Novel bioactive glass-AuNP composites for biomedical applications [625532]

Novel bioactive glass-AuNP composites for biomedical applications
K. Magyaria,⁎,T .N a g y – S i m o na, A. Vulpoia,R . A .P o p e s c ua,b,c, E. Licaretea, R. Stefanb,K .H e r n á d id,
I. Papucb,L .B a i aa,c,⁎⁎
aInterdisciplinary Research Institute on Bio-Nano-Sciences, Babes-Bolyai University, M. Kogalniceanu 1, 400084 Cluj-Napoca, Romania
bFaculty of Veterinary Medicine, University of Agricultural Science and Veterinary Medicine, 400372 Cluj-Napoca, Romania
cFaculty of Physics, Babes-Bolyai University, M. Kogalniceanu 1, 400084 Cluj-Napoca, Romania
dResearch Group of Environmental Chemistry, Department of Applied and Environmental Chemistry, Institute of Chemistry, University of Szeged, Rerrich B. tér 1., 6720 Szeged, Hungary
abstract article info
Article history:
Received 12 August 2016Received in revised form 30 December 2016
Accepted 13 March 2017
Available online 18 March 2017The bioactive glasses doped with gold nanoparticles (AuNPs) are very attractive materials due to their potential
in medical applications. In the present study Pluronic-nanogold hybrid nanoparticles were introduced during the
sol-gel route of the SiO 2-CaO-P 2O5glasses preparation. The obtained samples were characterized by UV –vis spec-
troscopy, X-ray diffraction, FT-IR spectroscopy, transmission electron and scanning electron microscopy and af-terwards they were investigated in terms of bioactivity, protein adsorption and cells viability. The in vitro
bioactivity assessment shows the increase of the number of agglomerated spherical shapes of apatite layers forall Au containing samples, but apatite like structure sizes are in fluenced by the AuNP content. Beside the spherical
shapes, three-dimensional flower-like nanostructures were observed on the surface of the glass with 0.2 mol%
Au
2O. Zeta potential and fluorescence spectroscopy measurements evidenced that the amount of serum albumin
adsorbed onto the composites surface increases with the AuNP content. FT-IR measurements point out that thesecondary structure of the adsorbed proteins presents few minor changes, indicating biocompatibility of the
AuNP doped glasses. The good proliferation rate of Human keratinocytes cells obtained in the presence of sam-
ples with 0.15 and 0.2 mol% Au
2O is comparable with the values achieved from free AuNP, fact that proves the
preservation of AuNP properties after their incorporation inside the bioactive glass matrices.
© 2017 Elsevier B.V. All rights reserved.Keywords:
Gold nanoparticlesBioactive glassesProtein adsorption
Cell viability
1. Introduction
Gold nanoparticles (AuNP) have a broad range of applications due to
their unique physical and chemical properties. Therefore, they could be
used in almost all medical applications such as diagnostics, therapy, pre-
vention and hygienic [1,2]. This applicability mostly depends on the par-
ticle size and shape, surface chemistry and charge [2,3]. The AuNPs with
1–2 nm diameters are toxic due to the possibility to irreversible binding
to the biopolymers in cells, the upper size limit of the penetration viathe
hematoencephalic barrier are between 5 and 20 nm and the colloidal
particles with 3 –100 nm diameters do not have toxic effect on cell cul-
tures [2]. Some promising properties, such as biocompatibility, facilesurface modi fication, good stability and optical properties [1],a n t i b a c t e –
rial effect [4]can be also exploited in tissue engineering uses.
Bioactive glasses based on SiO 2-CaO-P 2O5represent an important
group of biomaterials usable as scaffold in tissue engineering as a resultof their unique properties such as osteoconductive behavior and ability
to bond soft and hard tissue through the carbonated hydroxyapatite
layer (HA) that is formed when it is exposed to biological fluid[5–7].
By doping the bioactive glass with AuNP a material with multifunc-
tional character can be obtained. Beside bioactivity, the glass has the
possibility of releasing AuNP in a speci fic organ [8,9]. Furthermore, by
exploiting the versatile surface chemistry of AuNP glass functionalizedwith speci fic biomolecules can be produced [10].
In the literature, several papers reported about the obtaining of sili-
cate-based bioactive glasses doped with AuNP [1,8,11,12] . Due to the
poor solubility of gold, the limit of the concentration of AuNP from themelted glasses is lower compared to that from the glasses obtained by
the sol-gel method [1,13] . It was reported [8,11] that in the sol-gel de-
rived glasses the gold content was introduced only during the sol prep-aration and just as gold(III) chloride trihydrate (HAuCl
4·3H 2O) and theMaterials Science and Engineering C 76 (2017) 752 –759
⁎Corresponding author.
⁎⁎Correspondence to: L. Baia, Interdisciplinary Research Institute on Bio-Nano-Sciences,
Babes-Bolyai University, M. Kogalniceanu 1, 400084 Cluj-Napoca, Romania.
E-mail addresses: klara.magyari@ubbcluj.ro (K. Magyari), lucian.baia@phys.ubbcluj.ro
(L. Baia).
http://dx.doi.org/10.1016/j.msec.2017.03.138
0928-4931/© 2017 Elsevier B.V. All rights reserved.
Contents lists available at ScienceDirect
Materials Science and Engineering C
journal homepage: www.elsevier.com/locate/msec

AuNP in the glass matrix were obtained by thermal treatments [8,11] .
The size of the nanoparticles obtained using HAuCl 4·3H 2O was reported
to be greater than 25 nm, and also Aun+(n = 1 or 3) species together
with Au0in the form of isolated atoms were also present [8,11].
Jayalekshmi et al. [12]reported that gold nanoparticles incorporated
bioactive glass has been encapsulated in the chitosan-gelatin matrix via
a low temperature method. The AuNP were incorporated in the sol-gel
derived bioactive glass-polymer composite by functionalization viathe
amine linkages. Although they were found that these materials are bio-compatible, the size and shape of the AuNP were not studied. Addition-
ally, the use of Pluronic F127 for achieving the bioactive glass based
composites is expected to in fluence differently the morphological and
structural environment of the samples in comparison with the case ofreported gold nanoparticles incorporated bioactive glass [12].
Having in mind that the properties of AuNP depend on their size and
shape signi ficantly, the main purpose of the present study was the
obtaining of composites based on bioactive glasses with differentamounts of AuNP without having the presence in a considerable
amount of other gold species, i.e.their occurrence can be only the result
of AuNP presence. In the first step, spherical AuNP were obtained and
stabilized with Pluronic [14]. Afterwards, bioactive glasses doped with
the previously obtained AuNP were prepared using sol-gel methodand the newly obtained materials were structurally characterized by
means of X-Ray diffraction analysis (XRD) and FT-IR spectroscopy. Mor-
phological characterization of the free AuNP and the ones embedded
into the glass matrix was conducted using UV –vis absorption spectros-
copy and transmission electron microscopy (TEM). Further, the in vitro
bioactivity of the investigated samples was assessed in simulated bodyfluid (SBF). Taking into account that albumin is considered to greatly re-
duce the acute in flammatory response of biomaterials, its adsorption on
the material surface was used as an indicator regarding the material's
biocompatibility [15–17]. Thus, in order to predict the in vitro biocom-
patibility of the materials, the serum albumin adsorption on the com-posite surface was followed. Serum albumin is the most abundant
protein in the blood plasma and it is known to have a secondary struc-ture that contains about 55% αhelix with the remaining polypeptides
occurring in turns or flexible regions between the subdomains [18,19] .
It consists of three similar helical domains with eight pairs of double di-
sulfide bridges. Cysteine residue, located at position 34 is the single free
thiol group that is not involved in a disul fide bridge [20]. Finally, the cell
viability assay was performed on Human keratinocytes cells with the
main purpose of determining the optimal concentration of AuNP within
the glass system.
2. Materials and methods
2.1. Nanoparticle synthesis and stabilization with Pluronic acid
Gold nanoparticles were synthesized through the Turkevich-Frens
method [21]. Brie fly, hydrogen tetrachloroaurate-(III) trihydrate was
dissolved in ultrapure water at 10
−3M concentration and the solution
was brought to boil under continuous magnetic stirring. Further, 38.8
×1 0−3M trisodium citrate was rapidly added to the boiling HAuCl 4so-
lution at a 10:1 volume ratio. The boiling and stirring were continued for30 min, and then the colloid was cooled down to room temperature.
Fig. 1. Absorption spectra of as synthesized AuNP (black line) and AuNP stabilized with Pluronic F127 (red line) in aqueous solution (a) and TEM image of AuNP with Pluronic F127 (b).
(For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 2. UV–vis spectra of the 60SiO 2·(32-x)CaO·8P 2O5·xAu 2O (mol%) glass composite samples (a), and TEM images recorded from the sample with x = 0.15 mol% with different
magni fications (b) and (c).753 K. Magyari et al. / Materials Science and Engineering C 76 (2017) 752 –759

The obtained gold nanoparticles were stabilized with Pluronic F127
by simply mixing the colloidal solution with Pluronic F127 block copol-
ymer solution at a final concentration of 0.5 × 10−3M. Following mag-
netic agitation for ~20 min, the solution was left at room temperaturefor 24 h for the reaction to proceed. In order to remove the unbound
polymers from the solution, and to obtain concentrated nanoparticle so-
lution, the colloid was centrifuged for 30 min at 12000 rpm. One should
note that the entire AuNP-Pluronic colloids amount was embedded in-
side the bioactive network during the preparation process. Therefore,
the real content of Au can be considered as being the initially calculated
one.
2.2. Glass composite formation
The 60SiO
2·(32-x)CaO·8P 2O5·xAu 2O glass composites with x = 0;
0.05; 0.075; 0.1; 0.15 and 0.2 mol% (the gold amount is conventionally
indicated in the oxidic form Au 2O) were prepared by sol-gel route as de-
scribed in a previous study [16]. The precursors used were tetraethyl
orthosilicate (TEOS), triethyl phosphate (TEP) and calcium nitrate
tetrahydrate (Ca(NO 3)2·4H 2O), hydrolyzed in presence of nitric acid;
Reactants were added consecutively after 1-hour intervals, under con-tinuous stirring. Finally, there were added the solution of colloidal
gold nanoparticle, which was stirred 1 h. The solutions ( sol)w e r e
poured into closed containers that were kept at 37 °C until gelation(gel) was reached (~1 day). The resultant gels were aged 3 days at 37
°C and afterwards dried at 110 °C for 24 h. Material stabilization wascarried out at 600 °C/3 h. This temperature was determined bydifferential thermal analysis of the dried gels. All analyses were per-
formed on powder samples.
2.3. Assessment of the bioactivity
In order to check the bioactivity, the obtained powders were im-
mersed in SBF in closable conical polypropylene flasks that were placed
in an incubator at a constant temperature of 37 °C under static condition
and analyzed after immersion for 7 days. The SBF was prepared accord-
ing to Kokubo's protocol [22]. The solution was buffered at a pH of 7.4 at
37 °C. The weight of glass per volume of SBF used was 10 mg/mL foreach sample. After 7 days, the powders were filtrated, rinsed several
times with distillate water and dried.
2.4. Protein adsorption
Bovine serum albumin (BSA) (Sigma-Aldrich, molecular mass
66 kDa) solution with different concentrations was prepared in phos-
phate buffer solution at pH 7.4 to test the adsorption of protein on the
samples surface with the aid of several investigation methods.
For the fluorescence, zeta potential and FT-IR measurements the
glass powders were immersed in BSA solutions of 0.5 mg/mL,
1 mg/mL and 2 mg/mL, respectively, and afterwards placed in an incu-
bator at a constant temperature of 37 °C for 3 h.
BSA adsorption onto the glass samples was also assessed by zeta po-
tential measurement. The surface charge of the glass samples dispersed
Fig. 3. XRD patterns (a) and FT-IR spectra (b) of the 60SiO 2·(32-x)CaO·8P 2O5·xAu 2O (mol%) glass composites. The XRD pattern of gold is also included for comparison purpose.
Fig. 4. XRD patterns (a) and FTIR spectra (b) of the 60SiO 2·(32-x)CaO·8P 2O5·xAu 2O(mol%) glass composites after their immersion in SBF for 7 days. The XRD patterns of gold and HA as
well as FT-IR spectrum of HA is also included for comparison purpose.754 K. Magyari et al. / Materials Science and Engineering C 76 (2017) 752 –759

in ultrapure water was measured before immersion in BSA solution as
well as after separation from the unbound proteins by centrifugation.
The amount of the adsorbed BSA on different glass samples was
quantitatively determined by fluorescence spectroscopy. The unbound
BSA was first separated by centrifugation for 5 min at 3000 rpm and
thefluorescence spectra of the supernatants were collected. The quanti-
ty of BSA adsorbed onto the glass samples was calculated by subtractingthe BSA amount measured in the supernatant from the initial BSA
solutions.
The FT-IR spectra were measured on the glass powders removed
from protein solution, rinsed three times with distilled water, and
then dried at room temperature.
2.5. Cell viability
Human keratinocytes cells (HaCaT, Cell Line Service, Germany) were
cultured in Dulbecco's modi fied Eagle's medium (Lonza) supplemented
with 2 mM L-glutamine, Pen/Strep 100 U/mL and 10% FCS and incubated
in a humidi fied incubator with 5% CO 2atmosphere at 37 °C.
Cytotoxic effect of the glass samples was assayed using WST-1 dye
(water soluble tetrazolim salt, Millipore), assay based on the enzymatic
cleavage of the tetrazolium salt WST-I to formazan by mitochondrial de-
hydrogenases active in the living cells. Therefore, HaCaT cells were
seeded in a 96-well plate, at a density of 104cells/well. The following
day different amounts of glass samples were added to the test wellsand cells were placed in the incubator for additional 24 h. Each glasssamples concentration was tested in triplicate. Cells without glass sam-
ples were used as positive control. At the end of the incubation period,
medium was removed from all wells and 100 μL of fresh medium con-
taining 10% WST-1 solution were added to each well, cells being furtherincubated for another 60 min at 37 °C. Empty wells with medium con-
taining WST-1 reagent were used as blanks. After 60 min of incubation,
the absorbance was measured at 440 nm, using a microplate reader
(Flostar Omega, BMG, Germany). A reference wavelength was used at
650 nm.
2.6. Methods
2.6.1. UV –vis spectroscopy
The optical response of the prepared nanoparticles was character-
ized by means of absorption spectroscopy. Absorption spectra were re-
corded using a Jasco V-670 UV –Vis-NIR spectrometer with a 1 nm
spectral resolution. Absorption measurements of the glass sampleswere performed with an Analytic Jena Specord 250 plus UV –Vis spec-
trometer. The spectral resolution was of 2 nm.
2.6.2. Transmission electron microscopy
TEM images were recorded using a Tecnai F20 XTWIN field emission,
high resolution transmission electron microscope operating at an accel-
erating voltage of 200 kV and equipped with Eagle 4k CCD camera. Thesamples were suspended in distilled water and then added dropwise on
carbon film coated Cu grids.
2.6.3. X-ray diffraction
The X-ray diffraction analysis (XRD) was carried out on a Shimadzu
XRD 6000 diffractometer using CuK αradiation ( λ= 1.54 Å), with Ni-
filter. The diffractograms were recorded in 2 θrange from 10° to 80°
with a speed of 2°/min. The crystallites average size was calculated bythe Scherrer method.
2.6.4. FT-IR spectroscopy
The FT-IR absorption spec tra were recorded in refl ection con figuration
with a Jasco 6000 (Jasco, Tokyo, Japan) spectrometer, at room tempera-
ture, in the range 400 –4000 cm
−1;s p e c t r a lr e s o l u t i o no f4c m−1; using
the well-known KBr pellet technique. The FT-IR spectra of compositesafter protein adsorption were record ed using a Jasco IRT-5000 FT-IR mi-
croscope coupled to a Jasco FT-IR-6000 spectrometer in re flection confi g-
uration in the range 4000 –650 cm
−1at 4 cm−1resolution using the ×32
Cassegranian objective, imaging a sample area of ~50 × 50 μm. The re-
corded spectra were smoothed by 5-point Savitzky-Golay function.
Fig. 5. SEM images of 60SiO 2·(32-x)CaO·8P 2O5·xAu 2O(mol%) glass composites before
and after immersion in SBF for 7 days.755 K. Magyari et al. / Materials Science and Engineering C 76 (2017) 752 –759

2.6.5. Scanning electron microscopy
The SEM images were recorded on a Hitachi S-4700 Type II cold field
emission scanning electron microscope equipped with a Röntec QX2-
EDS spectrometer operating at an acceleration voltage of 30 kV.
Zeta potential values were measured by Malvern Instrument Zetaiser
Nano-ZS at 25 °C. The results were average from three measurements.
2.6.6. Fluorescence spectroscopy
Thefluorescence spectra of BSA were recorded using a Jasco LP-6500
spectro fluorimeter at 278 nm excitation wavelength in a quartz cell
with 2 mm path length.3. Results and discussion
3.1. Nanoparticles synthesis and characterization of the glass composite
structure
Stabilization of colloidal AuNP to prevent their undesired aggrega-
tion under biological condition can be achieved by encapsulation inbiocompatible polymeric shells [23]. Pluronic F-127 amphiphilic block
polymer is frequently used as stabilizing agent for different types ofnanoparticles due to its high biocompatibility and amphiphilic property
[14]. The free AuNP reveal a narrow plasmonic band in the visible region
centered at 518 nm, characteristic for individual spherical AuNP(Fig. 1 a). After stabilization with Pluronic a red-shift of 2 nm of the ab-
sorption maximum can be observed, which can be attributed to thechange of the refractive index in the close vicinity of the AuNP as a con-
sequence of the polymer adsorption. For the determination of size dis-
tribution and morphological feature of AuNP, TEM images were
recorded. One can observe small spherical AuNP with 12 ± 1 nm in di-
ameter ( Fig. 1 b).
These colloidal AuNP were introduced inside the glass matrix. In
order to get further insight about the size distribution and morphologi-
cal alternations of AuNP during the preparation and thermal treatment,
Uv–vis absorption spectra have been recorded ( Fig. 2 a). The consider-
able red-shift of the plasmonic band (from 520 to 536 nm) indicatesthe presence of larger nanoparticle size compared to the freshly pre-
pared colloidal AuNP. This behavior can be ascribed to the agglomera-
tion/aggregation of nanoparticles after their inclusion into the glass
matrix. Since the applied thermal treatment eliminates the polymercontent, one can expect the occurrence of an additional aggregation of
nanoparticles as a result of the high mobility of AuNP and low viscosity
of silicate glass matrix [11]. The broadening of the absorption band
suggests polydispersity of the AuNP incorporated into the glass matrix.TEM images con firm these results, thus one can infer that after the heat
treatment of the bioactive glasses a part of AuNP keeps the original sizeof 12 ± 1 nm ( Fig. 2 c), but a higher amount of nanoparticles with sizes
of about 100 nm also appears ( Fig. 2 b).
The XRD pattern of the sample without AuNP ( Fig. 3 a) exhibits
mainly amorphous characteristics with few crystallization germs at2θ–32° that can be associated with the formation of apatite like phase
[24]. The presence of gold nanocrystals into the glass matrix was con-
firmed by XRD pattern, where the diffraction lines can be associated
with (111), (200), (220) and (311) planes of a face centered cubicgold lattice ( Fig. 3 a)[25]. For the easy evidencing of the gold signature
in the XRD patterns of the investigated samples, a diffractogram of agold sample is also included as a reference [26]. The average crystallite
size of AuNP is 20.37 nm, as obtained from the Scherrer equation.
The FT-IR spectra of the glass composite samples have characteristic
absorption bands speci fic to a silicate network, and as expected, the
spectral characteristics are not in fluenced by the AuNP, due to their
low content ( Fig. 3 b). Speci fic
vibrational modes of Si \ \O\ \Si groups
can be identi fied as follow: Si \ \O\ \Si stretching (1080 and
1200 cm−1)[27,28] , non-bridging silicon-oxygen stretching
(Si\ \O\ \NOB) around 890 –975 cm−1[29],S i\ \O\ \Si bending
(800 cm−1)[27] and Si\ \O\ \Si rocking (~460 cm−1)[29].T h e
Fig. 6. Difference of zeta potential of glass composites, before and after BSA adsorption as a function of Au 2O amount (a). Adsorbed BSA on 1 mg glass composites obtained from
fluorescence spectra (b).
Fig. 7. FTIR spectra of 60SiO 2·(32-x)CaO·8P 2O5·xAu 2O(mol%) glass composites after BSA
adsorption.756 K. Magyari et al. / Materials Science and Engineering C 76 (2017) 752 –759

stretching vibrations of phosphate groups give rise to doublet at 565
and 604 cm−1[27,30] .
3.2. Assessment of bioactivity
To follow how the in vitro bioactivity of the gold content embedded
inside the bioactive glasses is in fluenced, the samples were immersed in
SBF. After one week of immersion the presence of self-assembled HA
can be observed. Thus, the presence of a HA crystalline phase is clearly
visible in the XRD patterns as evidenced for all samples by the re flection
recorded at 2 θangles of 25.4 (002) and 31.5 (211) ( Fig. 4 a). The wide
diffraction peak between 30° and 34° angles (2 θ) corresponds to the
overlapping of (112), (300) and (202) re flection planes of the well-crys-
tallized HA [31]. The overlapped component begins to be visible for thesample with x = 0.2. To easy associate apatite phase, XRD pattern of HAis included as a reference [32]. In the case of the FT-IR spectra recorded
after immersion in SBF, the presence of the doublet located around 604and 564 cm
−1becomes more accentuated. These two bands are attrib-
uted to P \ \O bending vibrations and they are characteristic bands of the
crystalline HA phase [27,33] .
Beside the above mentioned techniques, SEM images also con firm
the formation of a calcium phosphate layer on the samples surfaceafter their immersion in SBF, the HA is visible on all investigated glass'
surface ( Fig. 5 ). Although, an apatite layer is formed on all surfaces,
SEM images show differences regarding the formed layer morphologydepending on Au content, indicating the in fluence of the AuNP on the
sample bioactivity. As one can be observed from Fig. 5 , the presence of
AuNP is bene ficial in what regard the bioactivity of the samples, in the
Fig. 8. Distribution of secondary structure in lyophilized and adsorbed BSA onto 60SiO 2·(32-x)CaO·8P 2O5·xAu 2O(mol%) glass composites' surface and their distribution on secondary
structure in lyophilized adsorbed BSA onto glass composites' surface.757 K. Magyari et al. / Materials Science and Engineering C 76 (2017) 752 –759

case of glass with 0.2 mol% Au 2O, beside the spherical shapes, three-di-
mensional flower-like nanostructures can be observed.
3.3. Protein adsorption
BSA adsorption onto the samples was first qualitatively evaluated by
zeta potential measurements. To assess the surface charge modi fica-
tions of bioactive glasses with the increase of the AuNP content, the
zeta potential of glasses, before and after BSA adsorption, were mea-
sured in aqueous dispersion. Given that the studied glasses have zeta
potential values around −24 ± 3 mV and BSA molecules have higher
negative zeta potential at −17 mV [34], the relative zeta potential mod-
ification of glass composites after BSA adsorption was calculated ( Fig.
6a). It was found that with the increase of the AuNP content in the
glass composite, the difference of the zeta potential increases, due to
the higher amount of the adsorbed BSA.
In the next step, BSA adsorption was quantitatively evaluated by
means of fluorescence spectroscopy. The fluorescence intensities of
the BSA solutions after their separation from the glass sample compos-ites decrease with the increase of the AuNP content. Using the initial so-
lution of BSA, which was kept in the same condition, the weight of the
adsorbed BSA onto the sample surface was calculated by subtractingthe BSA quantity measured in the supernatant from the initial BSA solu-
tions ( Fig. 6 b). These results are in agreement with those obtained for
the zeta potential assessment regarding the amount of the adsorbedBSA that was found to increase with the increase of the AuNP content.
The reason is that the cysteine bonds to AuNP viaa strong Au-S interac-
tion[35]and BSA has one cysteinyl residue at position 34, and 17 disul-
fide bridges per molecule [36].
The albumin adsorption on the glass composite surfaces was con-
firmed by FT-IR spectroscopy by the appearance of absorption bands
characteristic for proteins: amide I (C \ \O stretch mode) at
1650 cm
−1,a m i d eI I( N \ \H in-plane bending mode) at 1550 cm−1
and amide III (C \ \N stretching and N \ \H bending modes) around
1400 cm−1(Fig. 7 )[37].
For the assessment of the changes of secondary structure conforma-
tion of BSA after its adsorption on the glass composite surface the
deconvoluted signals of the amide I absorption band from FT-IR spec-
trum were analyzed [17,37] . One should note that this band is the
most sensitive to conformational changes of the protein secondarystructure ( Fig. 8 ). The secondary structure of BSA is dominated by αhe-
lices (1649 –1657 cm
−1), with small amount of β-sheet (1618 –1641
and 1674 –1695 cm−1)a n d β-turn (1662 –1686 cm−1)s t r u c t u r e s [38].
The bands between 1608 and 1618 cm−1can be associated with the ex-
istence of β-sheet/amino acid side chain residues [39]. It is known that
both α-helix and β-sheet structures are stabilized by hydrogen bonds,
butα-helix is more flexible and more resistant to ambient conditions
[40]. Therefore, when the protein is attached on the surface, conforma-
tional changes occur, usually the β-sheets release their structure and go
to more flexible one [40]. Exactly this situation occurs after the BSA ad-
sorption, its secondary structure slightly changes by decreasing the β-
sheets amount. Whereas, the α-helix structure remains unchanged,
due to the fact that the three-dimensional protein structure is in flu-
enced and stabilized to a great extent by cysteine disul fide bridges
[36], which bond to AuNP. These results suggest that the samples with
AuNP preserve their biocompatibility.
3.4. Cell viability
As a next step, the cell viability in the in vitro studies was considered.
The viability of Human keratinocytes cells in the presence of the glass
composites is very close to or greater than 100%, indicating a good in
vitro tolerance ( Fig. 9 ). Therefore, the cytotoxic effect is almost absent
or minimal after 24 hour exposure in all diluted samples. Starting with
150μg/mL glass concentration, the proliferation of HACaT cells was pro-
moted by the samples with x = 0.15 and 0.2. These values arecomparable with proliferation of cells when the cell viability was testedon AuNP. The obtained results of proliferation rate of cells with AuNP
are in agreement with previous results reported by Lu et al. [41],
where was demonstrated that low concentration of gold nanoparticles(16– 51 nm) could stimulate keratinocytes cells proliferation. Thus,
these properties of AuNP are preserved when the glass matrix containsat least 0.15 mol% Au
2O.
4. Conclusions
The sol-gel method was successfully used to obtain bioactive glasses
doped with AuNP. The small spherical AuNP with 12 ± 1 nm diameter
were synthesized and stabilized by Pluronic F-127 amphiphilic block
polymer, and afterwards they were introduced in the sols. The material
stabilization was carried out at 600 °C and the results showed that be-
side the AuNP with size of 12 ± 1 nm nanoparticle aggregates withsizes about 100 nm were also formed. The XRD pattern indicates the
presence of both amorphous glass structure and gold nanocrystals.
After immersion in SBF, the presence of a self-assembled apatite layer
was identi fied on all investigated sample surfaces indicating a good in
vitro bioactivity of the investigated samples. Differences occurred in
the layer morphology with the increase of the AuNP content, namely
the sizes of agglomerated spherical shaped entities were found to be
greater when the samples contained AuNP. Only on the surface of the
glass with 0.2 mol% Au
2O, beside the spherical shapes, three-dimen-
sional flower-like nanostructures were observed.
The amount of the adsorbed serum albumin was increasing with the
increase of the AuNP content inside the glass matrix, due cysteine bondsto AuNP viaa strong Au-S interaction. Thanks to these interactions the
secondary structure of BSA after adsorption slightly changed by de-creasing the β-sheets amount, con firming a good biocompatibility of
the samples.
The viability of human keratinocytes cells after 24 h interaction with
investigated samples presented values very close to or greater than100%, indicating a good in vitro tolerance. However, the good prolifera-
tion rate of Human keratinocytes cells obtained on samples with 0.15and 0.2 mol% Au
2O are very close to the values obtained in the presence
of free AuNP, demonstrating thus the preservation of AuNP properties.
In summary, there were obtained AuNP embedded bioactive glass,
without the presence of other gold species, but with different sizes ofAuNP. However, the behavior of such obtained glass composites in bio-
logical environment seemed to be favorable.
Fig. 9. Viability of human keratinocytes cells after 24 h interaction with different
concentration of 60SiO 2·(32-x)CaO·8P 2O5·xAu 2O(mol%) glass composites and AuNP.758 K. Magyari et al. / Materials Science and Engineering C 76 (2017) 752 –759

Acknowledgements
This work was supported by a grant of the Romanian National Au-
thority for Scienti fic Research and Innovation, CNCS –UEFISCDI, project
number PN-II-RU-TE-2014-4-1597. K. Magyari wishes to thank for the
financial support provided by Babe ș-Bolyai University, project number
GTC_31798/2016.
References
[1]V. Aina, G. Cerrato, G. Martra, L. Bergandi, C. Costamagna, D. Ghigo, et al., Gold-con-
taining bioactive glasses: a solid-state synthesis to produce alternative biomaterials
for bone implantations, J. R. Soc. Interface 10 (2013) 20121040.
[2]L.A. Dykman, N.G. Khlebtsov, Gold nanoparticles in biology and medicine: recent ad-
vances and prospects, Acta Nat. 3 (2011) 34 –55.
[3]M.-C. Daniel, D. Astruc, Gold nanoparticles: assembly, supramolecular chemistry,quantum-size-related properties, and applications toward biology, catalysis, and
nanotechnology, Chem. Rev. 104 (2004) 293 –346.
[4]D. MubarakAli, N. Thajuddin, K. Jeganathan, M. Gunasekaran, Plant extract mediatedsynthesis of silver and gold nanoparticles and its antibacterial activity against clini-
cally isolated pathogens, Colloids Surf. B: Biointerfaces 85 (2011) 360 –365.
[5]A. Hoppe, N.S. Guldal, A.R. Boccaccini, A review of the biological response to ionic
dissolution products from bioactive glasses and glass-ceramics, Biomaterials 32
(2011) 2757 –2774.
[6]J.R. Jones, Review of bioactive glass: from Hench to hybrids, Acta Biomater. 9 (2013)4457 –4486.
[7]L.L. Hench, The story of bioglass, J. Mater. Sci. Mater. Med. 17 (2006) 967 –978.
[8]S. Simon, R. Ciceo-Lucacel, T. Radu, L. Baia, O. Ponta, A. Iepure, et al., Gold nanopar-ticles developed in sol-gel derived apatite-bioactive glass composites, J. Mater. Sci.
Mater. Med. 23 (2012) 1193 –1201.
[9]T. Radu, D. Benea, R. Ciceo-Lucacel, O. Ponta, S. Simon, Valence band dependence onthermal treatment of gold doped glasses and glass ceramics, J. Appl. Phys. 111
(2012) 034701.
[10] R.A. Sperling, P.R. Gil, F. Zhang, M. Zanella, W.J. Parak, Biological applications of gold
nanoparticles, Chem. Soc. Rev. 37 (2008) 1745 –2140.
[11] G. Lusvardi, G. Malavasi, V. Aina, L. Bertinetti, G. Cerrato, G. Magnacca, et al., Bioac-tive glasses containing Au nanoparticles. Effect of calcination temperature on struc-
ture, morphology, and surface properties, Langmuir 26 (2010) 10303 –10314.
[12] A.C. Jayalekshmi, C.P. Sharma, Gold nanoparticle incorporated polymer/bioactive
glass composite for controlled drug delivery application, Colloids Surf. B:
Biointerfaces 126 (2015) 280 –287.
[13] J. Sasai, K. Hirao, Relaxation behavior of nonlinear optical response in borate glassescontaining gold nanoparticles, J. Appl. Phys. 89 (2001) 4548.
[14] T. Simon, S.C. Boca, S. Astilean, Pluronic-Nanogold hybrids: synthesis and tagging
with photosensitizing molecules, Colloids Surf. B: Biointerfaces 97 (2012) 77 –83.
[15] L. Tang, J.W. Eaton, In flammatory response to biomaterials, Am. J. Clin. Pathol. 103
(1995) 466 –471.
[16] K. Magyari, L. Baia, A. Vulpoi, S. Simon, O. Popescu, V. Simon, Bioactivity evolution ofthe surface functionalized bioactive glasses, J. Biomed. Mater. Res. B Appl. Biomater.
103 (2015) 261 –272.
[17] K. Magyari, C. Gruian, B. Varga, R. Ciceo-Lucacel, T. Radu, H.-J. Steinhoff, et al., Ad-
dressing the optimal silver content in bioactive glass systems in terms of BSA ad-
sorption, J. Mater. Chem. B 2 (2014) 5799 –5808.
[18] X.M. He, D.C. Carter, Atomic structure and chemistry of human serum albumin, Na-ture 358 (1992) 209 –2015.
[19] E. Bramanti, E. Benedetti, Determination of the secondary structure of isomeric
forms of human serum albumin by a particular frequency deconvolution procedure
applied to Fourier transform IR analysis, Biopolymers 38 (1996) 639 –653.[20] S.
Sugio, A. Kashima, S. Mochizuki, M. Noda, K. K., Crystal structure of human serum
albumin at 2.5 Å resolution, Protein Eng. 12 (1999) 439 –446.
[21] G. Frens, Controlled nucleation for the regulation of the particle size in monodis-perse gold suspensions, Nat. Phys. Sci. 241 (1973) 20 –22.
[22] T. Kokubo, H. Takadama, How useful is SBF in predicting in vivo bone bioactivity?Biomaterials 27 (2006) 2907 –2915.
[23] J. Zhou, J. Ralston, R. Sedev, D.A. Beattie, Functionalized gold nanoparticles: synthe-sis, structure and colloid stability, J. Colloid Interface Sci. 331 (2009) 251 –262.
[24] S. Padilla, J. Roman, S. Sanchez-Salcedo, M. Vallet-Regi, Hydroxyapatite/SiO
2-CaO-
P2O5glass materials: in vitro bioactivity and biocompatibility, Acta Biomater. 2
(2006) 331 –342.
[25] Mechler A. Sirajuddin, A.A.J. Torriero, A. Nafady, C.-Y. Lee, A.M. Bond, et al., The for-mation of gold nanoparticles using hydroquinone as a reducing agent through a lo-calized pH change upon addition of NaOH to a solution of HAuCl
4,C o l l o i d sS u r f .A
Physicochem. Eng. Asp. 370 (2010) 35 –41.
[26] R.T. Downs, The RRUFF project: an integrated study of the chemistry, crystallogra-
phy, Raman and infrared spectroscopy of minerals, Program and Abstracts of the19th General Meeting of the International Mineralogical Association in Kobe,Japan, 2006 ( http://rruf finfo/gold/display=default/RRUFF ID: R0702791).
[27] J. Serra, P. Gonzalez, S. Liste, C. Serra, S. Chiussi, B. Leon, et al., FTIR and XPS studies ofbioactive silica based glasses, J. Non-Cryst. Solids 332 (2003) 20 –27.
[28] R. Veres, D.L. Tranda fir, K. Magyari, S. Simon, D. Barbos, V. Simon, Gamma irradiation
effect on bioactive glasses synthesized with polyethylene-glycol template, Ceram.
Int. 42 (2016) 1990 –1997.
[29] H. Aguiar, J. Serra, P. González, B. León, Structural study of sol –gel silicate glasses by
IR and Raman spectroscopies, J. Non-Cryst. Solids 355 (2009) 475 –480.
[30] S. Padilla, J. Roman, A. Carenas, M. Vallet-Regi, The in fluence of the phosphorus con-
tent on the bioactivity of sol-gel glass ceramics, Biomaterials 26 (2005) 475 –483.
[31] R.K. Singh, A. Srinivasan, Bioactivity of SiO
2–CaO–P2O5–Na2O glasses containing
zinc–iron oxide, Appl. Surf. Sci. 256 (2010) 1725 –1730.
[32] R.T. Downs, The RRUFF project: an integrated study of the chemistry, crystallogra-
phy, Raman and infrared spectroscopy of minerals, Program and Abstracts of the
19th General Meeting of the International Mineralogical Association in Kobe,
Japan, 2006 (O03-13, http://rruf finfo/general=HA/display=default/R060180 ,
RRUFF ID: R060180).
[33] R. Downs, The RRUFF project: an integrated study of the chemistry, crystallography,
Raman and infrared spectroscopy of minerals, Program and Abstracts of the 19th
General Meeting of the International Mineralogical Association in Kobe, Japan,
2006 (O03-13, http://rruf finfo/general=HA/display=default/R060180 ; RRUFF ID:
R050512).
[34] B. Jachimska, A. Pajor, Physico-chemical characterization of bovine serum albumin
in solution and as deposited on surfaces, Bioelectrochemistry 87 (2012) 138 –146.
[35] J.A. Carr, H. Wang, A. Abraham, T. Gullion, J.P. Lewis, l-Cysteine interaction with Au 55
nanoparticle, J. Phys. Chem. C 116 (2012) 25816 –25823.
[36] K. Nakamura, S. Era, Y. Ozaki, M. Sogami, T. Hayashi, M. Murakami, Conformational
changes in seventeen cystine disul fide bridges of bovine serum albumin proved by
Raman spectroscopy, FEBS Lett. 417 (1997) 375 –378.
[37] A. Barth, Infrared spectroscopy of proteins, Biochim. Biophys. Acta 2007 (1767)
1073 –1101.
[38] S. Nafisi, G. Bagheri Sadeghi, A. PanahYab, Interaction of aspirin and vitamin C with
bovine serum albumin, J. Photochem. Photobiol. B 105 (2011) 198 –202.
[39] S.Y. Lin, Y.S. Wei, T.F. Hsieh, M.J. Li, Pressure dependence of human fibrinogen corre-
lated to the conformational alpha-helix to beta-sheet transition: an Fourier trans-form infrared study microspectroscopic study, Biopolymers 75 (2004) 393 –402.
[40] S. Tunc, M.F. Maitz, G. Steiner, L. Vazquez, M.T. Pham, R. Salzer, In situ conformation-
al analysis of fibrinogen adsorbed on Si surfaces, Colloids Surf. B: Biointerfaces 42
(2005) 219 –225.
[41] S. Lu, D. Xia, G. Huang, H. Jing, Y. Wang, H. Gu, Concentration effect of gold nanopar-
ticles on proliferation of keratinocytes, Colloids Surf. B: Biointerfaces 81 (2010)406–411.759 K. Magyari et al. / Materials Science and Engineering C 76 (2017) 752 –759