Biomaterials 176 (2018) 106e121

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Biomaterials
journal homepage: www.elsevier.com/locate/biomaterials

Nanodiamonds as “artificial proteins”: Regulation of a cell signalling

system using low nanomolar solutions of inorganic nanocrystals
Lukas Balek a, Marcela Buchtova b, c, Michaela Kunova Bosakova a, d, Miroslav Varecha a, d,
Silvie Foldynova-Trantirkova e, f, Iva Gudernova a, Iva Vesela b, Jan Havlik g, h,
Jitka Neburkova g, i, Stuart Turner j, Mateusz Adam Krzyscik k, Malgorzata Zakrzewska k,
Lars Klimaschewski l, Peter Claus m, Lukas Trantirek e, Petr Cigler g, *, Pavel Krejci a, c, d, *
a
Department of Biology, Faculty of Medicine, Masaryk University, 62500, Brno, Czech Republic
b
Institute of Experimental Biology, Faculty of Sciences, Masaryk University, 62500, Brno, Czech Republic
c
Institute of Animal Physiology and Genetics of the CAS, 60200, Brno, Czech Republic
d
International Clinical Research Center, St. Anne's University Hospital, 65691, Brno, Czech Republic
e
Central European Institute of Technology, Masaryk University, 62500, Brno, Czech Republic
f
Institute of Biophysics of the CAS, 61265, Brno, Czech Republic
g
Institute of Organic Chemistry and Biochemistry of the CAS, 16610, Prague, Czech Republic
h
Faculty of Science, Charles University, 12840, Prague, Czech Republic
i
First Faculty of Medicine, Charles University, 12108, Prague, Czech Republic
j
EMAT, University of Antwerp, Groenenborgerlaan 171, B-2020, Antwerp, Belgium
k
Department of Protein Engineering, Faculty of Biotechnology, University of Wroclaw, 50-383, Wroclaw, Poland
l
Division of Neuroanatomy, Medical University of Innsbruck, A-6020, Innsbruck, Austria
m
Institute of Neuroanatomy and Cell Biology, Hannover Medical School, 30625, Hannover, Germany

a r t i c l e i n f o a b s t r a c t

Article history: The blocking of specific protein-protein interactions using nanoparticles is an emerging alternative to
Received 8 November 2017 small molecule-based therapeutic interventions. However, the nanoparticles designed as “artificial
Received in revised form proteins” generally require modification of their surface with (bio)organic molecules and/or polymers to
31 March 2018
ensure their selectivity and specificity of action. Here, we show that nanosized diamond crystals
Accepted 19 May 2018
(nanodiamonds, NDs) without any synthetically installed (bio)organic interface enable the specific and
Available online 21 May 2018
efficient targeting of the family of extracellular signalling molecules known as fibroblast growth factors
(FGFs). We found that low nanomolar solutions of detonation NDs with positive z-potential strongly
Keywords:
Nanodiamonds
associate with multiple FGF ligands present at sub-nanomolar concentrations and effectively neutralize
Cell signalling the effects of FGF signalling in cells without interfering with other growth factor systems and serum
FGF proteins unrelated to FGFs. We identified an evolutionarily conserved FGF recognition motif, ~17 amino
Fibroblast growth factor acids long, that contributes to the selectivity of the ND-FGF interaction. In addition, we inserted this
Nanotherapeutics motif into a de novo constructed chimeric protein, which significantly improved its interaction with NDs.
We demonstrated that the interaction of NDs, as purely inorganic nanoparticles, with proteins can
mitigate pathological FGF signalling and promote the restoration of cartilage growth in a mouse limb
explant model. Based on our observations, we foresee that NDs may potentially be applied as nano-
therapeutics to neutralize disease-related activities of FGFs in vivo.
© 2018 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license
(http://creativecommons.org/licenses/by/4.0/).

1. Introduction

Since the upswing of nanomedicine in the early 1990s, the use of

* Corresponding author.
E-mail addresses: cigler@uochb.cas.cz
(P. Krejci).
nanoparticles has strongly influenced the quality of treatment of
various diseases and pathogens. The low toxicity of some nano-
particles, their high surface to volume ratio and the possibility of
(P. Cigler), krejcip@med.muni.cz
polyvalent binding sites on their surface have enabled the

https://doi.org/10.1016/j.biomaterials.2018.05.030
0142-9612/© 2018 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
 L. Balek et al. / Biomaterials 176 (2018) 106e121
application of nanoparticles in targeted drug delivery systems, of
theranostic nanoparticles that perform simultaneous therapeutic
intervention and monitoring of the treatment, and of stimulus-
responsive systems based on hybrid nanoparticles with a broad
structural range [1,2]. Overcoming some of the current barriers in
cancer treatment, such as poor circulation times, drug resistance
and off-target toxicity, using nanotechnology-based systems cata-
lysed the development of a number of drug delivery nanosystems
that have already passed the translation phases and are currently
marketed as drugs [3]. More recently, promising new directions
utilizing nanoparticles for the selective binding or sequestration of
biologically active and regulatory compounds have also been
investigated. Such nanoparticles called “artificial proteins” [4] take
advantage of the remarkable similarity of some nanoparticles to
biomolecules, particularly to globular proteins. Artificial proteins
are however created to mimic chemically the protein surfaces and
typically do not contain proteins. The analogy between proteins
and artificial proteins involves not only their comparable overall
dimensions and surface charge but in general the analogous steric,
thermodynamic and kinetic behaviour of nanoscale structures in an
aqueous environment [5].
Similar to biomolecules, nanoparticles can thus bind, mimic, or
block specific proteins in living organisms [6]. The use of nano-
particles therefore offers attractive alternative opportunities to
small molecule-based therapeutic interventions. For example, the
selective binding of one component and consequent blocking of a
specific protein-protein recognition using non-toxic nanoparticles
can have a strong impact on the control of key disease-related
processes. However, owing to issues such as the lack of well-
defined binding pockets, developing nanoparticles that selectively
modulate protein-protein interactions remains highly challenging.
In the past decade, advanced experiments exploring the po-
tential of artificial proteins have demonstrated that a molecularly
designed nanoparticle interface can inhibit or regulate protein-
protein interaction in structurally diverse systems. The seminal
works in this field include the selective inhibition of cytochrome c
peroxidase and cytochrome c interaction using gold nanoparticles
of appropriate size coated with simple ligands terminated only
with carboxyl groups [7,8] and the selective disaggregation of prion
aggregates using dendrimeric nanoparticles, leading to the elimi-
nation of prion molecules from neuroblastoma cells [9]. The high
surface-to-volume ratio of nanoparticles has been later advanta-
geously used also for sequestration of target molecules using long
affinity DNA polymers attached to the nanoparticle surface [10],
and for the capture of a broad range of pathogens and toxins related
to sepsis from the blood using opsonin-modified magnetic nano-
beads [11,12]. Attachment of peptide fragments to gold nano-
particles was used for creation of artificial antibodies [13] and very
recently, an in vivo study on mice revealed the high potential of
nanoparticles for preventing receptor phosphorylation and down-
stream endothelial growth factor-dependent cell migration and
invasion into the extracellular matrix [14]. These and other exam-
ples [15] constitute strong evidence that the regulation of protein-
protein interactions using nanoparticles can be used to control key
biological processes.
The nanoparticles used for the selective control of protein-
protein interactions have always been decorated with a bio-
nanointerface consisting of (bio)organic molecules and/or poly-
mers. The organic functional groups presented on nanoparticles
and their flexible arrangement, however, can cause unwanted in-
teractions with immune cells [16], which are observed even for the
commonly used PEG coatings [17]. Here, we address a key task: is it
possible to achieve direct control of the crystalline nanoparticle
surface (without further organic modification) to obtain highly
selective protein binding that can effectively compete with natural
107
protein-protein interactions and mitigate their biological effects? In
practice, this task requires identifying pairs of proteins and inor-
ganic interfaces that show much stronger mutual interactions than
the formation of the protein corona, which occurs immediately
upon the exposure of a nanoparticle to a biological environment
[18].
In our screening, we identified such a specifically interacting
pair. We show here that one type of nanosized diamond crystals
(nanodiamonds, NDs) [19] without any synthetically installed
organic or bioorganic interface already display, at low nanomolar
concentration, extremely strong interactions with members of the
fibroblast growth factor (FGF) family of growth factors and mor-
phogens. The FGF system represents a major molecular system by
which cells sense their extracellular environment and respond to
communication signals during development, life and disease. Four
human FGF-receptors exist (FGFR1-4) which respond to commu-
nication signals delivered by at least 18 FGF ligands. The impor-
tance of FGFR signalling is further emphasized by evidence of their
role in disease. Many pathological conditions arise from aberrant
FGF/FGFR signalling, including several types of cancer, develop-
mental defects and metabolic disorders [20,21].
We show that NDs selectively neutralize FGF signalling (Fig. 1A)
without interference with serum proteins and other tested FGF-
unrelated growth factor systems, including EGF (epidermal
growth factor), NGF (nerve growth factor), TGFb1 (transforming
growth factor b1), IL6 (interleukin 6) and IFNg (g-interferon). We
identify the evolutionarily conserved FGF region responsible for the
selectivity of interaction with ND and discover the molecular
principles responsible for the unprecedented strength of the FGF-
ND interaction. Finally, we demonstrate that these purely protein-
inorganic nanoparticle interactions are strong enough to mitigate
pathological FGF signalling and promote the restoration of cartilage
growth in a mouse limb explant model.
2. Results and discussion
2.1. NDs with positive z-potential inhibit FGF signalling in cells
To test the nanoparticle-based inhibition of FGF signalling in
cells, we used NDs, which form a diverse group of carbon nano-
materials based on the diamond core [22,23]. NDs can be classified
into three basic types: detonation, high-pressure high-temperature
(HPHT), and chemical vapour deposition NDs. The detonation NDs
mostly used in this study are very small (<5 nm) nanoparticles that
can easily be excreted from the body by glomerular filtration [24].
They are tolerated long term and are non-toxic [25] even if applied
at multiple clinically relevant doses as drug delivery nanosystems
in rats and primates [26]. Because of the presence of both sp3 and
sp2 hybridized carbon atoms at the surface [27], detonation NDs
provide a wide range of anisotropic surface atomic arrangements
consisting only of C, O and H atoms [28,29]. The resulting surface
charges at facets [30] and uniquely organized interfacial water
layers [31,32] can lead to interfaces with opposite z-potentials
ranging approximately from
50 to þ50 mV [33e36]. This disparity
between z-potentials results in different affinities of particular
proteins to NDs and in their diverse adsorption dynamics, attach-
ments and conformations on the ND surface [37,38].
Based on these assumptions, we first focused on distinguishing
the dominant charge effects on the ND surface that contribute to
the inhibition of FGF signalling in cells. We selected three repre-
sentative types of ND: detonation NDs with either positive or
negative z-potentials (ND1 and ND-PL, respectively) and oxidized
ND-HPHT with negative z-potential (Fig. 1B). The particles showed
features characteristic for the particular ND types: elemental
composition and infrared spectra are presented in Table S1 and
108 L. Balek et al. / Biomaterials 176 (2018) 106e121

Fig. 1. General scheme of ND action in cell signalling and physico-chemical characteristics of ND particles. (A) The cartoon depicting the selective inhibition of FGF signalling by ND
particles described in this study. NDs bind FGF ligands preventing their association with transmembrane receptors (FGFR) and activation of signalling. (B) The z-potentials of the ND
particles used. ND-HPHT, high-pressure, high-temperature NDs (smallest isolated fraction); ND-PL and ND1, detonation NDs with negative and positive z-potentials, respectively.
(C) Size distributions of detonation NDs (positive z-potential) with decreasing mean size measured by QELS. ND1-ND3 originate from the same starting sample; their sizes gradually
decreased from ND1 to ND3 by ultracentrifugation (see Materials and Methods for details). (D) TEM micrograph of ND1. The diffraction ring pattern displays only diamond rings,
showing the structure. The scalebar corresponds to 100 nm. (E) High-resolution TEM micrograph depicting a detail of the ND1 particle. Scalebar corresponds to 2 nm. (F) Char-
acterization of NDs using z-potential and QELS measurements. While the z-potentials for ND-HPHT and ND-PL are negative, all other particles tested in the study show positive
values. The decrease in the peak maximum size for ND1-ND3 corresponds to the data shown for (C). The ND4 sample was prepared by the dialysis of ND1 against water, but no
significant change in z-potential or size was observed. ND5 is the supernatant from a control sample in which all particles were quantitatively removed from ND1 by high-speed
ultracentrifugation.

Figure S1 in Supplementary Material. We used rat chondrosarcoma
(RCS) cells, which express the cell surface FGF-receptors FGFR2 and
FGFR3 [39]. RCS cells respond to the activation of FGFR signalling,
via the addition of the prototypical FGF ligand FGF2, with a well-
characterized array of phenotypic changes. These changes include
potent growth arrest, loss of the extracellular matrix and induction
of premature senescence [40]. RCS cells offer a particular advantage
for inhibitor evaluation because only specific targeting of the FGF
pathway can restore cell growth, and thus inhibitor interference
with other, pro-growth pathways will not generate a positive
response in the growth arrest assay. We hypothesized that the
addition of NDs, which can potentially interact with FGF2 (a ligand)
or with FGFR2 and FGFR3 (receptors), would cause a concentration-
dependent reversal of the RCS growth arrest phenotype induced by
72 h of FGF2 treatment. While NDs with negative z-potential (ND-
PL or ND-HPHT) did not show any influence on FGF2 signalling,
ND1, which had positive z-potential, effectively reversed the cell
growth arrest in a concentration-dependent manner (Fig. 2A and
B). Thus, ND1 restored RCS growth specifically, i.e., without
simultaneous interference with serum-borne pro-growth signal-
ling pathways, such as PDGF (platelet-derived growth factor) and
insulin signalling.
The ND1 particles show structural features characteristic of
detonation NDs, as documented by transmission electron micro-
scopy (Fig. 1D and E). The diameter of the primary particles is on
average within a few nanometres; however, the NDs gently asso-
ciate in solution, resulting in a slightly larger size measured by
quasielastic light scattering (QELS). Because the observed effect of
ND1 on FGF2 signalling was most likely caused by the interaction of
proteins with the diamond surface, we anticipated that this phe-
nomenon might be size-dependent. We therefore separated frac-
tions of smaller NDs from ND1, obtaining samples ND2 and ND3
with decreased sizes and a narrower size distribution of particles
(Fig. 1C, F). The rescue of FGF-mediated RCS growth arrest by ND2
was slightly more effective than that by ND1, while a further
decrease in size (ND3) did not show additional improvement
(Fig. 2A). We therefore decided to use ND2 in most of the following
experiments.
In cells incubated with ND-HPHT and ND1-3, a minor but sta-
tistically significant drop in cell proliferation was found in cells
treated with ND only, compared to untreated controls (Fig. 2A, red
stars). We currently do not know what is responsible for this
phenotype. In cell cultures treated with higher concentrations of
NDs, we noted a precipitation of serum proteins (Fig. 2E). Serum
 L. Balek et al. / Biomaterials 176 (2018) 106e121
proteins occasionally precipitate in the DMEM media, which seems
to be enhanced by the NDs. It is unclear whether the precipitation
of serum proteins involved factors necessary for RCS proliferation.
It is important to mention that RCS cells grow exponentially and so
even the slight changes in the proliferation rate have profound
effect when the total cell numbers are determined at the end of the
5 days of the growth period. The viability of RCS cultures was
assessed by dye-exclusion assay with propidium iodide. No sig-
nificant ND2 effect on cell viability was found, as the RCS cultures
contained the percentage of dead cells below 1.5% in all experi-
mental conditions, demonstrating that neither FGF2 nor the NDs
cause significant cell death (not shown).
To exclude the potential influence of some impurities that might
be present in the starting colloidal solution of ND1 (for example,
traces of residual reagents used by the producer to purify the NDs
after detonation synthesis), we prepared two control samples. First,
we dialysed ND1 5x against water to remove any potential small-
molecule contaminants, obtaining ND4. Second, we quantitatively
removed all ND particles from ND1 using high-speed ultracentri-
fugation, obtaining the supernatant ND5. ND4 showed comparable
colloidal properties to those of ND1 and rescued FGF2-mediated
RCS growth arrest. No NDs were detectable by QELS in ND5 solu-
tion, which correspondingly produced no activity in the growth
arrest assay (Figs. 1F and 2C). These results indicate that the posi-
tively charged detonation ND present in ND1 is responsible for the
observed effect on FGF signalling.
To compare the efficacy of ND1-4 with an established approach
to the inhibition of FGF signalling, we performed the FGF2 growth
arrest assay with a small-molecule inhibitor of FGFR catalytic ac-
tivity, BGJ398 [41], which is currently being evaluated in clinical
trials for FGFR-driven cancer [42]. BGJ398 rescued the FGF2-
mediated growth arrest phenotype to a similar extent to that of
ND1-4 (Fig. 2D).
Similar to the growth arrest, the FGF2-mediated induction of
premature senescence in RCS chondrocytes, manifested as the
upregulation of the senescence markers lamin A/C and caveolin and
the downregulation of ID2 [43], was rescued by ND2 and by
dialysis-purified ND4 (Fig. 3A). The senescence associated (SA)-b-
galactosidase activity induced by FGF2 was also rescued by ND2
(Fig. 3B). To gain further insight into the mechanisms of the ND-
mediated inhibition of FGF signalling, we analysed the effect of
ND on the activation of FGFR signal transduction in RCS cells.
Treatment with FGF2 induced potent phosphorylation of well-
established mediators of FGF signalling, including ERK MAP ki-
nase, the adapter protein FRS2 involved in ERK pathway activation,
and the WNT pathway co-receptor LRP6, which is known to be
phosphorylated by FGF signalling during its interaction with WNT/
b-catenin pathway [44,45]. Fig. 3C shows that the FGF2-mediated
phosphorylation of ERK, FRS2 and LRP6 was inhibited by ND4.
Next, we monitored the ND effect on transcriptional activity of
FGF-ERK signalling in RCS cells. The cells were transfected with
vector expressing dTomato under the control of pKrox24 (MapErk)
promotor known to respond to ERK pathway activation [46]. The
cells were treated with FGF2 and ND2 for 24 h and dTomato fluo-
rescence was monitored by live cell imaging. Fig. 3D demonstrates
~65% suppression of FGF2-mediated pKrox24 (MapErk)dTomato
trans-activation by ND2. As activation of the ERK pathway mediates
both growth arrest and premature senescence in RCS cells [47], the
inhibition of ERK activation thus explains the ND-mediated rescue
of the FGF2 phenotypes in these cells.
Our data suggest that NDs can inhibit FGF signalling in cells by
interaction with FGF2 ligand or with its receptors FGFR2 and FGFR3.
Because FGFR3 overexpression in cells leads to its FGF ligand-
independent dimerization and activation, we tested whether this
process can be inhibited by NDs. Expression of wildtype FGFR3 or
109
its activating, disease associated mutant K650 M [48] in 293 T cells
lead to ligand-independent activation of FGFR3-ERK signalling,
which was not inhibited by ND2 (Fig. 3E).
Altogether, we demonstrate that ND particles with positive z-
potential (ND1-4), but not those with negative z-potential (ND-PL,
ND-HPHT), effectively inhibit FGF signalling in cells via protein-
nanoparticle interactions. Notably, all experiments were success-
fully performed in tissue culture medium containing 10% fetal
bovine serum (containing approximately 6 mg/ml proteins), while
the concentration of FGF was typically 6  105-fold lower (10 ng/
ml; 0.61 nM for FGF2) and the NDs were extremely diluted (10 mg/
ml, which corresponds approximately to 0.4 nM ND concentra-
tion). The exposure of inorganic nanoparticles [18] and particularly
of nanodiamonds [37,38,49e51] to this protein-rich environment
typically leads to the rapid formation of a strongly bound protein
corona. Thus, the observed interference with FGF signalling had to
be mediated by unusually strong protein-ND interactions to over-
come the concomitant ND interactions with serum proteins. In the
next set of experiments, we focused on the selectivity and struc-
tural nature of these interactions.
2.2. NDs inhibit signalling of multiple FGFs but not TGFb1, IL6, IFNg,
EGF and NGF
Having determined that NDs inhibit the activation of FGF sig-
nalling mediated by FGF2, we asked whether a similar effect might
be achieved for other ligands belonging to the FGF family. Among
the 18 existing FGF ligands, FGF1/2/4/5/6/8/9/16/17/18 can activate
ERK and cause growth arrest in RCS cells. However, only FGF2, FGF9
and FGF18 can achieve this effect alone, while all other FGFs require
stabilization with heparin due to their low intrinsic stability [52].
Because the addition of heparin could interfere with the ND-FGF
interaction, we tested the effect of NDs only on FGF9 and FGF18.
Similar to FGF2, the activation of ERK mediated by treatment with
FGF9 and FGF18 was rescued by ND2 (Fig. 4A). As shown before, RCS
cells cannot respond to FGF ligands belonging to the FGF7 family
(FGF7/10/22) due to the lack of the appropriate FGFR variants [52].
Thus, human breast carcinoma MCF7 cells were used to test the
effect of ND on FGF7/10/22 signalling. Fig. 4B demonstrates ERK
activation mediated by FGF7, FGF10 and FGF22 treatment in MCF7
cells, which was rescued by ND2. Altogether, we demonstrate that
the signalling of at least six different FGF ligands (FGF2/7/9/10/18/
22) can be inhibited by NDs.
Next, we asked whether NDs inhibit other signalling systems
unrelated to FGF. The tested systems were selected based on the
diversity of their mechanisms of ligand-receptor interactions and
on the diversity of their action in vivo. We thus determined the
effect of ND on the signalling of a morphogene (TGFb1), two un-
related cytokines (IL6, IFNg), and two unrelated growth factors
(EGF, NGF). In RCS cells, we found no effect of ND on the phos-
phorylation of SMAD2 transcriptional regulator in response to cell
treatment with TGFb1 [53], on the phosphorylation of STAT3
transcriptional regulator in response to IL6 treatment [54], or on
the phosphorylation of STAT1 in response to cell treatment with
IFNg [55] (Fig. 4C). These data indicate that NDs do not interfere
with TGFb1, IL6 and IFNg signalling. Similarly, in 293 T cells, no
effect of ND2 was observed on the activation of EGF signalling via
cell treatment with EGF or on the activation of NGF signalling
triggered by the NGF treatment of cells transiently expressing the
NGF-receptor TRKA (Fig. 4D). Together, we have demonstrated that
NDs do not interfere with the signalling of five different ligand-
receptor systems not related to FGFs.
110 L. Balek et al. / Biomaterials 176 (2018) 106e121

Fig. 2. NDs inhibit FGF2 signalling in cells. (A) RCS cells were treated in medium containing 10% FBS with FGF2 and five types of NDs differing in z-potential and particle size for
72 h; the FGF2-mediated growth arrest was quantified by crystal violet staining. Note the rescue of the growth arrest phenotype in cells treated with ND1-3. Data represent averages
 L. Balek et al. / Biomaterials 176 (2018) 106e121 111

2.3. Mechanism of action: NDs sequester multiple FGF ligands
In FGF signal transduction, the tyrosine phosphorylation of FRS2
is caused directly by active FGFRs [56]. Thus, the lack of FRS2
tyrosine phosphorylation in RCS cells treated with FGF2 and NDs
demonstrates that NDs inhibit the FGF signalling cascade at the
level of FGFR activation (Fig. 3B). The spontaneous FGFR3 dimer-
ization and activation was not however inhibited by NDs (Fig. 3E),
both suggesting that NDs inhibit FGF signalling at the level of
ligand-receptor interaction, possibly by the sequestration of FGFs.
We first determined the cellular localization of the FGF2,
labelled with DyLight550 [57] in the presence of ND2. In RCS
chondrocytes treated with FGF2-DyLight550 alone for 18 h, we
observed a cytosolic FGF2 accumulation, reflecting the FGFR-
mediated FGF internalization, which is well established [58]
(Fig. 5A and B). In contrast, binding on ND2 inhibited FGF2 inter-
nalization. Rather, the FGF2-DyLight550/ND2 complexes remained
outside of the cells, being retained in the abundant chondrocyte
extracellular matrix rich on sulfated proteoglycans (Fig. 5C). This
demonstrates that free FGFs reach the cells surface, bind to FGFRs
and are internalized by cells, while the FGF/ND complexes are held
in the extracellular matrix and cannot be internalized. The inhibi-
tory ND effect on FGF signalling thus lies in the sequestrations of
the FGF ligands from their cognate FGFR receptors. In another
words, NDs function as an extracellular FGF ligand-trap.
To ascertain this mechanism, we incubated various human re-
combinant FGFs with NDs in DMEM supplemented with 10% fetal
bovine serum (FBS) for 8 h, collected the NDs by ultracentrifugation
and analysed them for the presence of FGF by western blotting with
FGF-specific antibodies (Fig. 6A). First, we found the effective
depletion of medium FGF2 after incubation with ND4 (a dialyzed
version of ND1; Fig. 6B), which was confirmed by the lack of growth
arrest in RCS cells grown in the FGF2-depleted media (Fig. 6C).
Similar experiments carried out with other FGFs demonstrated an
efficient ND-mediated depletion of recombinant FGF1/4/8/10/17/
22, while less effective clearance was found in the case of the FGF
hormones FGF19, FGF21 and FGF23 (Fig. 6D). No significant ND-
mediated depletion of the recombinant signalling proteins IL6
and IFNg was found (Fig. 6E). Because interleukin 1b (IL1b) shares
evolutionary origins with FGFs [59], we tested IL1b in the depletion
studies. No ND-mediated depletion of IL1b was found (Fig. 6E),
demonstrating that sequence context is more important than sec-
ondary structure in the FGF-ND interaction.
Although we verified NDs as tight-binders of FGF1/2/4/8/10/17/
22, the expected non-covalent nature of the binding can lead to
reversibility of the ND-FGF interactions. Since all experiments were
performed in the presence of 10% serum, we were interested in
whether the absence of a serum protein corona might cause
stronger sequestration of FGF by NDs. We emphasize the extreme
mass ratio between serum proteins and FGFs: in pull-down ex-
periments (see above), we used approximately 6 mg/ml serum
proteins (corresponding to 10% FBS) and 100 ng/ml FGF, which
resulted in a mass excess of serum proteins by a factor of ~6  104.
This excess was even higher in cell experiments (~6  105), but the
FGF sequestration efficacy of the NDs was still very strong, as
demonstrated by the almost complete ND-mediated rescue of the
growth arrest (Fig. 2). To evaluate the contribution (either synergic
or competitive) of the serum proteins to the ND-FGF interaction
and the reversibility of this interaction, we utilized extreme wash
off conditions (denaturing Laemmli sample buffer containing 4%
SDS and 5% mercaptoethanol) and compared the FGF1-ND2 and
FGF2-ND2 interactions in DMEM/FBS, DMEM and phosphate buff-
ered saline (PBS) after this wash off. The time dependence of the
binding process was also determined by analysing the FGF content
in the samples (solution and ND) after a given time (0e300 ) fol-
lowed by addition of Laemmli sample buffer (Fig. 7A). While both
FGF1 and FGF2 were detectable in the presence of FBS (i.e., could be
washed off the ND2 by Laemmli sample buffer), incubation in
either PBS or DMEM led to yet stronger sequestration by ND2 and
the virtual absence of both FGFs in the samples. This observation
confirms a competition between serum proteins and FGF for
binding on the ND surface, and not a binding synergy. We also did
not observe differences between the points in the 0e300 time scale
(time point 00 is the sample taken immediately after mixing the
FGFs with ND2), which implies the very rapid adsorption of both
FGFs on the NDs (Fig. 7A).
Interestingly, we observed even tighter binding for FGF22. We
found a complete lack of detectable FGF22 in all tested fractions,
i.e., input, medium after incubation, supernatant and pellet after
centrifugation and Laemmli sample buffer wash off (Fig. 6D).
Moreover, FGF22 was not released from pelleted NDs even after
incubation with lysis buffers containing up to 10% Triton X-100 or
Igepal CA-630, combined with sonication. No FGF22 signal was
detected even when FGF22 was added directly to an ND2-con-
taining solution of DMEM/FBS mixed with Laemmli buffer (1:1)
(data not shown). These data suggest a very strong association of
FGF22 with NDs, regardless to the presence of a protein corona
derived from FBS. To rule out the possibility that NDs somehow
degrade FGFs, thus destroying the antibody epitope needed for
detection by western blot, we incubated ND2 with FGF2 labelled
with Cy5.5 dye (FGF2-Cy5.5) and analysed the resulting complexes
by confocal microscopy. Fig. 7B shows the association of FGF2-Cy5.5
with ND2, whereas no Cy5.5 signal was detected in ND2 incubated
with free Cy5.5 or with unlabelled FGF2. Because the putative ND-
mediated degradation of FGF2 would release the Cy5.5, our data
suggest an association of intact FGF2 with ND2.
We next used z-potential measurements to characterize the
binding of FGF2 to ND2. The solution of ND2 in deionized water
showed z-potential 43.2 ± 4.2 mV and after mixing with solution of
FGF2 it changed to 26.8 ± 1.3 mV (data not shown). The pH of the
resulting solution was 5.7, which is far below the isoelectric point of
FGF2 (pI ¼ 11.2) and corresponds to the positively charged, pro-
tonated state of the FGF2. After alkalization of the solution to pH
12.0 (i. e. above the pI), where most of the protein functional groups
are deprotonized and the protein charge is switched to negative, we
observed correspondingly a dramatic reduce of the z-potential by
50 mV. The final value
24.2 ± 1.0 mV resulting from charge switch
of the protein clearly indicates the presence of the FGF2 on ND2
surface. In contrast, the z-potential of ND2 (without FGF2) at pH
12.0 was
5 mV only.
Finally, we employed circular dichroism (CD) to determine
whether association with NDs causes unfolding of FGF2, thus
interfering with conformation of its secondary structure. There
were small changes in far UV CD spectra between FGF2 alone and

of eight biological replicates with the indicated SD (*p < 0.05, **p < 0.01, ***p < 0.001, Student's t-test). The results represent four (n) independent experiments. The statistics on
differences in growth of untreated cells versus cells treated with NDs only is highlighted in red. (B) Microscopic appearance (phase contrast, 200x) of RCS cells treated with FGF2 and
ND1 for 72 h. Note the smaller colonies with enlarged cells in culture treated with FGF2 (10 ng/ml); this phenotype is rescued by ND1 (10 mg/ml). (C) The effect of ND4 (i.e.,
repeatedly dialysed ND1 solution) and the supernatant obtained by quantitative removal of the NDs from ND1 by ultracentrifugation (ND5) on the FGF2-mediated RCS growth
arrest, demonstrating that only ND but not any possible soluble impurity in their solution inhibits FGF2 signalling. (D) The effect of ND1-4 (10 mg/ml) on FGF2-mediated RCS growth
arrest compared to a chemical inhibitor of FGFR kinase activity BGJ398 (10 nM). (E) The precipitation of serum proteins triggered by NDs in cell cultures treated with ND2 for 72 h
(black arrows). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
112 L. Balek et al. / Biomaterials 176 (2018) 106e121
 L. Balek et al. / Biomaterials 176 (2018) 106e121 113

Fig. 4. NDs inhibit FGF signalling but not TGFb1, IL6, IFNg, EGF or NGF signalling. (A) RCS or (B) MCF7 cells were treated in medium containing 10% FBS with 20 ng/ml FGF2, FGF7,
FGF9, FGF18 and with 10 ng/ml FGF10 or FGF22 either alone or together with 10 or 15 mg/ml ND2 for 1 h, and ERK phosphorylation (p) was analysed by western blot. The total ERK
levels serve as a loading control; n, number of independent experiments. (C) RCS cells were treated with 20 ng/ml TGFb1, 50 ng/ml IL6 or 50 ng/ml IFNg for 10 min and analysed for
the phosphorylation (p) of SMAD2, STAT1 and STAT3 by western blot. (D) 293 T cells were treated with 20 or 50 ng/ml EGF alone or together with NDs for 5 min, transfected with
vector expressing human wild-type TRKA for 24 h, and treated with 20 ng/ml NGF alone or in the presence of ND2 for 1 h. Cells were analysed for ERK phosphorylation (p) with the
total ERK serving as a loading control.

FGF2 complexed with ND2 (Fig. 7C), suggesting that the FGF2's
secondary structure is preserved upon binding to ND2. Both CD
spectra showed a minimum at around 205 nm, typical of b-sheet-
rich proteins of the b-II type, and a positive peak between 220 and
240 nm, with the maximum centered near 229 nm, in clear contrast
to unfolded FGF2, induced by 4 M urea at 80  C.
2.4. The heparin binding site (HBS) is involved in FGF interaction
with NDs
Fig. 6 demonstrates the efficient ND-mediated depletion of FGFs
belonging to the paracrine FGF subfamily (FGF1/2/4/8/10/17/22),
while less effective depletion was found for endocrine FGFs (FGF19/
21/23). Because the selectivity of ND for a specific group of proteins
can be caused by the presence of a common type of binding
domain, we focus here on its identification. FGF19/21/23 act as
metabolic hormones regulating phosphate waste, the metabolic
adaptation to starvation, and glucose homeostasis [60]. The slow
metabolic action of endocrine FGFs dictates their persistence in
circulation for prolonged periods, without association with pro-
teoglycans at the endothelial cell surface. Although both paracrine
and endocrine FGFs share a central conserved core of ~120 amino
acids, all endocrine FGFs lack the consensus motif required for
heparin binding (heparin binding site, HBS), which is typical of
paracrine FGFs (Fig. 8A) [61]. The less effective ND-mediated
depletion of FGF19, FGF21 and especially FGF23 (Fig. 6D), there-
fore, indicates that the strong FGF-ND interaction observed for
paracrine FGFs might be partially mediated by the HBS.
To address the role of the HBS in paracrine FGF-ND interaction,
we generated a chimeric FGF23 protein, herein referred to as

Fig. 3. NDs inhibit the FGF2-mediated activation of FGFR signalling and the induction of senescence. (A) RCS cells were treated in medium containing 10% FBS with 20 ng/ml FGF2,
10 mg/ml ND2 or ND4, and 10 nM BGJ398. The effects of inhibitors on the FGF2-mediated induction of the senescence markers lamin A/C and caveolin was determined by western
blot 72 h later. The reduction in ID2 expression, which correlates with premature senescence in RCS cells, is also shown [43]. Actin served as a loading control; n, number of
independent experiments. (B) Cells were treated by FGF and ND2 for 48 h and the activity of senescence associated (SA) b-galactosidase was determined. Note the induction of
senescence by FGF2 which is rescued by ND2. (C) Cells were treated with FGF2 and ND4 for the indicated times, and the ND effect on the FGF2-mediated phosphorylation (p) of
LRP6, FRS2 and ERK members of FGFR downstream signalling pathways was determined by western blot. The total levels of each molecule and actin serve as loading controls. (D)
Transactivation of pKrox24 (2xD-E)dTomato reporter73 in RCS cells, determined by live cell imaging of dTomato fluorescence over 24 h. Cells were transfected with pKrox24
(MapErk)dTomato fluorescent reporter for 12 h before treatment with FGF2 and ND2 for 24 h. The signal recorded in two wells (four independent positions each) was averaged and
graphed (S.D. in grey). (E) 293 T cells were transfected with vectors expressing wildtype (WT) FGFR3 and its activating mutant K650 M, and treated with ND2 for 24 h. Cells were
analysed for indicated molecules by WB. Note the phosphorylation (p) of K650M-FGFR3 and ERK which is not counteracted by ND2. Similarly, ND2 does not interfere with activation
of ERK by WT FGFR3.
114 L. Balek et al. / Biomaterials 176 (2018) 106e121

Fig. 5. ND/FGF complexes do not enter cells. RCS cells were transfected with myrAKT-GFP to label cytoplasm and membrane and treated with FGF2-DyLight550 and ND2 48 hours
later. For the dual treatment, FGF2 was pre-incubated with ND2 in a serum-free media for 30 minutes. Cells were fixed 18 hours later. (A) Representative pictures showing FGF2
localization. First row, maximal projections from the top view; middle and bottom rows, orthogonal views from the Z-stack. The FGF2 and ND2 concentrations used to treat cells are
5 and 10 times higher compared to the rest of the study, due to technical difficulties in recording the DyLight550 fluorescence at low FGF2/ND2 doses. Note the prevalent
accumulation of the FGF2/ND2 complexes outside the cells, with limited entry to the RCS cytosol, compared to the intracellular accumulation of FGF2 in cell treated with FGF2 alone.
Scalebar, 10 mm. (B) The intracellular FGF2 was quantified as detailed in Material and Methods. Alternatively to the myrAKT-GFP transfected RCS cells, RCS with the Flag-tagged
endogenous FGFR3 [94] were used and the intracellular moiety was defined by the Flag immunostaining. The red line shows the background fluorescence intensity of the
FGF2-naïve cells. The graph compiles two experiments using myrAKT-GFP RCS cells and two experiments using Flag-FGFR3 RCS cells (mean±SEM, ***p<0.001, ANOVA). (C) RCS
chondrocyte cultures growing for 72 hours were stained with alcian blue to visualize the abundant cartilaginous matrix. (For interpretation of the references to colour in this figure
legend, the reader is referred to the Web version of this article.)

FGF23-HBS, in which the FGF2 HBS was inserted into the corre-
sponding location in the FGF23 sequence (Fig. 8B). We transfected
293 T cells with vectors expressing wt FGF23 and FGF23-HBS,
released the expressed FGF23 and FGF23-HBS into the culture
medium by snap freezing, and compared the ND-mediated clear-
ance of both proteins. The addition of HBS significantly improved
the sequestration of FGF23 on ND2, demonstrating that the C-ter-
minal region of paracrine FGFs involving HBS contributes to the
specific interaction between FGFs and NDs (Fig. 8C and D). How-
ever, in the same time, the observations of different capacities of
FGF2 and IFNg (Fig. 6), which are both known to be strong heparin
binders [62e64], to bind NDs are suggestive that mechanism of
NDs binding to paracrine FGFs is likely to be distinct from that of
the heparin binding. In this particular case, the difference in the
binding capacity could be explained by different 3D folding topol-
ogies of these proteins [cf. PDB ID: 4OEF (FGF2) and 1HIG (IFNg)],
which would imply an important role of 3D structure of paracrine
FGFs in ND binding. However, the observation of dramatic differ-
ences in ND binding to FGF2 and IL1b (Fig. 6), having essentially
identical 3D structures [65], indicates that it is sequence compo-
sition in the C-terminal region rather than the topological context
that contributes to the observed selectivity. Cooperativity between
the FGF structure and the sequence context in the C-terminal re-
gion of paracrine FGFs thus provide plausible explanation for the
observation of only partial recapitalization of the FGF2 binding
capacity by the FGF23-HBS (Fig. 8C and D).
2.5. NDs inhibit FGFR signalling in mouse limb explant cultures
The structural selectivity and tight binding of certain FGFs to
NDs suggest their potential use as therapeutics for FGF-related
diseases. To test the efficacy of NDs in intervening with FGF-
related pathological processes in complex biological systems, we
utilized an established model based on limb explants isolated from
E18 mouse CD1 embryos [39]. When isolated from the embryos, the
limb explants continue their growth programme for several days in
culture, driven by the intrinsic signalling of several pathways such
as TGF/BMP, WNT and hedgehog, which act in concert to ensure
proper chondrocyte proliferation and differentiation [66]. The limb
explant model thus allows evaluation of the specificity and efficacy
of the ND-mediated inhibition of FGF signalling in a complex tissue
environment where many protein interactions must remain intact
to allow normal growth. Similar to RCS chondrocytes, the activation
of FGFR3 in limb explants inhibits their growth via impairing both
proliferation and differentiation of the limb chondrocytes [39,67].
This effect stems from the physiological role of FGFR3 as a negative
regulator of skeletal growth [68].
Continuous treatment with FGF2 for 8 days resulted in signifi-
cant inhibition of the limb explant growth accompanied by a
reduction in hypertrophic cartilage, as evidenced by changes in the
microanatomical appearance of the growth plates and the dimin-
ished expression of the hypertrophic chondrocyte marker collagen
10 (Fig. 9A and B). These effects were reversed by treatment with
ND2 or ND3 (4 nM solution, 0.1 mg/ml). NDs partially rescued the
FGF2 growth-inhibitory effect, including restoration of the hyper-
trophic cartilage (Fig. 9C). Importantly, normal growth and tissue
microanatomy were found in limb explants cultured with ND2 and
ND3 alone for 8 days, suggesting minimal influence of the NDs on
non-FGF-related signalling pathways regulating limb growth.
 L. Balek et al. / Biomaterials 176 (2018) 106e121 115

Fig. 6. NDs sequester multiple FGF ligands (A) Outline of FGF pull-down experiments. Human recombinant FGFs were incubated with NDs in DMEM supplemented with 10% FBS for
8 h, and the NDs were then collected by centrifugation (100,000 rcf, 2 h). Supernatant and pellet samples were analysed for the presence of FGF by western blot with specific
antibodies. The input samples and samples taken after 8-h incubation but before centrifugation serve as controls. (B) Effective depletion of FGF2 from culture medium by ND4.
Please note the lack of FGF2 signal in the medium supernatant after incubation with ND4. (C) No growth arrest was detected in RCS cells grown in the latter medium supernatant for
72 h, in contrast to the media supernatant containing FGF2 only, which caused normal growth arrest. (D) Depletion of soluble recombinant FGF1, FGF4, FGF8, FGF10, FGF17, FGF19
and FGF21-23 by ND4 in DMEM supplemented with 10% FBS. The depletion is demonstrated by the lack of FGF signals in the supernatant fraction in the medium containing ND4.
Note the less effective clearance of FGF23 by ND4 and the predominant clearance of dimeric (d.) FGF21, as opposed to FGF21 monomer (m.). Also note the complete disappearance of
FGF22 from all analysed samples containing ND4, indicating the very strong binding of FGF22 to ND4 and leading to the disappearance of detectable FGF22 from all tested fractions.
(E) No significant depletion of recombinant IL6, IFNg and IL1b by ND2. IL6, IFNg and IL1b were detected by western blot with specific antibodies.

3. Conclusions previous studies utilizing nanoparticles modified with an organic

interface, we focused on the identification of strong and selective
Here, we have reported a hitherto unexplored approach to interactions between the inorganic interface and proteins. We
selectively regulating protein-protein interaction using purely found that detonation NDs with positive z-potential can uniquely
inorganic nanoparticles in a biological environment. In contrast to interact with members of the FGF growth factor family, which
116 L. Balek et al. / Biomaterials 176 (2018) 106e121

Fig. 7. The dynamics of ND-FGF binding. (A) Recombinant FGF1 and FGF2 were added for the indicated times to PBS, DMEM or DMEM supplemented with 10% FBS. The solution
sample was taken after the indicated times and analysed for FGF by western blot. (B) ND2 was incubated with FGF2 labelled with Cy5.5 (FGF2-Cy5.5), with non-labelled FGF2 or
with free Cy5.5 for 8 h, ND2 was collected by centrifugation and analysed by confocal microscopy. Note the association of FGF2-Cy5.5 with ND2 in contrast to no association with
free Cy5.5. Corresponding phase contrast images are also shown (Ph2, lower panel), scale bar 50 mm. (C) Circular dichroism spectra of ND2, FGF2, ND2 mixed with FGF2 and
unfolded FGF2. Spectra were acquired in phosphate buffer at 20  C and concentration 0.39 mg/ml for both FGF2 and ND2. Unfolded FGF2 was obtained by the incubation of the
protein in 4 M urea in phosphate buffer at 80  C for 2 h. Each spectrum represents an accumulation of three scans. Data are representative for three independent experiments (n).
Note that binding on ND2 does not cause FGF2 unfolding.

represents one of the major signalling systems regulating cell
functions during development, life and disease. We observed that
ND colloids diluted to low nanomolar concentrations could
sequester FGFs at their biologically relevant concentrations (~10 ng/
ml; 0.61 nM), compete with their interaction with FGFR and miti-
gate their inhibitory effects on cell growth in concentration-
dependent manner. This regulation of protein-protein interaction
was effective in a biological environment containing other proteins
(at concentrations higher by a factor of ~6  105) that form a protein
corona on the ND. The regulation was also highly selective: we did
not observe any influence on other signalling systems unrelated to
FGF, such as TGFb, IL6, IFNg, EGF and NGF. The quality of this
interaction is similar to that of the highly potent biomolecular in-
teractions evolved in nature. We further investigated the nature of
the interactions between FGF and ND, identifying the involvement
of the heparin binding site in the selective recognition of paracrine
FGFs by ND. We confirmed this hypothesis by generating a chimeric
FGF23-HBS protein, in which we artificially inserted the FGF2
heparin binding site into the corresponding location in the FGF23
sequence. FGF23-HBS showed much stronger affinity to ND than
did the wild-type FGF23. Finally, thanks to the extremely tight FGF-
ND binding, we were able to demonstrate that highly diluted 4 nM
ND colloids are able to mitigate pathological FGF signalling and
promote the restoration of cartilage growth in the mouse limb
explant model. The interaction of FGFs with ND is apparently
unique among the family of carbon nanomaterials: previous works
demonstrated no adsorption of FGF2 to multiwall carbon nano-
tubes, fullerenes, and graphite [69].
Notably, we observed no manifestations of ND toxicity, which is
consistent with previous studies utilizing NDs as delivery agents for
a broad range of therapies [70,71], including successful clinical
trials [72]. We are optimistic regarding the potential use of NDs in
the treatment of diseases, as their enormous potential for
improving patient care and personalized medicine has already
been demonstrated by D. Ho and co-workers [73e77] as well as by
other teams [78e83]. Applications in regenerative medicine also
show a great potential [73,84e90]. Our findings expand the spec-
trum of medicinal applications of NDs and suggest an unprece-
dented pathway for the control of signal transduction in biological
systems using NDs as artificial regulatory proteins.
4. Material and methods
4.1. Chemicals, solvents, ND characterization and preparation of ND
colloids
Sodium hydroxide, nitric acid and sulfuric acid were purchased
from Penta (Czech Republic) in p. a. quality. Deionized water was
used for the preparation of all colloids and particle treatments. The
dispersion of detonation NDs in water (NanoAmando) was supplied
by the Nanocarbon Research Institute (Japan). HPHT ND (MSY
0e0.05) was supplied by Microdiamant (Switzerland). A sample of
oxidized detonation NDs (PL-D-G01) was obtained from Plasma-
chem, Germany. All ND samples were dispersed by ultrasonication.
Typically, 20 mg of ND was mixed with 2 ml of deionized water
(MiliQ) in a plastic vial and sonicated for 30 min in an ice bath using
Cole Parmer 750 W probe sonicator with a tapered sonotrode
microtip. The output was set to 35% amplitude with a 1 s pulse
duration. Before cell experiments, the samples were shortly redis-
persed using Branson Sonifier 150 (6 W, 11  2 s impulses, sample
 L. Balek et al. / Biomaterials 176 (2018) 106e121 117

Fig. 8. The heparin binding site (HBS) is involved in FGF interaction with NDs. (A) Schematic representation of FGF2 and FGF23. Central core sequence (~120 amino acids) conserved
among all members of the FGF family and HBS conserved among endocrine and paracrine FGFs. (B) Sequence alignment of human (paracrine) FGF2 and (endocrine) FGF23 at the
location of the FGF2 HBS. Residues required for the binding of heparin to FGF2 [95] are highlighted in red. In the chimeric FGF23-HBS protein, the GRAKRAFLPGMNPPPYS segment of
the wild-type FGF23 was replaced by the KRTGQYKLGSKTGPGQK segment corresponding to the FGF2 HBS. (C) FGF23 and FGF23-HBS were expressed in 293 T cells, released via
freezing at
80  C and incubated with ND2 for 8 h. ND2 was then collected by centrifugation. Supernatants and pellet samples were analysed for FGF23 by western blot (arrow). The
input samples and samples taken after 8 h of incubation but before centrifugation serve as controls. Note the more efficient depletion of FGF23-HBS by ND2 (supernatant fraction)
than that of wt FGF23. (D) The FGF23 signal in supernatant samples obtained from five (n) independent experiments was quantified by densitometry and graphed. Data from
individual western blots were expressed as a percentage of the control (FGF23 alone). Numbers represent averages from five experiments with the indicated standard deviation
(SD). Statistically significant differences are indicated (**p < 0.01, ANOVA). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web
version of this article.)

solution 2 ml). Samples ND2, ND3 and ND5 were prepared using
centrifugation in a Beckman Coulter Le80 K ultracentrifuge with an
SW 41 Ti swinging bucket rotor. For other preparations, an
Eppendorf Microcentrifuge 5430 was used. Gravimetric determi-
nation of ND concentration: The analysed sample (1000 ml) was
evaporated in a pre-weighted 1.5 ml Eppendorf tube using a Lab-
conco Centrivap system. The concentrations of the solutions were
calculated from the weight of the solid residues. HPHT NDs were
oxidized by air in a Thermolyne 21100 tube furnace at 510  C for 5 h.
The NDs were subsequently treated with a mixture of HNO3 and
H2SO4 (90  C, 3 days), with 1 M NaOH (80  C, 1 h) and finally with
1 M HCl (80  C, 1 h). Between treatments, the NDs were separated
by centrifugation at 5000 rcf (20 min). After HCl treatment, the
diamonds were 3x centrifuged (5000 rcf, 20 min; 7000 rcf, 30 min;
20,000 rcf, 30 min) and redispersed using a tip sonicator. The
brown colloid remaining after the last centrifugation was diluted
with water to yield a solution of ND-HPHT (1 mg/ml). ND-PL:
Plasmachem detonation diamond powder (3 mg) was mixed with
3 ml of water in a plastic tube and sonicated for 60 min using a
probe sonicator in an ice bath. The obtained colloid was filtered
using a 0.45 mm glass microfibre syringe filter. ND1: The dispersion
of detonation ND NanoAmando (10 ml, 5.0% aqueous solution) was
diluted with water to a concentration of 10 mg/ml, sonicated for
10 min and filtered using a 0.2 mm PVDF syringe filter. ND2: ND1
colloidal solution (40 ml, 10.3 mg/ml) was centrifuged in an ultra-
centrifuge (15,000 rcf, 2 h). The supernatant was isolated and
filtered using a 0.2 mm PVDF syringe filter, yielding ND2 colloid
(26 ml, 7.8 mg/ml). The ND3: ND2 colloidal solution (13.2 ml,
7.8 mg/ml) was centrifuged in an ultracentrifuge (30,000 rcf, 2 h).
The top 2/3 of the supernatant was isolated and filtered using a
0.2 mm PVDF syringe filter, yielding ND3 colloid (7 ml, 4.0 mg/ml).
ND4: ND1 colloidal solution (1 ml, 10 mg/ml) was dialysed 5x
against water in 6e8 kDa dialysis tubing (Spectra/Por), filtered
using a 0.2 mm PVDF syringe filter and diluted, yielding ND4 colloid
(9 ml, 1.0 mg/ml). ND5: ND1 colloidal solution (13.2 ml, 1 mg/ml)
was centrifuged in an ultracentrifuge (200,000 rcf, 2 h). The top 2/3
of the supernatant was isolated, diluted by adding the same
amount of water and centrifuged once more (200,000 rcf, 2 h). The
118 L. Balek et al. / Biomaterials 176 (2018) 106e121

Fig. 9. NDs inhibit FGF signalling in limb explant cultures. (A) Tibias isolated from E18 mice embryos were cultured in medium containing 10% FBS, 50 ng/ml of FGF2, and 100 mg/ml
of ND2 or ND3 for 8 days. (B) Histological sections of representative tibias were stained with haematoxylin/eosin (HE). Note the marked FGF2-mediated reduction in hypertrophic
cartilage (H), visualized by collagen 10 in situ hybridization (sections counterstained with eosin, E). This phenotype is rescued by NDs. Cj, chondro-osseous junction. Scale bar,
100 mm. (C) The tibia length differences at isolation and after 8 days of incubation (D length) were determined and graphed. Note the partial rescue of the growth-inhibitory FGF2
phenotype mediated by NDs. Statistically significant differences are highlighted (Student's t-test, ***p < 0.001). The results are a compilation of six independent experiments; n, the
number of tibias analysed.

top 1/3 of the supernatant was isolated and filtered using a 0.2 mm
PVDF syringe filter, yielding a colourless ND5 solution. The ultra-
centrifugation of FGF and ND tissue culture media solutions
(100,000 rcf, 2 h) was conducted with 5 ml media from each con-
dition using a SW55 Ti rotor and Optima XPN 90 K Ultracentrifuge
(Beckman Coulter, Brea, CA). The molar concentrations of NDs were
calculated using spherical approximation of particles and diameter
28 nm estimated by QELS for ND1 (Fig. 1F). QELS and z-potential
measurements were performed with 1 mg/ml NDs water using the
Zetasizer Nano ZS system (Malvern) at room temperature. For
measurement of z-potential in presence of FGF2, aqueous solutions
of ND2 (25 mg/ml) and FGF2 (25 mg/ml) were mixed in volume ratio
1:1 (slow addition of ND2 to FGF2). The pH was adjusted using
NaOH. An aberration-corrected FEI titan “cubed” microscope
operated at 80 kV was used for high-resolution TEM imaging and
for diffraction work. For high-resolution TEM measurements, the
ND4 sample (1 mg/ml) was used.
Cell growth, senescence and viability assays, and western blot
(WB).
RCS, 293 T and MCF7 cells were cultivated in DMEM (Sigma-
Aldrich, St. Louis, MO) supplemented with 10% FBS and penicillin/
streptomycin (Life Technologies, Carlsbad, CA). For growth assays,
2.5  102 RCS cells per well were seeded in 96-well plates and
grown for 5 days. Cell numbers were determined by crystal violet
staining as described previously [91]. Cellular senescence was
determined using Senescence b-Galactosidase Staining Kit, ac-
cording to manufacturer's protocol (Cell Signalling, Beverly, MA).
For propidium iodide (PI)-exclusion assay, RCS cells were plated
into 24-well plates at 20,000 cells/well, and treated with FGF2, ND2
or camptothecin 48 h later. The entire well content, including
floating cells, was pelleted 48 h later, and mixed with 1 mg/ml PI.
The percentage of non-viable, PI-positive cells was immediately
analysed using Cytomics FC 500 flow cytometry system (Beckman
Coulter, Inc., CA, USA) at channel FL3 (emission at 620 nm). 10,000
non-gated cells per sample were analysed. Camptothecin and
propidium iodide were obtained from Sigma-Aldrich. FGF1, FGF2,
FGF4, FGF7, FGF8, FGF9, FGF10, FGF17, FGF18, FGF19, FGF21, FGF22,
FGF23, EGF, NGF, TGFb1, IL1b, IL6, and IFNg were obtained from RnD
Systems (Minneapolis, MN); BGJ398 was obtained from Sell-
eckchem (Houston, TX). For WB, cells were extracted into Laemmli
sample buffer (0.125 M Tris-Cl pH 6.8, 20% glycerol, 4% SDS, 0.005%
bromophenol blue). Extracts were resolved by SDS-PAGE, trans-
ferred onto a PVDF membrane and visualized by chem-
iluminescence (Thermo Scientific, Rockford, IL). The following
antibodies were used: lamin A/C, ID2, caveolin, LRP6, pFRS2,
pFGFR3, ERK1/2, pERK 1/2, actin, pSTAT1, STAT1, pSTAT3, STAT3,
SMAD2, and pSMAD2 (Cell Signalling); FRS2 (Santa Cruz Biotech-
nology, Santa Cruz, CA); pLRP6 (Millipore, Billerica, MA); V5 (Invi-
trogen); FGF2 (Sigma-Aldrich); and FGF1, FGF4, FGF8, FGF10, FGF17,
FGF19, FGF21, FGF22, FGF23, IL1b, IL6, and IFNg (RnD Systems).
Media with FGF2 and/or ND were preincubated for 8 h at 37  C and
5% CO2.
 L. Balek et al. / Biomaterials 176 (2018) 106e121
4.2. FGF2-Cy5.5 and FGF2-DyLight550 preparation,
immunocytochemistry, confocal microscopy and life cell imaging
The 18 kDa isoform of His-tagged rat FGF2 was expressed in
E. coli BL21 (DE3-trx) after IPTG induction and growth at 30  C.
Recombinant 18 kDa FGF2 was purified as previously described [92]
with the following modification: after the first purification of the
His-tagged protein on Ni-beads, the protein was eluted and an
additional purification step performed on heparin sepharose. After
high-salt elution with 1.5 M NaCl, the protein was dialysed against
phosphate buffered saline at 4  C overnight. Subsequently, recom-
binant FGF2 was fluorescently labelled with Cy5.5 maleimide
mono-reactive dye (Amersham) according to the manufacturer's
protocol. Microscopic images were acquired using a Carl Zeiss LSM
700 confocal microscope. FGF2-DyLight550 was prepared as
described before [57]. The Flag immunocytochemistry was per-
formed as described before [93]. Time-lapse cell imaging was
conducted using an automated incubation microscope BioStation
CT (Nikon, Tokyo, Japan). Phase contrast and dTomato fluorescence
images were automatically acquired every 15 min during a 24 h
period. Images were processed and analysed in Nikon BioStation CT
2 software. For the FGF2-DyLight550 imaging, the 3D images were
collected, and a single 2 mm section showing the strongest signal in
the red channel obtained by excitation using the 555 nm laser was
selected. The cell boundaries were manually defined, and the in-
tegrated intensity of the fluorescence in this area was calculated.
Images were exported into Microsoft Publisher for the preparation
of publication figures.
4.3. Vectors, transfection, CD measurements and preparation of the
FGF23-WT and FGF23-HBS
Cells were transfected using FuGene 6 transfection reagent ac-
cording to manufacturer's protocol (Promega). The pcDNA3.1. vec-
tors expressing FGFR3 and TRKA were described before [73], vector
carrying myrAKT-GFP was obtained from J. Chung. CD spectra were
recorded in 200e300 nm wavelength range in phosphate buffer
(25 mM H3PO4, pH 7.3) at 20  C on a Jasco J-815 spectropolarimeter.
Spectra were acquired at protein concentration 0.39 mg/ml
(4.5  10
5 M) and 0.39 mg/ml of NDs using 1 mm cuvette with a
slit width set to 2 nm, at a scan rate of 200 nm/min. Each spectrum
represented an accumulation of three scans. The synthetic genes for
human FGF23-WT and FGF23-HBS, inserted into the pcDNA3.1
vector were purchased from Thermo Fisher Scientific, and the
plasmid DNAs were verified by sequencing. The 293 T cells were
transfected with FGF23-WT or FGF23-HBS for 24 h. Proteins were
released from the cells into the medium by freezing (
80  C/4 h),
and cell debris was removed by centrifugation (15,000 g for
30 min). Samples were incubated at 37  C in a 5% CO2 incubator for
8 h and then centrifuged at 100,000 g for 2 h. The top 0.5 ml frac-
tions were prepared as supernatant samples, and the bottoms of
centrifugation tubes with or without ND were washed with
Laemmli buffer and analysed as pellets. All samples were analysed
for FGF23 by WB.
4.4. Limb explant culture, histology and collagen 10 in situ
hybridization
Tibias obtained from E18 mouse embryos were grown for 8 days
at 37  C in DMEM supplemented with ND2 or ND3 (100 mg/ml) and
FGF2 (50 ng/ml), with daily media changes. The length of the tibias
was measured at the beginning of the experiment (day 0) and at the
end of cultivation (day 8) in Axio Vision (Zeiss, Germany). Tibia
histology, alcian blue staining and collagen 10 in situ hybridization
were performed as described previously [39]. Animal experiments
119
were reviewed and approved by the Institutional Animal Care and
Use Committee at the Institute of Animal Physiology and Genetics
AS CR (Libechov, Czech Republic; 213/2011).
5. Data availability
The raw/processed data required to reproduce these findings
cannot be shared at this time as the data also forms part of an
ongoing study.
Acknowledgements
We thank Miriam Minarikova and Hella Brinkmann for their
excellent technical assistance. Supported by Ministry of Education,
Youth and Sports of the Czech Republic (KONTAKT II LH1523; Na-
tional Program of Sustainability II projects LQ1605 and CEITEC 2020
(LQ1601)); project SYMBIT (CZ.02.1.01/0.0/0.0/15_003/0000477),
financed by ERFD; Agency for Healthcare Research of the Czech
Republic (15-33232A, 15-34405A); Czech Science Foundation
(GA17-09525S, 17-12075S); National Science Centre grant 2014/12/
W/NZ1/00457. The work of P.C. and J.N. was supported from Eu-
ropean Regional Development Fund; OP RDE; Project: “Chemical
biology for drugging undruggable targets (ChemBioDrug)” (No.
CZ.02.1.01/0.0/0.0/16_019/0000729).
Appendix A. Supplementary data
Supplementary data related to this article can be found at
https://doi.org/10.1016/j.biomaterials.2018.05.030.
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Supplementary Material
Table S1. Elemental composition of the ND sample. The elemental analysis was performed
using CHN PE 2400 Series automatic analyzer. Note the characteristic content of N (~2 %) in
both detonation NDs (ND1 and ND-PL), which contrasts with the low N content in ND-HPHT.
The origin of the particles is reflected also in higher H content in both detonation NDs.
%C %H %N
ND1 88.05 1.18 2.11
ND-PL 81.99 1.16 2.20
ND-HPHT 91.16 0.63 0.10

Figure S1. Infrared spectra of NDs. The spectra were recorded on Nicolet 6700FT-IR spectrometer
(Thermo Scietific, Waltham, MA USA) purged by dry nitrogen and equipped by a standard MIR source,
KBr beam splitter and DTGS detector using DRIFTS technique. For measurements, the original samples
without any dilution using KBr were used. All spectra were obtained as results of 256 scans in spectral
region 3800-400 cm-1 with a spectral resolution 2 cm−1 and a Happ-Genzel apodization function was
applied. Data processing was performed using OMNIC software. The characteristic signals of surface
groups are marked by dashed lines.