slow and sustain release as demonstrated by cytotoxicity assay. Moreover,
presented results also indicates the significant yet differential binding capacity of other tested
drugs (i.e., gentamycin, vancomycin, amoxicillin, tetracycline and ciprofloxacin) to F-GNPs,
though they found to be low in comparison to fetuin-A-drugs conjugates. Notably, the drug to
protein binding also dictates the drug residing time in the blood. Hence the above work is
anticipated to offer a significant insight on how serum proteins can interact with different
chemical nature molecules within the biological sphere. Further studies must be conducted in
order to introduce F-GNPs into other biomedical applications.
Conflict of Interest
Authors declare no conflict of interest.
Acknowledgments
The author (IM) thankfully acknowledges the Higher Education Commission for the
financial support as Merit scholarship under the indigenous Ph.D. program (117-13097-BM7-
273). Indirect financial support from Higher Education Commission (HEC No. 20-
1339/R&D/09; No. 20-3891/NRPU/R&D/HEC/13) and Pakistan Science Foundation
(PSF/Res/S-KU/Bio-422) to SAA is also acknowledged.
Author Contributions
Conceived and designed the experiments: SAA. Performed the experiments: IM SA MRS
AA AH SAA. Analyzed the data: SAA IM SA MRS AA. Contributed reagents/materials/analysis
tools: SAA MRS AA AH. Wrote the paper: SAA IM.
Supplementary Data
Provided in a separate file.
References
[2] D. Agudelo, G. Bérubé, H.A. Tajmir-Riahi, An overview on the delivery of antitumor drug
[4] A.M. Sakwe, R. Koumangoye, S.J. Goodwin, J. Ochieng, Fetuin-A ({alpha} 2HS -
Glycoprotein) is a major serum adhesive protein that mediates growth signaling in breast
[5] N. Zaidi, S. Nusrat, F.K. Zaidi, R.H. Khan, pH-dependent differential interacting
mechanisms of sodium dodecyl sulfate with bovine serum fetuin: a biophysical insight. J.
[6] K.M. Dziegielewska, W.M. Brown, S.J. Casey, D.L. Christie, R.C. Foreman, R.M. Hill and
N.R. Saunders, The Complete cDNA and Amino Acid Sequence of Bovine Fetuin. its
homology with a2hs glycoprotein and relation to other members of the cystatin
[8] Y. Dautova, D. Kozlova, J.N. Skepper, M. Epple, M.D. Bootman, D. Proudfoot, Fetuin-A
chronic heart failure. J Clin Lab Anal. 2017 [Epub ahead of print].
[10] Y.S. Shim, M.J. Kang, Y.J. Oh, J.W. Baek, S. Yang, I.T. Hwang, Fetuin-A as an
[11] W.T. Chang, C.H. Wu, L.W. Hsu, P.W. Chen, J.R. Yu, C.S. Chang, W.C. Tsai, P.Y. Liu,
associated with geriatric sarcopenia and cardiac hypertrophy. Sci Rep. 23 (2017) 40996.
[12] M.M. Cai, E.R. Smith, S.G. Holt, The role of fetuin-A in mineral trafficking and
[13] D.A. Chistiakov, A.N. Orekhov, Y.V. Bobryshev, Vascular smooth muscle cell in
in uremic rats with hyperphosphatemia. Am J Physiol Renal Physiol 309 (2015) F744-54.
[16] J.F. Trepanowski, J. Mey, K. Varady, Fetuin-A: a novel link between obesity and related
[18] P.A. Price, J.E. Lim, The inhibition of calcium phosphate precipitation by fetuin is
22144-22152.
Dechent Fetuin-B, a liver-derived plasma protein is essential for fertilization. Dev. Cell.
25 (2013) 106-112.
[21] J.X. Liu, Y.H. Zhai, F.S. Geng, J.H. Xia, J.F. Gui, Molecular characterization and
expression pattern of fetuin-B in gibel carp (Carassius auratus gibelio). Biochem. Genet.
46 (2008) 620-633.
[22] S. Vijayakumar, S. Ganesan, Gold nanoparticles as an HIV entry inhibitor. Curr. HIV Res.
10 (2012) 643-646.
[23] A.S. Thakor, J. Jokerst, C. Zaveleta, T.F. Massoud, S.S. Gambhir, Gold nanoparticles: a
[25] A.G. Patel, S.H. Kaufmann, How does doxorubicin work? Elife 1 (2012) e00387.
[26] S.N. Khan, A.U. Khan, An in silico approach to map the binding site of doxorubicin on
the binding sites of antibiotic drugs doxorubicin and N-(trifluoroacetyl) doxorubicin with
[28] T.Y. Chen, Studies of the stability, protein binding and pharmacokinetics of doxorubicin
using a new assay method. (1984) Open Access Master's Theses. University of Rhode
[29] I. Vhora, S. Patil, P. Bhatt, A. Misra, Protein- and peptide-drug conjugates: an emerging
drug delivery technology. Adv. Protein Chem. Struct. Biol. 98 (2015) 1-55.
[32] M.M. Bradford, A rapid and sensitive method for the quantitation of microgram quantities
of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72 (1976) 248-
254.
[33] S.A. Ali, Z.H. Zaidi, A. Abbasi, Oxygen Transport Proteins: I. Structure and organization
ofhemocyanin from scorpion (Buthus sindicus). Comp. Biochem. Physiol. B. 112 (1995)
225-232.
[34] U.K. Laemmli, Cleavage of structural proteins during assembly of the head of
[35] S.A. Ali, D.C. Yang, T.N. Jackson, E.A. Undheim, I. Koludarov, K. Wood, A. Jones,
W.C. Hodgson, S. McCarthy, T. Ruder, B.G. Fry, Venom proteomic characterization and
[36] S.A. Ali, T.N. Jackson, N.R. Casewell, J. Visser, D.H. Low, S. Rossi, K. Baumann,
[37] A.H. Wardi, G.A. Michos, Alcian blue staining of glycoproteins in acrylamide disc
[38] S.A. Ali, M. Alam, A. Abbasi, E.A.B. Undheim, B.G. Fry, H. Kalbacher, W. Voelter,
[39] M. Ateeq, M.R. Shah, N. ul Ain, S. Bano, I. Anis, Lubna, S. Faizi, M.F. Bertino, S.
Sohaila Naz, Green synthesis and molecular recognition ability of patuletin coated gold
[41] A. Hameed, S.A. Ali, A.A. Khan, S.T. Moin, K.M. Khan, J. Hashim, F.Z. Basha, M.I.
micellization, and density functional theory studies. RSC Adv. 4 (2014) 64128-64137.
[42] M.R. Shah, S. Ali, M. Ateeq, S. Perveen, S. Ahmed, M.F. Bertino, M. Ali, Morphological
analysis of the antimicrobial action of silver and gold nanoparticles stabilized with
ceftriaxone on Escherichia coli using atomic force microscopy. New J. Chem. 38 (2014)
5633-5640.
[43] L. Whitmore, B.A. Wallace, DICHROWEB, an online server for protein secondary
structure analyses from circular dichroism spectroscopic data. Nucleic Acids Res. 32
(2004) 668-673.
[44] J.J. Irwin, B.K. Shoichet, ZINC – A free database of commercially available compounds
[45] J.J. Irwin, T. Sterling, M.M. Mysinger, E.S. Bolstad, R.G. Coleman, ZINC: a free tool to
[47] A. Grosdidier, V. Zoete, O. Michielin, Fast docking using the CHARMM force field with
1025.
[49] E.F. Pettersen, T.D. Goddard, C.C. Huang, G.S. Couch, D.M. Greenblatt, E.C. Meng, T.E.
Ferrin, UCSF Chimera--a visualization system for exploratory research and analysis. J.
[50] W.M. Brown, K.M. Dziegielewska, N.R. Saunders, D.L. Christie, P. Nawratil, W. Müller-
Esterl, The nucleotide and deduced amino acid structures of sheep and pig fetuin.
Common structural features of the mammalian fetuin family. Eur. J. Biochem. 205 (1992)
321-331.
[51] Ashida, H., Yamamoto, K. and Kumagai, H., Trypsin inhibitory activity of bovine fetuin
[52] S.K. Chaturvedi, E. Ahmad, J.M. Khan, P. Alam, M. Ishtikhar, R.H. Khan,
Elucidating the interaction of limonene with bovine serum albumin:a multitechnique appr
[54] C. Wang, I. Lascu, A. Giartosio, Bovine serum fetuin is unfolded through a molten
semiconductor thin films: optical characterization. Adv. Funct. Mater. 17 (2007) 347-
354.
cationic nanoparticles for controlled delivery of plasmid DNA. Int. J. Pharm. 343 (2007)
247-254.
[57] A. Barth, Infrared spectroscopy of proteins. Biochim. Biophys. Acta. 1767 (2007) 1073-
1101.
[58] S. Link, M.A. El-Sayed, Size and temperature dependence of the plasmon absorption of
[59] C.H. Bae, S.H. Nam, S.M. Park, Formation of silver nanoparticles by laser ablation of a
silver target in NaCl solution. Appl. Surf. Sci. 197 (2002) 628-634.
fibrinogen and fetuin subunits by element mass spectrometry coupled to capillary liquid
Budapest, Hungry.
[63] F. Naseem, R.H. Khan, S.K. Haq, A. Naeem, Characterization of molten globule state of
[64] N. Gull, S. Chodankar, V.K. Aswal, P. Sen, R.H. Khan, Kabir-ud-Din, Spectroscopic
studies on the interaction of cationic surfactants with bovine serum albumin. Colloids
[66] F. Kratz, Albumin as a drug carrier: design of prodrugs, drug conjugates and
Fig. 3(a) Comparative differential analysis of the purified bovine fetuin-A and contaminant BSA
(from Fig. 2b) on 10% SDS-PAGE stained with 0.5% Alcian glycoprotein stain followed by
0.2% colloidal commassie blue G-250. (b) 12% SDS-PAGE electro-blot of the two gel extracted
bands of bovine fetuin-A (from Fig. 3a) used in N-terminal sequence identification. M is the
known molecular weight markers.
Fig. 4. Monodispersity and hydrodynamic radius (RH) analysis of the purified fetuin-A in 50 mM
Tris-saline buffer, pH7.2. Experimental conditions (a), and comparison of finally obtained
results (b) of purified BSA (top) and bovine fetuin-A (bottom) indicating highly pure,
monodispersed signals. (c) Far-UV CD analysis of the purified fetuin-A and BSA (0.1 mg.ml-1)
in 10 mM phosphate buffer, pH7.2.
Fig. 5(a) UV/Vis spectra of fetuin-A conjugated GNPs. Insert: gold solution (transparent) and
synthesized fetuin-A conjugated GNPs (pink). (b) Comparative FT-IR spectra in the region of
4000-500 cm-1 of fetuin-A alone and fetuin-A conjugated GNPs. See "materials and methods"
for details.
Fig. 6. Atomic force microscopic (AFM) analysis of fetuin-A conjugated GNPs. 3D topography
(a) and particles size distribution (b), while (c) illustrate the radius of hydration and
monodispersity established by DLS analysis.
Fig. 7. Stability profile of fetuin-A conjugated GNPs. Monitoring the effect of heat after 2 h (a),
salt (NaCl) concentration (b) and variable pH2-12 (c) after 24 h incubations.
Fig. 8. Effect of doxorubicin on far-UV (a: 190-250 nm) and near-UV (b: 250-330 nm) CD
spectra of purified fetuin-A (0.1 mg.ml-1) with variable concentrations of drug (20 - 240 μM).
Fig. 9. UV/Vis absorption spectra of fetuin-A (2 μg.μL-1) (a) and fetuin-A conjugated GNPs (b)
in the absence and presence of different concentrations of doxorubicin (20 - 240 μM). The same
samples have also been subjected to measure the florescence emission spectra excited at 280 nm
(c and d, respectively). Stock solution for Dox and fetuin-A is prepared in Milli-Q water and in
20 mM Tris-saline buffer pH7.4, respectively.
Fig. 10. Quantitative interaction studies of Dox and other antimicrobial drugs with fetuin-A (2
μg.μL-1) and fetuin-A conjugated GNPs as detected by quenching effect on specific fluorescence
intensity at 560 nm. Significant difference (P<0.05) to original response has been observed by
one way ANOVA followed by Bonferroni post hoc test. Drugs studies include; 1, vancomycin, 2,
amoxicillin, 3, tetracycline, 4, ciprofloxacin, 5, gentamycin, and 6, doxorubicin at a fixed
concentration of 240 μM.
Fig. 11. Quenching effect of doxorubicin (40 - 120 μM) on fluorescence intensity of fetuin-A
conjugated GNPs (a) and fetuin-A alone (b) in human blood plasma. See "materials and
methods" for details.
Fig. 12. 3D structural modeling of bovine fetuin-A and molecular docking analysis with
doxorubicin. (a) The ribbon model represents the structure of fetuin-A as α-helix (red), β-sheets
(cyan) and loop regions (grey) of protein. N-/C-terminal residues, D1-D3 domains, and only
Trp51 residue present in fetuin-A are also marked. Maximum doxorubicin molecules bound to the
loop regions of fetuin-A in blind docking (b). (c) Top view of the same visualized by rotating the
molecule at 90° towards the reader. Zoom insert: the best fit Dox-fetuin-A complex.