SiO2 nanoparticles as platform for delivery of nucleoside triphosphate analogues into cells

Article abstract of DOI:10.1016/j.bmc.2012.11.057

A system for delivery of analogues of 2′-deoxyribonucleoside triphosphate (dNTP) based on SiO₂ nanoparticles was proposed. A simple and versatile method was developed for the preparation of SiO ₂-dNTP conjugates using the 'click'-reaction between premodified nanoparticles containing the azido groups and dNTP containing the alkyne-modified γ-phosphate group. The substrate properties of SiO ₂-dNTP were tested using Klenow fragment and HIV reverse transcriptase. Nucleoside triphosphates being a part of the SiO₂-dNTP nanocomposites were shown to be incorporated into the growing DNA chain. The rate of polymerization with the use of SiO₂-dNTP or common dNTP in case of HIV reverse transcriptase differed insignificantly. It was shown by confocal microscopy that the proposed SiO₂-dNTP nanocomposites bearing the fluorescent label penetrate into cells and even into cellular nuclei.

Full text of DOI:10.1016/j.bmc.2012.11.057

Bioorganic & Medicinal Chemistry 21 (2013) 703–711
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
SiO2 nanoparticles as platform for delivery of nucleoside triphosphate
analogues into cells
Svetlana V. Vasilyeva a, , Vladimir N. Silnikov a, Natalia V. Shatskaya b, Asya S. Levina a,
⇑
Marina N. Repkova a, Valentina F. Zarytova a
a Institute of Chemical Biology and Fundamental Medicine, Lavrent’ev Ave., 8, Novosibirsk 630090, Russia
b Institute of Cytology and Genetics, Lavrent’ev Ave., 10, Novosibirsk 630090, Russia
a r t i c l e i n f o
a b s t r a c t
A system for delivery of analogues of 20-deoxyribonucleoside triphosphate (dNTP) based on SiO2 nano-
particles was proposed. A simple and versatile method was developed for the preparation of SiO2–dNTP
conjugates using the ‘click’-reaction between premodified nanoparticles containing the azido groups and
Article history:
Received 7 September 2012
Revised 20 November 2012
Accepted 24 November 2012
Available online 11 December 2012
dNTP containing the alkyne-modified c-phosphate group. The substrate properties of SiO2–dNTP were
tested using Klenow fragment and HIV reverse transcriptase. Nucleoside triphosphates being a part of
the SiO2–dNTP nanocomposites were shown to be incorporated into the growing DNA chain. The rate
of polymerization with the use of SiO2–dNTP or common dNTP in case of HIV reverse transcriptase dif-
fered insignificantly. It was shown by confocal microscopy that the proposed SiO2–dNTP nanocomposites
bearing the fluorescent label penetrate into cells and even into cellular nuclei.
Keywords:
SiO2 nanoparticles
Nucleoside triphosphates
‘Click’ chemistry
Delivery into cells
Substrate properties
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
considered as an ideal system for delivering drugs, but their use is
limited because of serious drawbacks, namely: the high instability
Abnormal nucleosides are known to be used as etiotropic anti-
viral preparations.1,2 However, the active antiviral form is not
nucleoside analogues themselves but their 50-triphosphates, which
are major substrates for DNA and RNA viral polymerases. The main
reason of using high doses of nucleoside analogues as antiviral
preparations is a low level of their intracellular accumulation, the
necessity of their intracellular phosphorylation, drug metabolism
and deactivation, and rapid clearance from the bloodstream. More-
over, the phosphorylation of nucleoside analogues with cellular en-
zymes to form corresponding 50-triphosphates is much less
efficient as compared to natural nucleosides. This also leads to
the use of high concentrations of nucleoside substrate, and thus
to an increase in toxicity.
This problem could be solved by using phosphorylated forms of
nucleoside analogues: mono- or, better, triphosphates. Unfortu-
nately, it is well known that nucleoside triphosphates are hardly
internalized by cells when applied alone. Therefore, the search
for efficient vehicles for nucleoside triphosphates remains an ur-
gent problem.
and time consuming preparation.4 The polymeric drug delivery sys-
tem for analogues of nucleoside triphosphates was proposed in the
work of Vinogradov et al.5 A noncovalent complex of the nucleoside
50-triphosphate analogue with polyethyleneimine was created and
placed in the nanogel (polymer consisting of polyethylene glycol
and polyethylenimine). The delivery of nucleoside triphosphates
analogues with this system was shown to be quite high; however,
the nanocomposite appeared to be more toxic by two orders of
magnitude than the non-phosphorylated nucleoside analogue.
The cytotoxicity of the delivery system is related to its cationic nat-
ure6,7 with its ability to damage membranes8,9 and bind important
cellular components.10
The only known successful applications of dNTPs are covalent
phospholipide conjugates of anti-HIV drug, 30-azidothymidine
(AZT), which demonstrate a higher bioavailability and a lower tox-
icity than AZT of itself.11
To address the problem of drug transportation into cells, nano-
biotechnology is quite actively involved. The integration of biolog-
ical and nonbiological materials resulting in functional devices is
an interdisciplinary multiscale task of great practical importance.12
A variety of nanomaterials are recently proposed as vehicles for
drug delivery.13 The main important requirements for such mate-
rials are the ability to penetrate into eukaryotic cells, low toxicity,
biocompatibility, biodegradation, etc. Nanoparticles of titanium
dioxide and silicon dioxide are attracted special attention due to
There are different approaches to create the delivery systems for
nucleoside triphosphates. One of the first systems to deliver nucle-
oside analogues into cells were liposomes.3 Liposomes could be
⇑
Corresponding author. Tel.: +7 383 3635183; fax: +7 383 3635182.
E-mail address: svetlana2001@gmail.com (S.V. Vasilyeva).
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.11.057

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S. V. Vasilyeva et al. / Bioorg. Med. Chem. 21 (2013) 703–711
their biocompatibility, stability and the possibility of modifying
the surface for the immobilization of various compounds.14,15
The effectiveness of penetration of nanoparticles depends on
their size and the cell type. It was shown that SiO2 nanoparticles
of 40–70 nm in size penetrate into the Hep-2 and Caco-2 cells cells
and in their nuclei.16,17 Some experiments demonstrated the abil-
ity of SiO2 nanoparticles to biodegradation.15,18
The goal of this work was to design nanocomposites consisting
of analogues of 20-deoxyribo nucleoside triphosphates (dNTP)
immobilized onto SiO2 nanoparticles and to study their substrate
properties in reactions catalyzed by DNA polymerases and their
ability to penetrate into eukaryotic cells.
N-(3-propynyloxypropyl trifluoroacetamide linker group23 was at-
tached to the C5 position of 20-deoxyuridine followed by the depro-
tection of the amino group and introduction of the fluorescein
residue (Scheme 3). Target 5-{3-[3-(fluorescein-5-yl-thiourei-
do)propoxy]
ynyl)oxypropyl]-gamma-amino}-triphosphate
prop-1-ynyl}-20-deoxyuridine-50-{N-[3-(prop-1-
was obtained
3
from 3a with a yield of 70%. The structures of the final products
2, 3 were confirmed by 1H and 31P NMR and mass spectrometry
(MS).
Nanoparticles containing the azide groups (1) were prepared by
the treatment of SiO2–NH2 nanoparticles with N-hydroxy-
succinimide ester of azidohexanoic acid (N-SEHA) (1b, Scheme
4A), which was synthesized according to Scheme 4B. Compound
1 has the characteristic azide absorption band in the IR spectra in
contrast to the unmodified nanoparticles. (IR spectra of SiO2–NH2
(IR SiO2–NH2) and product 1 (IR 1) see in Supplementary data).
The azido group band of azidohexanoic acid (AHA) of a high
intensity is observed in the range of 2286.75–1998.78 cmꢀ1. An
approximately 10-fold excess of N-SEHA over the amino groups
in SiO2–NH2 was shown to be sufficient for the successful conver-
sion of the amino groups into the azido groups (Table 1, IR AHA, IR
Samples 1–3 in Supplementary data). The evaluation of the amount
of the amino groups with picric acid25 before and after the reaction
showed that ꢁ85% of the amino groups were modified. Not all ami-
no groups in SiO2–NH2 are, probably, accessible for the reaction
due to steric hindrances that results in the non-quantitative yield
of the reaction. The capacity of the prepared azido modified nano-
2. Results and discussion
To use SiO2 nanoparticles as platform for delivery of dNTP ana-
logues into cells we suggested a simple and versatile method of
their covalent attachment to nanoparticles: the ‘click’-reaction be-
tween premodified nanoparticles containing the azido groups and
dNTP containing the alkyne-modified gamma-phosphate group.
Among various carbon–heteroatom bond formation reactions,
the copper catalyzed Huisgen 1,3-dipolar cycloaddition is the pre-
mier example of click chemistry known for its high degree of selec-
tivity and stability.19 Moreover, the resulting triazole ring itself has
been shown to possess a variety of biological functions including
anti-HIV, antibacterial, and potent antihistamine activity.20–22 We
took advantage of this click chemistry to develop the drug delivery
system. Scheme 1 illustrates the copper(I)-catalyzed azide–alkyne
cycloaddition (CuAAC) reaction between premodified nanoparti-
cles containing the azide groups (1) and nucleoside triphosphates
particles for the azido groups was evaluated to be 0.43 lmol/mg.
The yield of the ‘click’-reaction between the azido-modified
nanoparticles and alkyne-containing compounds was evaluated
by the disappearance of the signal of the azido group in the IR spec-
trum. The reaction between the azido-modified nanoparticles 1
and the 3-aminopropoxypropynyl linker group (L) was carried
out as the control experiment (6, Scheme 4C). The IR spectrum of
product 6 showed the absence of the azido group signal (compare
IR 6 and IR 1 in Supplementary data) that confirmed the formation
of the covalent bond between the linker and the nanoparticles. A
partial disappearance of the azido group band was shown when
containing the alkyne-modified
in the formation of the desired nanocomposites (4 or 5).
Introduction of the alkyne group into the -phosphate of dNTP
c-phosphate (2 or 3) that resulted
c
was performed using the 3-aminopropoxypropynyl linker (L,
Schemes 2 and 3).23 Activation of the terminal phosphate group
was achieved in the presence of the nucleophilic catalyst.24 Despite
the facts that this reaction is not universal, depends on the nature
of conjugating components, and sometimes is accompanied by a
large number of side products, the yields of the desired products
were satisfactory (52% for (2) and 64% for (3a), Schemes 2 and 3).
To study the penetration of the proposed nanocomposites into
cells, we synthesized fluorescent-labeled dUTPFlu (3) by the
introduction of the fluorescein residue in compound 3a. The
the azido-nanoparticles were treated by alkyne-modified c-phos-
phate 2 or 3 under the ‘click’ reaction condition (IR 1 and 4, IR 1
and 5 in Supplementary data). Usually, the yield in the azide–al-
kyne Huisgen’s cycloaddition reaction for small molecules exceeds
80%;26–28 however, the kinetics of this reaction for small and large
Scheme 1. Synthesis of nanocomposites SiO2–dCTP and SiO2–dUTPFlu via the copper(I)-catalyzed azide–alkyne cycloaddition (CuAAC) reaction between the premodified
nanoparticles containing the azide groups and nucleoside triphosphates containing the alkyne-modified gamma-phosphate.

S. V. Vasilyeva et al. / Bioorg. Med. Chem. 21 (2013) 703–711
705
Scheme 2. Synthesis of the alkyne-modified dCTP—20-deoxycytidine-50-{N-[3-(prop-1-ynyl)oxypropyl]-gamma-amino}-triphosphate. Yield 52%.
Scheme 3. Synthesis of alkyne-modified dUTPFlu bearing the fluorescent label—5-{3-[3-(fluorescein-5-yl-thioureido)propoxy]prop-1-ynyl}-20-deoxyuridine-50-{N-[3-(prop-
1-ynyl)oxypropyl]-gamma-amino}-triphosphate. Overall yield 45%.
Scheme 4. (A) Synthesis of the azidomodified SiO2 nanoparticles. Overall yield 63%. (B) Synthesis of the N-hydroxysuccinimide ester of 6-azidohexanoic acid (N-SEHA). (C)
Synthesis of the SiO2–L–NH2 by ‘click’-reaction. Yield approximately100%.

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S. V. Vasilyeva et al. / Bioorg. Med. Chem. 21 (2013) 703–711
mer as it was in the absence of dCTP (compare lanes 4 and 2 in
Fig. 1). This means that all dCTP in the SiO2–dCTP nanocomposite
is strongly associated with nanoparticles and does not go into
solution.
molecules significantly differs from each other. As reported, the
yield of this reaction is reduced below 50% when two polymers
are involved in the cycloaddition reaction.29 The yield is expected
to decrease even more when the accessibility of the azido groups
on solid particles is limited due to steric hindrances. The yield of
the attachment of triphosphate 2 to nanoparticles evaluated by
measuring the optical density of the resulting product SiO2–dCTP
dissolved in 0.2 M NaOH was 58%. The capacity of SiO2–dCTP for
Then we compared the kinetics of polymerization in the reac-
tion mixtures containing four native dNTPs or three native dNTPs
and SiO2–dCTP instead of dCTP. Both polymerases were used in
these experiments (Figs. 2 and 3). Concentrations of dCTP in the
nanocomposite and each of the native triphosphates were the
same. It is seen from the kinetic curves that the reaction rate is
slightly lower in the case of using SiO2–dCTP instead of dCTP
although the initial primer disappears in 1–1.5 h in all cases. As
can be expected, the use of SiO2–dCTP instead of dCTP leads to
some retardation of the reaction at the steps of the formation of
19-mer and 23-mer, that is immediately before the involvement
of SiO2–dCTP in the reaction. This is particularly clearly seen from
Figure 2B at the initial reaction stages. Interestingly, that in the
case of Klenow fragment, the initial reaction rate when using
SiO2–dCTP instead of dCTP significantly differs from that when
using four native triphosphates (Fig. 2), while in the case of RT
HIV (virus polymerase), this difference is not so pronounced
(Fig. 3). It offers hope that the proposed nanocomposites will be
better recognized by virus DNA polymerases than cellular DNA
polymerases.
the triphosphate was evaluated to be 0.25 lmol/mg. Altogether,
these data confirmed the successfulness of the Huisgen’s 1,3-dipo-
lar cycloaddition reaction for the preparation of the SiO2–dNTP
nanocomposites.
It was shown by the Small-Angle X-ray Scattering (SAXS) meth-
od30,31 that the size of the initial SiO2–NH2 nanoparticles (up to
22–24 nm) insignificantly differed from that of the final SiO2–dNTP
nanoparticles (up to 25–27 nm), that is the difference was not
more than 2–3 nm (see diagrams of the particles size distribution
in Supplementary data). This is a reasonable result considering that
the size of dNTP with the linker (4–5 nm) is much lower than the
size of one nanoparticle and that this molecule can be not neces-
sarily positioned perpendicular to the surface of the particle.
The prepared nanocomposites were studied for their ability to
be substrates for DNA polymerases. The property of the synthe-
sized SiO2–dCTP along with other native dNTPs to be involved in
the growing DNA chain was tested on a model system consisting
of a 30-mer DNA template, 16-mer DNA primer. Klenow fragment
(DNA polymerase I from Escherichia coli) and HIV reverse transcrip-
Thus, the results show the availability of the SiO2–dNTP as a
substrate for DNA polymerases.
The next step of our investigation was to show whether the pro-
tase were used as DNA polymerases.
posed nanocomposites can penetrate into cells. The SiO2–dUTPFlu
nanocomposite 5 bearing the fluorescent label at the 5 position
of 20-deoxyuridine was synthesized for this purpose. Figure 4 dem-
onstrates the image of HeLa cells after incubation with this nano-
composite. It is seen that the fluorescent label is uniformly
distributed within the cells. It should be noted that the nanocom-
posite penetrates not only into the cellular cytoplasm but even in
nuclei without any transfection agents or auxiliary procedures.
Figure 1 demonstrates the results of polymerization in the system
with Klenow fragment. The use of all four native triphosphates
led to the formation of the full length 26-mer oligonucleotides
(Fig. 1, lane 1). When only three native triphosphates of guanosine,
adenosine, and thymidine were used, the chain of the synthesized
DNA fragment was limited by a truncated 19-mer fragment accord-
ing to the template-primer structure (Fig. 1, lane 2). The replace-
ment of dCTP by SiO2–dCTP in the mixture of triphosphates
provided the polymerization similar to the use of four natural tri-
phosphates, although some amount of the 19-mer fragment was
still observed (compare lanes 3 and 1 in Fig. 1). The use of superna-
tant from the SiO2–dCTP suspension led to the formation of the 19-
3. Conclusions
In this work we used the click chemistry to develop the delivery
system for 20-deoxyribonucleoside triphosphates. Our data
confirmed the successfulness of the Huisgen’s 1,3-dipolar cycload-
dition reaction for the preparation of the SiO2–dNTP nanocompos-
ites. The fact that polymerase can recognize and incorporate
nucleoside triphosphates in such nanobiocomposites into a grow-
ing DNA chain is of great importance. The results demonstrated a
possibility of the utilization of SiO2 nanoparticles as vehicles for
the delivery of nucleoside triphosphates analogues into cells. It
was shown that the proposed SiO2–dNTP nanocomposites pene-
trated into eukaryotic cells and even in cellular nuclei. This makes
it possible to use the nanobiocomposites bearing nucleoside tri-
phosphate analogues as promising therapeutic drugs.
4. Experimental section
4.1. General
Figure 1. Electrophoresis in 20% PAAG of the extension products in the template
(30-mer)/primer (16-mer) system in the presence of Klenow fragment (1 ꢂ 10ꢀ3 U)
and four dNTPs (lane 1), three native dNTP except dCTP (lane 2), three native
dNTP + SiO2–dCTP (lane 3), three native dNTP + supernatant from SiO2–dCTP
suspension (lane 4), template (lane 5), primer (lane 6). 10ꢀ6 M for the template,
The following reagents were used: 2,20-dithiodipyridine, fluo-
rescein isothiocyanate isomer I, N-hydroxysuccinimide, butyl ace-
tate, tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine (TBTA),
copper(II) sulfate pentahydrate, and (+)-Sodium
talline (Sigma–Aldrich, USA); 6-bromohexanoic acid (Alfa Aesar);
1 ꢂ 10ꢀ6
M
for the primer, 1 ꢂ 10ꢀ5
M for dNTPs; SiO2– dCTP —0.07 mg/ml
(1 ꢂ 10ꢀ5 M for triphosphate) reaction time 60 min, 37 °C. Spots were visualized
L-ascorbate crys-
with Stains All.

S. V. Vasilyeva et al. / Bioorg. Med. Chem. 21 (2013) 703–711
707
Figure 2. Electrophoresis in 20% PAAG of the extension products in the template (30-mer)/[32P]primer (16-mer) system at 37 °C in the presence of Klenow fragment
(5 ꢂ 10ꢀ3 U) and three native dNTP + SiO2–dCTP (A) or four native dNTP (B) kinetic curves of these processes in the presence of four native dNTP (1) or three native
dNTP + SiO2–dCTP (2) concentrations: 4 ꢂ 10ꢀ6 M for the template, 1 ꢂ 10ꢀ6 M for the primer, 1 ꢂ 10ꢀ5 M for dNTPs; 0.07 mg/ml for SiO2–dCTP (1 ꢂ 10ꢀ5 M for dCTP) (C).
Figure 3. Electrophoresis in 20% PAAG of the extension products in the template (30-mer)/[32P]primer (16-mer) system at 37 °C in the presence of HIV RT (2 U) and three
native dNTP + SiO2–dCTP (A) or four native dNTP (B) kinetic curves of these processes in the presence of four native dNTP (1) or three native dNTP + SiO2–dCTP (2)
concentrations: 4 ꢂ 10ꢀ6 M for the template, 1 ꢂ 10ꢀ6 M for the primer, 1 ꢂ 10ꢀ5 M for dNTPs; 0.07 mg/ml for SiO2–dCTP (1 ꢂ 10ꢀ5 M for dCTP) (C).
triphenylphosphine (Fluka Chemie AG, Buchs, Switzerland);
dicyclohexylcarbodiimide (Merck, Germany); SiO2–NH2 nanoparti-
cles (SkySpring Nanomaterials, Inc., USA); deoxynucleoside
triphosphates (ICBFM, Russia). 5-(3-Aminopropoxyprop-1-ynyl)-
20-deoxycytidin-50-triphosphate, 3-propynyloxypropylamine, 20-
deoxycytidine-50-triphosphate (trimbuthylammonium salt) and
1 M TEAAc buffer (pH 7) were acquired from ‘NanoTech-S’ LLC
(Novosibirsk, Russia). All other reagents were from Sigma–Aldrich
(Milwaukee, WI, USA). Organic solvents were dried and purified by
standard procedures. 1H and 31P NMR spectra were recorded on
Bruker AV-400 and AV-300 spectrometers.
The chemical shifts (d) are reported in ppm relative to the resid-
ual solvent signals. In case of 31P, an external standard of 85%
H3PO4 was used. The coupling constant (J) values are expressed
in Hertz (Hz) and spin multiples are given as s (singlet), d (dou-
blet), t (triplet), q (quartet), m (multiplet), app.t (apparent triplet),
and br (broad singlet). Matrix-assisted laser desorption/ionization-
time of flight (MALDI-TOF) mass spectra were registered on a Re-
flex III spectrometer (Bruker Daltonics, Germany) in a positive
detector mode with dihydroxybenzoic acid as a matrix. UV-absorp-
tion spectra were recorded on a UV-1800 spectrophotometer (Shi-
madzu, Japan). The presence of the azide functional group was
registered by a Bruker Tensor 27 spectrometer (IR-spectra). SAXS
patterns from the samples of sols of SiO2–NH2, SiO2–dCTP, and
SiO2–dUTP(Flu) nanoparticles were measured on the small-angle
X-ray diffractometer Kristalloflex-805 (Siemens, Germany). The
product yields (Schemes 2 and 3) were evaluated as described in
the figure legends. Aliquots of the reaction mixtures were taken
off at the appropriate time, 10-fold diluted with water or ethanol,
centrifuged, and analyzed by analytical anion exchange HPLC or
analytical reverse phase HPLC. The analytical anion exchange chro-
matography was performed on a Milichrom-4 chromatograph
(Econova, Russia) using a 2.5 ꢂ 60 mm column packed with Polysil
SA, 15 lm (Vector, Russia). A linear gradient (flow rate 50 lL/min)
from 0 to 0.8 M of K2HPO4/KH2PO4 (pH 7.0) was used. Preparative
anion exchange chromatography was performed on DEAE Sepha-
dex A-25, 40–120 l, (Pharmacia Fine Chemicals, Sweden). Analyt-
ical reverse phase HPLC was performed using a Milichrom A-02
chromatograph (Econova, Russia) and a 2 ꢂ 75 mm column packed
with ProntoSIL 120-5C18 AQ (Bischoff, Leonberg, Germany). Pre-
parative reverse phase chromatography was performed on Polygo-
prep C18, 50–100
l (Macherey-Nagel, Germany). Thin layer
chromatography was carried out using Alufolien Kieselgel 60
F254 plates (Merck, Germany) in appropriate solvent mixtures
and was visualized by UV irradiation, ninhydrin (for amine
groups), or cystein/aqueous sulphuric acid (for nucleoside-contain-
ing compounds).
4.2. Synthesis of 6-azidohexanoic acid 1a
The synthesis of 6-azidohexanoic acid 1a was carried out as de-
scribed in32 (Scheme 4B) from the corresponding bromoacid,
DMSO was removed by evaporation with butyl acetate that in-
creased the yield of the desired products by 20%. DMF can be also

708
S. V. Vasilyeva et al. / Bioorg. Med. Chem. 21 (2013) 703–711
Figure 4. Confocal laser scanning microscope images of HeLa cells after incubation with SiO2–dNTUFlu. (a) nuclei stained with DAPI blue (405 nm); (b) fluorescein-labeled
nanocomposites, green (488 nm); (c) cell membranes stained with Cell Mask Plasma Membrane Stain (543 nm), (d) superposition of channels (a–c).
used as a solvent. After the removal of residual solvent by evapora-
tion at 56 °C, the yield of the final product 1a was 78%. Spectral
characteristics of the product correspond to those previously
described.32
spectra of the samples 1–3, respectively are presented in Supple-
mentary data.
Synthesis of SiO2–NH–CO–(CH2)5–N3 for subsequent experi-
ments was carried out using eightfold excess of N-SEHA over the
amino groups in SiO2–NH2. 0.2 M N-SEHA (200
amide) was added to the SiO2–NH2 suspension (20 mg, 0.5
l
l in dimethylform-
mol/
4.3. Synthesis of succinimidyl 6-azidohexanoate 1b (Scheme 4B)
l
mg for the amino groups) in 0.6 ml of a water/dimethylformamide
6-Azidohexanoic acid (1.2 g, 7.64 mmol) was added to a solu-
tion of N-hydroxysuccinimide (966 mg, 8.4 mmol) in dichloro-
methane (10 mL). The dicyclohexylcarbodiimide solution (1.73 g,
8.4 mmol) in dichloromethane (4 mL) was then added, and the
reaction mixture was left overnight at room temperature. Dicyclo-
hexylurea was removed by filtration, and the filtrate was concen-
trated under reduced pressure to yield white crystals (1.84 g, 95%
yield). Spectral characteristics of 1b: 1H NMR (CDCl3, 400 MHz):
d (CHCl3 = 7.26 ppm), 3.29 (t, 2H, CH2N3), 2.85 (dd, 4H, Suc), 2.63
(t, 2H, CH2COOH), 1.77 (m, 2H, N3CH2CH2), 1.63 (m, 2H,
CH2CH2COOH), 1.51 (m, 2H, N3CH2CH2CH2); IR: N@N@N 2090 cm
mixture (1:1) (the first addition). In 30-min intervals, the second
and the third portions of N-SEHA (by 100 ll) were added to the
reaction mixture. After stirring the mixture for 30 min at room tem-
perature, it was centrifuged; supernatant was removed, and the
particles were washed by acetone (3 ꢂ 1 ml) and ether (1 ꢂ 1 ml)
and air-dried.
The amount of the amino groups before and after the treatment
of SiO2–NH2 was evaluated by the picric acid method.25 The yield
of the reaction was evaluated to be 85%.
4.5. Synthesis of 20-deoxycytidine-50-{N-[3-(prop-1-
ynyl)oxypropyl]-gamma-amino}-triphosphate 2
ꢀ1
.
4.4. Synthesis of azidomodified nanoparticles 1 (Scheme 4A)
2,20-Dithiodipyridine (198 mg, 0.9 mmol, 5 equiv) and triphen-
ylphosphine (236 mg, 0.9 mmol, 5 equiv), in DMSO (150 lL each)
SiO2–NH2 nanoparticles (2 mg, 0.5
l
mol/mg for the amino
were added to a solution of 20-deoxycytidine-50-triphosphate (trib-
uthylammonium salt; 0.18 mmol) in DMSO (3 mL). After stirring
the reaction mixture for 30 min, the solution of 3-propynyloxypro-
groups) was suspended in 0.2 ml of a water/dimethylformamide
mixture (1:1), followed by the addition of N-hydroxysuccinimide
ester of 6-azidohexanoic acid (N-SEHA) (0.65, 2.5 or 10 mg, i.e.,
2.5, 10, or 40-fold excess over the amino groups in nanoparticles).
After stirring the mixture for 1 h at room temperature, it was cen-
trifuged; supernatant was removed, and the particles were washed
by acetone (3 ꢂ 0.1 ml) and ether (1 ꢂ 0.1 ml) and air-dried. IR
pylamine23 was added (200
lL, 1.8 mmol, 10 equiv), and the mix-
ture was incubated at rt for 2 h. The resulting product was
purified by anion exchange chromatography on DEAE Sephadex
A-25. The elution was performed with a linear gradient from 20%
EtOH to 1 M NH4HCO3 in 20% EtOH (150 mL each). Appropriate
fractions were collected and evaporated. The residue was co-evap-

S. V. Vasilyeva et al. / Bioorg. Med. Chem. 21 (2013) 703–711
709
orated several times with aqueous ethanol to remove traces of the
buffer. Target 20-deoxycytidine-50-{N-[3-(prop-1-ynyl)oxypropyl]-
gamma-amino}-triphosphate 2 was precipitated by the addition
of 6% NaClO4 in acetone to the aqueous solutions of the product.
The yield was 0.094 mmol (52%). 31P (121.5, D2O) d: ꢀ0.83 (d, 1P,
P , J 20.9), ꢀ10.94 (d, 1P, P , J 20.9), ꢀ22.59 (t, 1P, Pb, J 18.6). 1H
(m, 4H, O–CH2–CH2–CH2–N (L1), O–CH2–CH2–CH2–N (L2)); 3.49
(m, 2H, O–CH2–CH2–CH2–NH–Flu (L1)); 2.82 (m, 2H, O–CH2–CH2–
CH2–N (L2); 2.10 (m, 2H, H20); 1.89 (m, 2H, O–CH2–CH2–CH2–
N(L1)); 1.66 (m, 2H, O–CH2–CH2–CH2–N(L2)). LC–MSD-Trap-XCT,
[M+H]+: calcd 1063.81[M+H]+; found 1063.4. calcd 1069.8
[M+Li]+; found 1069.4.
c
a
(300, D2O) d: 8.0 (d, 1H, H(6), J 7.4); 6.36 (app.t, 1H, H10, J 6.86);
6.32 (d, 1H, H(5), J 7.4); 4.61 (m, 1H, H40); 4.18 (m, 5H, H30, O–
CH2–C„C, O–CH2–CH2–CH2–NH); 3.64 (t, 2H, O–CH2–CH2–CH2–
NH, J 6.43); 3.00–2.93 (m, 2H, H50); 2.47–2.27 (m, 2H, H50); 1.80–
1.76 (m, 2H, O–CH2–CH2–CH2–NH). LC–MSD-Trap-XCT, [M+H]+:
calcd 579.09 [M+H]+, found 579.08; calcd 601.09 [M+Na]+, found
601.08; calcd 623.09 [M+2Na]+, found 623.07; calcd 645.09
[M+3Na]+, found 645.07.
4.8. Synthesis of SiO2–dCTP 4 by ‘click’-reaction (Scheme 1)
Azidomodified nanoparticles 1 (4 mg, 1.72
pended by sonication in 200
L H2O. Solution of 20-deoxycyti-
dine-50-{N-[3-(prop-1-ynyl)oxypropyl]-gamma-amino}-triphos-
phate 2 (10 mM in H2O, 344 L, 3.44 mol, 2 equiv) was added to
the suspension followed by the addition of copper(II) sulfate
pentahydrate (50 mM stock solution in H2O, 44 L), tris[(1-ben-
zyl-1H-1,2,3-triazol-4-yl)methyl] amine (50 mM stock solution in
DMSO, 44 L), 1 M TEAAc buffer (pH 7, 20 L), and sodium ascor-
bate (freshly prepared 0.1 M solution in water, 64 L). The reaction
lmol) were sus-
l
l
l
l
4.6. Synthesis of 5-(3-aminopropoxyprop-1-ynyl)-20-
deoxyuridine-50-{N-[3-(prop-1-ynyl)oxypropyl]-gamma-
amino}-triphosphate 3a
l
l
l
mixture was degassed with argon for 2 min and stirred at room
temperature for 24 h. The modified SiO2-bound triphosphate was
centrifuged, supernatant was removed. The modified nanoparticles
were successively washed with aqueous 0.1 M NaCl, water, and
diethyl ether, followed by air-drying. The yield of the click product
was approximately evaluated by IR spectra to be more than 50%,
(see IR 1 and 4 in Supplementary data). More precisely the yield
of the attachment of triphosphate 2 to nanoparticles evaluated
by measuring the optical density of the resulting product SiO2–
dCTP dissolved in 0.2 M NaOH. It was 58%. (UV spectrum: (0.2 M
2,20-Dithiodipyridine (33 mg, 0.15 mmol), and triphenyl-
phosphyne (39 mg, 0.15 mmol) in DMSO (50 lL each) were added
to a solution of 5-(3-trifluoroacetamidopropoxyprop-1-ynyl)-20-
deoxyuridine-50-triphosphate23 (tributhylammonium salt; 0.031
mmol) in anhydrous DMSO (0.507 mL). After stirring the reaction
mixture for 30 min, the 3-propynyloxypropylamine23 solution
was added (50 mg, 0.44 mmol), and the mixture was incubated
at rt for 2 h. The resulting product was purified as in the case of
compound 2 (see above). The protected 50-triphosphate was sub-
jected to the standard deprotection protocol (ammonia treatment
for the trifluoroacetic protective groups). After the deprotection,
resulting 5-(3-aminopropoxyprop-1-ynyl)-20-deoxyuridine-50-{N-
[3-(prop-1-ynyl)oxypropyl]-gamma-amino}-triphos phate 3a was
precipitated by the addition of 6% LiClO4 in acetone to the aqueous
NaOH), kmax/nm (e): 260 (7400)). Another way to evaluate the effi-
ciency of modification was to compare the amount of triphosphate
2 added to the particles and its amount in combined supernatants
after the treatment and washing by optical density (UV spec-
trum:water, kmax/nm (e): 260 (7400). The extent of the attachment
solution of the product. UV spectrum: (water), kmax/nm (
e
): 280
of triphosphate 2 evaluated by the ratio of (I0–Is)/I0ꢃ100% (I0 and Is
are the values of the optical density of 2 in the initial solution and
in supernatant, respectively) was 55%.
(14,400). According to the UV-spectral data, the yield was
0.020 mmol (64%). 31P (121.5, D2O) d: ꢀ0.28 (d, 1P, P , J 20.82),
c
ꢀ10.66 (d, 1P, P , J 20.82), ꢀ21.28 (t, 1P, Pb, J 19.78). 1H (300,
a
D2O) d: 7.86 (m, 1H, H(6)); 6.16 (m, 1H, H10); 4.42 (m, 1H, H40);
4.27 (s, 2H, O–CH2–C„C–(L1)); 3.98 (m, 5H, H50, O–CH2–C„C–
(L2), H30); 3.41 (m, 4H, O–CH2–CH2–CH2–N (L1), O–CH2–CH2–
CH2–N (L2)); 2.92–2.75 (m, 4H, O–C2–CH2–CH2–N (L1), O–CH2–
CH2–CH2–N (L2)); 2.16 (m, 2H, H20); 1.74–1.60 (m, 4H, O–CH2–
CH2–CH2–N(L1, L2)).
4.9. Synthesis of SiO2–dUTPFlu 5 by ‘click’-reaction (Scheme 1)
Azidomodified nanoparticles 1 (2.5 mg, 1 lmol) were ultrason-
ically suspended in 100 lL H2O. Solution of 5-{3-[3-(fluorescein-
5-yl-thioureido)propoxy]prop-1-ynyl}-20-deoxyuridine-50-{N-[3-
(prop-1-ynyl)oxypropyl]-gamma-amino}-triphosphate 3 (10 mM
in H2O, 543
lowed by the addition of copper(II) sulfate pentahydrate
(50 mM stock solution in DMSO, 22 L), tris[(1-benzyl-1H-1,2,3-
triazol-4-yl)methyl] amine (50 mM stock solution in DMSO,
22 L), 1 M TEAAc buffer (pH 7, 15 L), and sodium ascorbate
(freshly prepared 0.1 M solution in water, 40 L, 4 mol). The
lL, 5.043 lmol) was added to the suspension, fol-
4.7. Synthesis of 5-{3-[3-(fluorescein-5-yl-
thioureido)propoxy]prop-1-ynyl}-20-deoxyuridine-50-{N-[3-
(prop-1-ynyl)oxypropyl]-gamma-amino}-triphosphate 3
l
l
l
5-(3-Aminopropoxyprop-1-ynyl)-20-deoxyuridine-50-{N-[3-
(prop-1-ynyl)oxypropyl]-gamma-amino}-triphosphate 3a (10 mg,
0.014 mmol) was dissolved in 1 M carbonate–bicarbonate buffer,
pH 9.0 (0.8 mL), followed by the addition of the fluorescein isothi-
ocyanate solution (55 mg, 0.14 mmol) in a DMSO/DMF mixture
(1:1, v/v, 0.40 mL). The reaction mixture was stirred for 2 h, the
reaction products were then precipitated with 6% LiClO4 in acetone
(14 mL), centrifuged, and the precipitate was washed with acetone
(3 mL) and ester. The target compound was purified by the reverse
phase chromatography on Polygoprep C18 in a gradient of EtOH (0–
10%) in water and precipitated with 6% LiClO4 in acetone. Rf = 0.17
(PriOH-conc. NH3–H2O, 7:1:2). The yield of 3 was 70%. 31P (121.5,
D2O) d: ꢀ0.39 (d, 1P, P , J 20.35), ꢀ10.77 (d, 1P, P , J 20.35),
l
l
reaction mixture was degassed with argon for 2 min and stirred
at room temperature for 24 h.
The modified nanoparticles containing the bound fluorescent-
labeled triphosphate was centrifuged, supernatant was removed.
Modified nanoparticles were successively washed with aqueous
0.1 M NaCl, water, and diethyl ester, followed by air-drying. The
yield of the click product evaluated by IR spectra was ꢁ40%, see
IR 1 and 5 in Supplementary data. The yield of the modification
evaluated by optical density of the resulting product SiO2–dUTPFlu
5 dissolved in 0.2 M NaOH. It was 50%. (UV spectrum: (0.2 M
NaOH), kmax/nm (e): 280 (14,400)).
c
a
ꢀ21.96 (t, 1P, Pb, J 20.35). 1H (300, D2O) d: 7.80 (s, 1H, H(6));
7.56 (m, 1H, Flu4); 7.50–7.47 (m, 1H, Flu6); 7.24–7.21 (m, 1H,
Flu7); 7.01 (m, 2H, Flu40,50), 6.54–6.50 (m, 2H, Flu10,30), 6.39–6.33
(m, 2H, Flu70,20), 5.98 (m, 1H, H10); 4.82 (m, 1H, H40); 4.30 (s, 2H,
O–CH2–C„C–(L1)); 3.90 (m, 5H, H50, O–CH2-C„C–(L2), H30); 3.66
4.10. Synthesis of SiO2–L–NH2 6 by ‘click’-reaction (Scheme 4C)
Azidomodified nanoparticles 1 (0.7 mg, 0.28
pended by sonication in 50 L H2O. 3-Propynyloxypropylamine
(11 mg 0.1 mmol) was added to the suspension followed by the
lmol) were sus-
l

710
S. V. Vasilyeva et al. / Bioorg. Med. Chem. 21 (2013) 703–711
addition of copper(II) sulfate pentahydrate (50 mM stock solution
in DMSO, 22 L), tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]
amine (TBTA, 50 mM stock solution in DMSO, 22 L), 1 M TEAAc
buffer (pH 7, 10 L), and sodium ascorbate (freshly prepared
0.1 M solution in water, 32 L, 3.2 mol). The reaction mixture
was degassed with argon for 2 min and stirred at room tempera-
ture for 24 h. The modified nanoparticles containing the bound
aminolinker (L) were centrifuged, and successively washed with
aqueous 0.1 M NaCl, water, and diethyl ester, followed by air-dry-
ing. The yield of the ‘click’-product was evaluated by IR spectra
approximately100%, (see IR 6 in Supplementary data).
acetone (500
solved in water (10
l
L), centrifuged, washed with acetone, dried, dis-
L), and subjected to electrophoresis in 20%
l
l
l
PAAG. After electrophoresis, the gel was dried in a FB GD 45 Gel
dryer (Fisher Sscientific, USA) and scanned on a Molecular Imager
FX-PRO Plus (Bio-Rad, USA). The reaction yields were evaluated
using the Gel-Pro Analyzer 4.0 program (MediaCybernetics, United
States) by determining the integral optical density (IOD) of spots in
each lane (Figs. 2 and 3).
l
l
l
4.13. Penetration of SiO2–dUTPFlu into HeLa cells
HeLa cells were cultured on glass cover slips (Invitrogen, USA)
in RPMI media supplemented with 5% fetal calf serum (5%), penicil-
4.11. Primer extension reaction
lin and streptomycin (100 lg/ml, each) at 37 °C in a humidified
Klenow fragment and 10ꢂ SE Klenow buffer containing 500 mM
Tris–HCl, pH 7.6, 100 mM MgCl2, and 50 mM DTT (Sibenzyme, Rus-
sia) or HIV RT and 10ꢂ RT buffer containing 500 mM Tris–HCl, pH
8.3, 750 mM KCl, 30 mM MgCl2, and 50 mM DTT (Applied Biosys-
tems, USA) were used in our experiments.
incubator under ambient pressure air atmosphere containing 5%
CO2. At approximately 70% confluence, cells were treated with
the SiO2–dUTPFlu nanocomposite (2 nmol/ml) for 24 h and fixed
with formaldehyde (3.7%) for 10 min, followed by washing with
PBS. Nuclei and cell membranes were stained with DAPI and Cell
Mask Plasma Membrane Stain (Molecular Probes, Invitrogen,
USA), respectively for 10 min. After washing the cells with PBS,
they were visualized by confocal laser scanning LSM 510 UV META
microscope (Carl Zeiss, Inc., Germany). Laser lines were 405 nm
(for nuclei, DAPI blue), 488 nm (for fluorescein-labeled nanocom-
posites, green), and 543 nm (for cell membranes, red) (Fig. 4).
The mixture of the primer (16-mer, 10ꢀ4 M, 4
(30-mer, 10ꢀ4 M, 4
L), and 10ꢂ Klenow buffer (8
with water to 80 L, kept at 95 °C for 5 min and cooled to room
lL), the template
l
lL) was adjusted
l
temperature for 30 min. The resulting duplex was used for the
extension reactions.
The extension reaction was carried out in a mixture (20
l
L) con-
L), Klenow frag-
L of each of dNTP (four native dNTPs
taining the above duplex (4 lL), Klenow buffer (2 l
ment (1 ꢂ 10ꢀ3 U), and 2
l
Acknowledgments
(mixture 1), or three native dNTP except dCTP (mixture 2), or three
native dNTP + SiO2–dCTP (mixture 3), or three native dNTP + super-
natant from SiO2–dCTP suspension (mixture 4)). The final concen-
trations of the components in each reaction mixture were 4 ꢂ 10
ꢀ6 M for the template, 1 ꢂ 10ꢀ6 M for the primer, 1 ꢂ 10ꢀ5 M for
dNTPs; 0.07 mg/ml for SiO2–dCTP (1 ꢂ 10ꢀ5 M for triphosphate in
this nanocomposite). The reaction was carried out at 37 °C for
Authors thank S. I. Baiborodin for the assistance in the use of
confocal fluorescent microscopy and F. V. Tuzikov for the assis-
tance in the analysis of the size of nanoparticles. The work was
supported by RFBR Grant no. 11-04-01408-a.
Supplementary data
60 min 3 M LiClO4 (30 lL) was added to each reaction mixture. In
case of the presence of SiO2–dCTP, reaction mixtures were centri-
fuged (14,000ꢂg, 5 min) for separation of SiO2 nanoparticles. The
reaction products from each reaction mixture were precipitated
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2012.11.057.
with acetone (500
dissolved in water (10
l
L), centrifuged, washed with acetone, dried,
References and notes
lL), and subjected to electrophoresis in
20% PAAG. After electrophoresis, the gel was stained with Stains
All, dried in a FB GD 45 Gel dryer (Fisher Scientific, USA), and
scanned (Fig. 1).
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