We study experimentally the photoluminescence of small glycine-coated silver nanoparticles and their application as the bio-imaging markers of the neural stem cells. In addition we study nanoparticle’s toxic effects on the neural stem cells. Glycine-coated silver nanoparticles were synthesized using a thermal reduction of silver nitrate in a glycine matrix and size-separated via centrifugation. The properties of the nanoparticles
were characterized using transmission electron microscopy, extinction and photoluminescence spectroscopy. Our results indicate that the nanoparticles have deleterious effects on the cells and showed an amplified increase in their death rates. In fixed cells the particles penetrate the membranes within an hour and 25 minutes of incubation, but do not penetrate into the body of the cell

Almemar, Z.

Dmytruk, I.M.

Kotko, A.V.

Kravets, V.V.

Pinchuk, A.O.

SocioEconomic Challenges Journal (Sumy State University)

PROCEEDINGS OF THE INTERNATIONAL CONFERENCE  
NANOMATERIALS: APPLICATIONS AND PROPERTIES 
Vol. 4 No 2, 02NABM02(4pp) (2015) 
 
 
2304-1862/2015/4(2)02NABM02(4) 02NABM02-1  2015 Sumy State University 
Fluorescent Glycine-Coated Silver Nanoparticles as Bio-Imaging  
Agents for the Neural Stem Cells 
 
V.V. Kravets1,2, Z. Almemar1, A.V. Kotko3, I.M. Dmytruk2, A.O. Pinchuk1, * 
 
1 University of Colorado Colorado Springs, 1420, Austin Bluffs Prkw., 80918, Colorado Springs, CO, USA 
2 Taras Shevchenko National University of Kyiv, 2 Academick Glushkov Prospect, Kyiv, 03127, Ukraine 
3 I.M. Frantsevich Institute for problems of Material Science, 3 Krzhizhanovsky str., 03680, Kyiv, Ukraine 
 
(Received 27 May 2015; published online 26 August 2015) 
 
We study experimentally the photoluminescence of small glycine-coated silver nanoparticles and their 
application as the bio-imaging markers of the neural stem cells. In addition we study nanoparticle’s toxic 
effects on the neural stem cells. Glycine-coated silver nanoparticles were synthesized using a thermal re-
duction of silver nitrate in a glycine matrix and size-separated via centrifugation. The properties of the na-
noparticles were characterized using transmission electron microscopy, extinction and photoluminescence 
spectroscopy. Our results indicate that the nanoparticles have deleterious effects on the cells and showed 
an amplified increase in their death rates. In fixed cells the particles penetrate the membranes within an 
hour and 25 minutes of incubation, but do not penetrate into the body of the cell. 
 
Keywords: silver nanoparticles, stem cells, toxicity, bio-imaging, surface plasmon resonance, photolumi-
nescence 
 
 PACS number(s): 78.55.Hx, 78.67.Bf 
 
 
                                                          
* apinchuk@uccs.edu 
1. INTRODUCTION 
 
Recent advances in nanobiotechnology have demon-
strated nanoparticles (NPs) as potential drug delivery 
agents and luminescent biological labels for bio-
imaging [1,2]. Consequently, the ability to track and 
image the fate of NPs starting from the systemic down 
to the sub-cellular level is an essential factor in drug 
delivery system. In addition, monitoring morphological 
and functional alterations in the cells due to the thera-
peutic delivery is also gaining interest [3]. The devel-
opment of NPs and their bio-imaging applications sig-
nifies the need to study, examine, and monitor their 
toxicity [4]. In this study, we demonstrated the pene-
tration of glycine-coated silver NPs through the mem-
brane of the neural stem cells (NSCs). We found that 
glycine-coated NPs induce primarily apoptotic response 
in time-studies. In unison, these NPs were found to 
inhibit the proliferation of NSCs. Overall, the function-
al NPs could potentially be used for controlled drug 
release and fluorescence imaging of biological cells. 
The literature indicates that a considerable amount 
of research has focused on the generation and develop-
ment of NPs, however the study of silver NPs in bio-
imaging of the NSCs is a novel approach.  In this study, 
we establish that NSCs tend toward the 45% increased 
cell death when treated with silver NPs. Our studies 
indicate that NPs penetrate through the cell mem-
branes, however not into their body. 
The growing research interest in NSCs is driven by 
their possible application as a therapeutic tool in neu-
rodegenerative disorders caused by the loss of neural 
cells. NSCs exist in the developing central nervous 
system and are capable of differentiating into mature 
neurons and in some cases non-neuronal cells called 
neuroglia cells [5]. C17 cells (derived from developing 
mouse cerebellum) are the primary mammalian cell 
line used in these experiments. C17s are adherent cells 
and readily undergo apoptosis when environmental 
conditions are not ideal [6].  
Small noble metal NPs have been extensively stud-
ied because of their unique optical and electronic prop-
erties [7, 8] which lead to their numerous applications 
in biochemistry and medicine.  Namely, metal NPs 
offer opportunity for advancements in bio-imaging [9], 
improvement of solar cells [10], information storage 
[11], and other. 
We synthesized silver NPs through the thermal re-
duction in glycine matrix. The size separation was 
performed using centrifugation. The success of the 
synthesis and size separation is supported by the 
transmission electron microscopy (TEM) and optical 
extinction and photoluminescence (PL) spectroscopy 
characterization. The data supporting NPs characteri-
zation is presented in the following section. 
 
2. MATERIALS AND METHODS 
 
2.1 Nanoparticle synthesis and characterization 
 
Silver nitrate (100mg) and glycine (1g) were mixed 
and dissolved in 10 ml of distilled water. The water was 
then removed by evaporation with a rotoevap in order 
to prepare the solid phase mixture.  Thermal reduction 
of the solid phase mixture was completed at 445 K. The 
reduction of silver nitrate to solid silver was indicated 
as color of the mixture change from white to dark 
brown. To purify the product we sonicated and then 
filtered it with 2 micrometer filter. Next, the product 
was centrifuged with different relative centrifugal forc-
es (500g – 16000g). 
The samples containing NPs of different sizes were 
characterized by TEM to determine shapes and sizes of 
particles and confirm the efficiency of size separation 
by centrifugation. The size distribution of selected 
 V.V. KRAVETS, Z. ALMEMAR, A.V. KOTKO, I.M. DMYTRUK, A.O.PINCHUK PROC. NAP 4, 02NABM02 (2015) 
 
 
02NABM02-2 
sample is shown in Fig.1. The JEOL JEM-100CX elec-
tron microscope was used for the size characterization. 
Sample which contained smallest NPs of mean diame-
ters of 9 nm was later used in the bio-imaging and 
toxicity studies. Most of the particles had a spherical 
shape. 
Extinction and emission spectra of the sample are 
described in the section 3.1. 
 
 
 
Fig. 1 –TEM of silver nanoparticles of different sizes.  
 
2.2 Cell culture  
 
The C17.2 cell line adopted from the cerebellum of 
newborn mice are capable of differentiating into neu-
rons and are very sensitive to factors in their surround-
ing environments. The adherent C17.2 cell line was 
supplied by Dr. Evan Y. Snyder from Harvard Medical 
School, Boston, MA. C17.2 neural stem cells (NSC). 
Cells were grown in Dulbecco's modified Eagle's medi-
um (DMEM) and supplemented with HEPES, 
Pen/Strep, L-Glutamine, 10% FBS, and 5% Normal 
Horse Serum. Cells were cultured in normal/unaltered 
FBS media for five-weeks. Once 2×106 to 5×106 cells per 
ml of medium was grown, the cells were lysed and di-
vided. The cells were diluted to a factor of 1:200, and 
life stage passages 13-18 were used for these studies. 
 
2.3 Cell line treatment  
 
Neural stem cells were isolated and resuspended in 
the appropriate media. 1×106 cells/mL of the cell sus-
pension was used for manual cell counting. The re-
maining cell suspension was resuspended to 36×106 
cells per ml. For viability tests, NSCs were treated with 
glycine-coated silver NPs and localization tests were 
performed using 1% paraformaldehyde as the primary 
fixative to cease all metabolic activities in the NSCs.  
 
3. RESULTS 
 
3.1 Extinction and photoluminescence of silver 
nanoparticles 
 
Here we present extinction and PL emission spectra 
of the particles (shown on Fig.2). Extinction spectra (a) 
reveal surface-plasmon-resonance-related (SPR) peak, 
situated at 425 nm.  
The spectral range of the PL emission (Fig.2 b) coin-
cides with the SPR-related extinction spectral range. It 
can be rationalized by two theories: first, the interband 
transitions in silver, enhanced by the SPR, or second, 
the plasmon emission itself.  First theory is based on 
the idea of the interaction of the photons (emitted via 
the interband transition of the electron) with the elec-
tric field created by the oscillating free electrons (SPR) 
[12]. Second theory is based on radiative relaxation of 
the surface plasmon resonance [13]. 
Peak position in the PL spectrum (Fig.2 b) is slight-
ly red-shifted with respect to the extinction spectral 
peak (Fig.2 a). It was found at 448 nm. The red shift 
may be explained by the loss of energy via non-
radiative relaxation of the electrons prior to the emis-
sion. 
 
 
 
Fig. 2 – (a) Extinction (optical density) and 
(b) Photoluminescence spectra of nanoparticles of mean diam-
eter of 9 nm. 
 
Similarity of the extinction and PL emission spectra 
illustrates the fact that their mechanisms are related to 
the SPR.  
We picked the sample with the smallest NPs for bio-
imaging, because small particles have greater chance to 
penetrate through the cell membranes. Next section 
describes the fluorescence confocal microscopy of the 
neural stem cells incubated with the silver NPs. 
 
3.2 Bio-imaging  
 
We studied penetration of the nanoparticles into the 
fixed and live cells. As a control we used a sample of 
cells, without the particles to monitor healthy cells under 
 FLUORESCENT GLYCINE-COATED SILVER NANOPARTICLES AS BIO-IMAGING AGENTS ... PROC. NAP 4, 02NABM02 (2015) 
 
 
02NABM02-3 
preferable conditions. Second sample of cells was kept 
under the same conditions, but in addition contained 
nanoparticles. The cells in the control sample were ad-
hered to the well and the cell division could be monitored 
as shown in Fig. 3. 
 
 
 
 
 
Fig. 3 – Confocal fluorescent and transmission image overlay 
of the control sample of neural stem cells without NPs. No 
background fluorescence is present. Cell division can be 
observed.  
 
Unlike in the control sample (Fig.3), cells in the 
sample with NPs weren’t adhered to the surface of the 
well shortly after the incubation, they maintained near-
spherical shapes during the experiment, and no cell 
proliferation was observed. The fluorescence signal due 
to NPs can be seen on Fig. 4b. NPs have caused toxic 
effect on the cells. The untreated NSCs show about 45% 
more viable cells than the NP-treated cells. The figure 
below illustrates the pronounced difference between 
the blank and the treated NSCs. 
 
Fig. 4 – Confocal fluorescence and transmission image overlay 
of the sample of neural stem cells (a) without NPs and (b) with 
NPs.  
 
We also studied NPs penetration through the cell 
membranes of the fixed cells in 3D (Fig.5). The experi-
ment was performed over 1 hour 25 minutes. Particles 
penetrated the cell membrane, but were not observed 
in the body of the cell (dark area in the center of the 
Fig.5a). It is possible that longer exposure to the NPs 
would allow them to go deeper onto the cell body. The 
so-called “Z-stack” on Fig.5a shows 9 focal planes, ob-
tained within 1 micrometer from each other. The com-
bination of all z-planes together (top view of a cell) is 
shown on (b).  
 
 
a b 
 
Fig. 5 – Confocal fluorescence microscopy of (s) Z-stack of the 
NSC with nanoparticles (a), Top view of all layers collected (b) 
 
4. CONCLUSIONS 
 
We showed that glycine-coated silver nanoparticles of 
mean diameters of 9 nm have surface-plasmon-
a) 
b) 
a) 
b) 
c) 
 V.V. KRAVETS, Z. ALMEMAR, A.V. KOTKO, I.M. DMYTRUK, A.O.PINCHUK PROC. NAP 4, 02NABM02 (2015) 
 
 
02NABM02-4 
resonance-related photoluminescence. They can pene-
trate membranes of fixed neural stem cells, and hence 
can serve as bio-imaging probes in fluorescence micros-
copy. The particles also cause morphology, proliferation 
and viability changes to the live cells, mostly causing cell 
death. This finding potentially can be used for targeted 
cell destruction.  
AKNOWLEDGEMENTS 
 
Authors are grateful to Todd Linden for help with 
preparation of nanoparticles. We thank to the BioFron-
tiers Institute and NATO (SFPP-984617) grants for 
partial financial support. 
 
 
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Fluorescent Glycine-Coated Silver Nanoparticles as Bio-Imaging Agents for the Neural Stem Cells

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Fluorescent Glycine-Coated Silver Nanoparticles as Bio-Imaging Agents for the Neural Stem Cells

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