A designed 5-fluorouracil-based bridged silsesquioxane as an autonomous acid-triggered drug-delivery system

Article abstract of DOI:10.1002/chem.201301081

Two new prodrugs, bearing two and three 5-fluorouracil (5-FU) units, respectively, have been synthesized and were shown to efficiently treat human breast cancer cells. In addition to 5-FU, they were intended to form complexes through H-bonds to an organo-bridged silane prior to hydrolysis-condensation through sol-gel processes to construct acid-responsive bridged silsesquioxanes (BS). Whereas 5-FU itself and the prodrug bearing two 5-FU units completely leached out from the corresponding materials, the prodrug bearing three 5-FU units was successfully maintained in the resulting BS. Solid-state NMR ( ²⁹Si and ¹³C) spectroscopy show that the organic fragments of the organo-bridged silane are retained in the hybrid through covalent bonding and the ¹Ha NMR spectroscopic analysis provides evidence for the hydrogen-bonding interactions between the prodrug bearing three 5-FU units and the triazine-based hybrid matrix. The complex in the BS is not affected under neutral medium and operates under acidic conditions even under pH as high as 5 to deliver the drug as demonstrated by HPLC analysis and confirmed by FTIR and ¹³Ca NMR spectroscopic studies. Such functional BS are promising materials as carriers to avoid the side effects of the anticancer drug 5-FU thanks to a controlled and targeted drug delivery. My name is Bond, H-Bond: A new 5-fluorouracil (5-FU) prodrug, showing cytotoxicity against breast cancer cells, was successfully maintained through H-bonds to an organobridged silsesquioxane (BS). The resulting complex is not affected under physiological conditions but operates under lysosomal pH to deliver the prodrug (see figure). The designed 5-FU prodrug/BS is a promising autonomous pH-sensitive material carrier, which avoids the side effects of the anticancer drug due to a controlled and targeted release. Copyright

Full text of DOI:10.1002/chem.201301081

DOI: 10.1002/chem.201301081
A Designed 5-Fluorouracil-Based Bridged Silsesquioxane as an Autonomous
Acid-Triggered Drug-Delivery System
Simon Giret,[a] Christophe Thꢀron,[a] Audrey Gallud,[b] Marie Maynadier,[b]
Magali Gary-Bobo,[b] Marcel Garcia,[b] Michel Wong Chi Man,[a] and Carole Carcel*[a]
Abstract: Two new prodrugs, bearing
two and three 5-fluorouracil (5-FU)
units, respectively, have been synthe-
sized and were shown to efficiently
treat human breast cancer cells. In ad-
dition to 5-FU, they were intended to
form complexes through H-bonds to an
organo-bridged silane prior to hydroly-
sis-condensation through sol–gel proc-
esses to construct acid-responsive
bridged silsesquioxanes (BS). Whereas
5-FU itself and the prodrug bearing
two 5-FU units completely leached out
from the corresponding materials, the
prodrug bearing three 5-FU units was
successfully maintained in the resulting
BS. Solid-state NMR (29Si and 13C)
spectroscopy show that the organic
fragments of the organo-bridged silane
are retained in the hybrid through co-
valent bonding and the H NMR spec-
troscopic analysis provides evidence for
the hydrogen-bonding interactions be-
tween the prodrug bearing three 5-FU
units and the triazine-based hybrid
matrix. The complex in the BS is not
affected under neutral medium and op-
erates under acidic conditions even
under pH as high as 5 to deliver the
drug as demonstrated by HPLC analy-
sis and confirmed by FTIR and
13C NMR spectroscopic studies. Such
functional BS are promising materials
as carriers to avoid the side effects of
the anticancer drug 5-FU thanks to a
controlled and targeted drug delivery.
1
Keywords: cancer · drug delivery ·
drug design · hydrogen bonding ·
sol–gel processes
Introduction
deliver antimicrobial or therapeutic substances to the target
site among which mesoporous silica nanoparticles
(MSN)[1e,2] appear as one of the most promising strategies
that is being adopted by many researchers. Indeed, MSN
have superior features compared with organic polymers ex-
hibiting highly ordered porous structure, tunable pore size,
pore volume, and high surface areas, which can be exploited
as reservoirs for active molecules. Moreover, silica provides
mechanical and chemical stabilities, efficient protection for
the payloads as well as biocompatibility and is non-toxic
when in the amorphous state.[3] These MSNs are mostly
being developed for medical applications,[1g,4] and a main ob-
jective is to achieve specifically drug administering to target
cells. Quite recently, striking achievements have been high-
lighted with newly synthesized stimuli-responsive MSN
nanomachines to deliver cargo molecules. To obtain efficient
nanosystems, MSNs have been functionalized with organic
groups that can act as caps, gatekeepers, valves, and so
on,[1g,4a–c], which can be operated under an appropriate stim-
uli (pH, redox potential, magnetic, etc.) to deliver the cargo
molecules after pore openings.[5]
In the past few decades, several kinds of nanomaterials have
been developed for application in biological and medical
fields, such as sensors, cell markers, and drug-delivery sys-
tems.[1] The latter is particularly one of the most exciting
challenges to take up and it is now established that well-de-
signed drug carriers with controlled delivery can overcome
some issues of conventional therapy and enhance the thera-
peutic performance of a given drug. For a maximum thera-
peutic efficiency, an optimal amount of the agent must be
delivered without premature release to the targeted cell.
This would avoid the use of any excess of the drug, which
would reduce general toxicity and would cause minimal side
effects. Several approaches have since been developed to
[a] S. Giret, C. Thꢀron, Dr. M. Wong Chi Man, Dr. C. Carcel
Laboratory Architectures Molꢀculaires et
Matꢀriaux Nanostructurꢀs
Institut Charles Gerhardt Montpellier
(UMR 5253 CNRS-UM2-ENSCM-UM1)
ENSCM, 8 rue de lꢁꢀcole normale
Since its introduction in the last two decades, bridged sil-
sesquioxanes (BS)[6] have fascinated scientists because they
are hybrid materials with a high content of organic groups
linked regularly to the silica matrix and can thus combine
both the properties of the latter with those of the organic
components.[7] Owing to the wide variety of organics and to
their intrinsic properties that can be introduced in BS, our
group has been exploring their uses in several application
fields such as catalysis,[8] solid-state lighting and luminescent
34296 Montpellier (France)
E-mail: carole.carcel@enscm.fr
[b] A. Gallud, Dr. M. Maynadier, Dr. M. Gary-Bobo, Dr. M. Garcia
Institut des Biomolꢀcules Max Mousseron
UMR 5247 CNRS, Universitꢀ Montpellier 1
Universitꢀ Montpellier 2, ENSCM
Facultꢀ de Pharmacie, 15 Avenue Charles Flahault
34093 Montpellier Cedex 05 (France)
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.201301081.
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FULL PAPER
solar concentrators[9] or for selective ion uptake.[10] In BS,
porosity can be created, for example, by cleavage of the co-
valently bonded sacrificial organics in the silica matrix[11] or
by using surfactants to create ordered mesopores. In the
latter case, periodic mesoporous organosilicates (PMOs)[12]
are obtained in which either the organics are located in the
walls from which they can be randomly dispersed[6b,7a] or
well-arranged; in the latter case, a hierarchical crystalline
hybrid silica is formed.[13] A recent review gives a compila-
tion of different applications of PMOꢁs, for example, as cata-
lysts and adsorbents for which the high surface area and po-
rosity are the bare essentials.[14] Consequently, like MSN,
PMOs have also been used as drug-delivery systems[15] and
it was shown that the morphology of the material (hollow
spheres vs. solid spheres)[15a] as well as the organic functions
inside the walls (compared with functionalized MSN)[15b]
could improve or affect their drug-release properties. To
date, the use of PMOs in this field is still in its infancy be-
cause they are not easy to synthesize in a pure state without
avoiding the use of mixing the organobridge precursor with
other alkoxysilane precursors such as tetramethylorthosili-
cate (TMOS) and tetraethylorthosilicate (TEOS),particular-
ly when the bridging organic unit is voluminous.[16]
(donor–acceptor–donor) pattern H-bonded with cyanuric
acid (CA) bearing the complementary ADA (acceptor–
donor–acceptor) patterns (9H bonds).
It was shown that the templating CA molecule could com-
pletely be removed from the material upon acid treatment
and interestingly even under a mild acidic medium (pH 5.5).
It is known that pH values vary significantly in different tis-
sues and cellular compartments. While it is close to neutral
in blood and normal tissues (pH 7.4), it is lightly acidic in tu-
moral tissues (pH 6.8) and more acidic in endosomal (pH 6–
5.5) and lysosomal (pH 5.0–4.5) compartments.[22] We thus
decided to investigate this pH-sensitive BS and see whether
it is likely to be developed as a drug platform for controlled
drug-delivery. We chose 5-fluorouracil (5-FU) as the drug
candidate as it bears an adequate ADA pattern, which
makes it liable to associate with the DAD pattern of P1
(Figure 2). Moreover, it is commonly used as an anticancer
Herein, we used another concept to design new BS-based
drug carriers in which high porosity and high surface area
are not required features. The BS is synthesized with the
corresponding bridge organosilane precursor in the presence
of a non-silylated molecule, which is held by weak H-bond-
ing interactions in the resulting BS. This molecule plays the
double role of the templating agent and the drug itself. The
drug release (in this case the templating molecule) can be
achieved by applying an acidic pH that cleaves the H-bonds.
We recently reported a new family of BS synthesized
from monosilylated complementary base-pair derivatives in
which the two organic units are linked together (molecular
recognition) by H-bonding to form the bridging unit.[17]
Solid-state 1H NMR spectroscopic investigations revealed
the preservation of the molecular recognition sites[18] and
the preserved H-bonds are even shown to play an important
role in the nanostructuring of a ureidopyrimidinone-based
BS.[19] These original BS are prone to applications and thus
a molecular-imprinted BS[20] and more recently a pH-sensi-
tive BS have been reported.[21] The latter was designed from
a bis-silylated triazine derivative P1 (Figure 1) with a DAD
Figure 2. Schematic representation of the H-bonding interaction between
P1 and 5-FU.
drug in the clinical treatment of several solid cancers, such
as breast, colorectal, liver, and brain cancer. Due to its high
rate of metabolism in the body, a high concentration of 5-
FU is needed to improve its therapeutic effect. A continu-
ous administration of 5-FU is thus required, which repre-
sents a serious drawback since a concentration above a cer-
tain limit produces severe side effects. To overcome this
issue, controlled and targeted release of 5-FU is needed.
Thus, the strategy to construct a BS holding a 5-FU unit by
molecular recognition patterns is quite appealing because a
direct and controlled release of the drug in the cell will be
self-triggered by the acidic pockets of the cells.
To evaluate the role of the amount of H-bonds on the sta-
bility of this new system and consequently on the release
rate of the drug under neutral and acidic conditions, we con-
sidered two new compounds (2 and 3), with two and three
5-FU units, respectively (Figure 3) for which we also studied
the in vitro toxicity towards human breast cancer cells.
Results and Discussion
The objective of this work was to make use of a bridged sil-
sesquioxane BS that can hold 5-FU at neutral pH (like in
Figure 1. Structure of precursor P1.
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C. Carcel et al.
Figure 3. Structures of new 5-FU-based prodrugs 2 and 3 and their H-bonding association with P1.
blood medium) to create a new type of carriers with no pre-
mature release of the drug and to demonstrate that its direct
and controlled release can be triggered by the acidic pH
close to those found in the acidic compartments of cells.
Syntheses and studies of 2 and 3: In addition to 5-FU, which
is used as drug model, two new 5-FU prodrugs (2 and 3)
with two and three 5-FU units, respectively, linked through
an ester function to an alkyl core were synthesized
(Figure 3). These linkers can be cleaved chemically or enzy-
matically[23,24] to generate 5-FU and/or 5-fluorouracil-acetic
acid (5-FUA also referred to as compound 1 in this work)
whose biological activities are known.[25]
Syntheses: Compounds 2 and 3 were prepared as described
in Scheme 1. Compound 1 (5-FUA) was obtained according
to a known procedure[23a] by treating 5-FU with chloroacetic
Scheme 1. Synthesis of compounds 2 and 3.
acid in alkaline solution at 608C. Adding an excess of thion-
yl chloride to compound 1 led to the formation of the corre-
sponding acyl chloride. The latter was then treated with 2,2-
dimethylpropane-1,3-diol and with 1,1,1-tri(hydroxymethyl)-
propane to afford 2 and 3, respectively.
Investigations on the association of 5-FU and compounds 2
and 3 with P1: We first investigated the association feasibili-
ty between 5-FU and the BS precursor P1 by performing
liquid 1H NMR spectroscopic studies in [D6]DMSO
(Figure 4). In the 1H NMR spectrum of 5-FU, it was not pos-
ꢀ
ꢀ
sible to distinguish between the two protons, H N3 and H
N1, because they exist as a single broad peak at d=
11.1 ppm. On the addition of one equivalent of P1, these
two H separated into two distinct and well-resolved peaks at
ꢀ
Figure 4. Liquid 1H NMR spectrum in [D6]DMSO of 5-FU, P1, and com-
plex 5-FU/P1 (1:1).
d=10.9 and 11.8 ppm. This implies that the proton of H N3
is involved in the H-bonding process with the DAD face of
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A Designed 5-Fluorouracil-Based Bridged Silsesquioxane
FULL PAPER
P1, which confirms the complex formation between P1 and
5-FU.
Similarly to 5-FU, the N3 proton of 5-FU units in 2 and 3
was used to probe the H-bonding interactions. As shown in
Figure 5a, the probe proton of 2 appears as a sharp peak at
d=12.0 ppm. In this case, since 2 bears two 5-FU units, a 1/
this, cell cultures were treated by increasing doses of 2 and 3
(from 5 to 50 mm). For comparison, cell cultures were also
treated in the same conditions with compound 1 and 5-FU.
All compounds induced a decrease in the cell number com-
pared to control cells, which are treated with equivalent vol-
umes of the vehicle (DMSO) (Figure 6a). This effect was
Figure 6. Cytotoxicity of drugs. a) Images of human MCF-7 breast cancer
cells incubated for 72 h in the absence (control, C) or presence of the
drug (50 mm). The scale bar (top image) represents 30 mm; b) Effect of in-
creasing concentrations (from 5 to 50 mm) of (pro)drugs on MCF-7 cell
growth. Values are mean ꢁSEM of three independent experiments.
confirmed by a MTT assay, which demonstrated that the
four molecules induced a decrease in cell growth (Fig-
ure 6b). The dotted line on the graph represents the level of
cell seeding at the beginning of the experiment. At the high-
est concentration tested (50 mm) 5-FU, the commercial drug,
induced a 66% of cell number decrease that is close to max-
imal complete cell growth inhibition. In this experiment the
known derivative 5-FUA (compound 1) induced only 24%
inhibition. In the case of 2 and 3, they induced a 29 and
44% decrease in cell number, respectively, and are therefore
less efficient than the drug 5-FU but more efficient than
compound 1, which is in agreement with the number of
units contained in each.
Figure 5. Liquid 1H NMR spectra of complex between a) compound 2
and P1, and b) compound 3 and P1 in [D6]DMSO.
2 molar equivalent of P1 was used to form the complex
(Figure 3). Following the addition of P1, no significant shift
was observed but this peak broadens and the chemical shift
ranges from d=11.7 to 12.3 ppm. Similar observations were
made in the spectrum of 3 (Figure 5b), which exhibits a
sharp singlet at d=12 ppm and broadens (from d=11.5 to
12.5 ppm) on addition of 1/3 molar equivalent of P1. As in
the case of 5-FU this change can be attributed to H-bonding
Cell-cycle analysis: Since 5-FU is converted in the cell into
different cytotoxic metabolites that interfere with DNA syn-
thesis[26] leading to cell cycle arrest and apoptosis, com-
pounds 2 and 3 were studied by using fluorescence-activated
cell sorting (FACS) and compared to 5-FU and 5-FUA. In
this experiment, MCF-7 cells were treated with 5-FU, 5-
FUA, 2 and 3 (50 mm) during 24 h. Figure 7 shows that 5-FU
induces cell cycle arrest in the S phase (54 instead of 21%
in control cells), an increase in apoptosis (11 instead of
1.5% in control cells) and a total disappearance of G2/M
ꢀ
formation of the H N3 of the 5-FU prodrugs with the sily-
lated precursor P1.
In vitro toxicity: The biological potential of the newly syn-
thesized prodrugs of 5-FU (2 and 3) was established by a cy-
totoxic assay on human breast cancer cells (MCF-7). For
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C. Carcel et al.
tone successively) and drying (at room temperature) proc-
esses, the hybrid was characterized using routine solid-state
techniques for analyses.
Material BS1 using precursor P1 and 5-FU: Subsequently,
we considered the synthesis of the corresponding bridged
silsesquioxane with 5-FU. In this case, an equimolar amount
of P1 and 5-FU was used and the hydrolysis–condensation
was performed under similar reaction conditions as for BS0
leading to a white gel BS1 after one day. Figure 8a depicts
the solid-state FTIR spectra of 5-FU, BS0, and BS1, respec-
tively. The IR spectrum of 5-FU shows distinct vibrations
between 1700 and 1800 cmꢀ1 and unexpectedly, these are
not found in the IR spectrum of BS1. Instead, vibration
bands with similar intensities to those of BS0 can be seen
and both spectra of BS0 and BS1 can be completely super-
imposed (Figure 8a), which suggests that BS1 like BS0 is
free of 5-FU. This was confirmed by 13C solid-state NMR
Figure 7. Cell-cycle analysis. MCF-7 cells treated for 24 h with DMSO
alone (control, C) or supplemented with 5-FU, 5-FUA, 2, or 3 (50 mm),
respectively.
phase. Whereas cells treated with 5-FUA are arrested in the
S phase (43%), a cell population escaped this arrest (9% in
G2/M). This could explain, at
least in part, the lower efficien-
cy of 5-FUA observed in
Figure 6.
Concerning
com-
pounds 2 and 3, a significant
decrease was noted in the G0/
G1 phases (41 and 37%, re-
spectively) in comparison with
the control (56%). We can ob-
serve the strongest proportion
of cells in the apoptotic subG1
phase (24 and 33%, respective-
ly) when treated with 2 and 3.
It is also interesting to note a
quasi-total disappearance of
G2/M phase in cells treated
with 2 and 3, which is compara-
ble to 5-FU. Finally, these re-
sults are in accordance with the
cytotoxicity data in Figure 6
demonstrating an efficiency of
2 and 3 between those of 5-FU
and 5-FUA.
Synthesis, characterization and
studies of materials BS0–BS3:
Blank material BS0 using only
precursor P1 without drug mol-
ecule: A blank experiment was
realized by using only P1 to
give the hybrid material BS0,
which will serve for compari-
son. P1 was dissolved in DMSO
and the hydrolysis–condensa-
tion was performed with a nu-
cleophilic catalyst (fluoride
anion), which leads to a white
gel BS0 after one day. After
Figure 8. a) FTIR spectra of BS1 (c), BS0 (g), and 5-FU (a); b) 13C Solid-state NMR spectra of BS0,
washing (water, EtOH and ace- BS1, and BS1A.
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A Designed 5-Fluorouracil-Based Bridged Silsesquioxane
FULL PAPER
analysis (Figure 8b). Indeed, we compared the 13C NMR
spectrum of BS0 with that of BS1 and additionally with that
of BS1A obtained by acid-treatment of BS1 to deliver any
5-FU that could be present in this hybrid. No typical chemi-
cal shifts of 5-FU could be observed in the spectrum of BS1
and no chemical shift or intensity variation of C=O and C=
N peaks was visible between the spectra of BS0 and BS1
(Figure 8b).[21] Instead all three
Considering BS3, interestingly the FTIR spectrum exhib-
its two characteristic vibration bands (1710 and 1755 cmꢀ1)
of 3, whose intensities remain strictly the same even after
washing several times with water (Figure 9). Moreover a
broad and intense band appears ranging from 950 to
ꢀ1
ꢀ ꢀ
1100 cm , which is attributable to the Si O Si vibration,
significant of a highly condensed hybrid. In addition, HPLC
spectra could completely be
overlapped. In addition, no 5-
FU was detected by HPLC in
the solution recovered after the
acidic treatment and washing of
BS1. We therefore concluded
that the drug molecule, 5-FU,
was not retained in the material
during the hydrolysis–condensa-
tion step. It may be due to the
amount of water used, which
can cleave the complex during
the hybrid preparation and thus
explains the release of all 5-FU
in solution. Indeed it has al-
ready been reported that the
H-bonds with 5-FU are sensi-
Figure 9. FTIR spectroscopy analysis of BS3 (b), BS0 (g), compound 3 (a), and BS3A (c).
tive to small molecules such as
water and thus appears as an
issue to retain it in the resulting BS during the hydrolysis–
analysis revealed only traces of 3 in the recovered washing
solution from freshly prepared BS3 showing that the former
is well retained in the solid material. Thus, a high loading
rate of 3 (ꢂ20 weight%) is expected in BS3.
condensation process.[27] We confirmed this while perform-
1
ing the liquid H NMR spectroscopic investigations with P1
and 5-FU and showed that the addition of traces of water
was sufficient enough to break the H-bonds since the two
BS3 was then treated with an acid solution (pH 2) leading
to BS3A. In the corresponding IR spectrum (Figure 9), the
two characteristic bands of 3 (1710 and 1755 cmꢀ1) com-
ꢀ
ꢀ
singlets of N3 H and N1 H completely disappeared (see
the Supporting Information S1). This result is different from
the one we reported with the cyanuric acid[21] and may be
due to the fluorine atom, which weakens the complex. We
therefore decided to synthesize two new 5-FU prodrugs (2
and 3) with two and three ADA patterns, respectively. Com-
pounds 2 and 3 would afford more H-bonding and should
induce a stronger complex with P1 such that they can be
maintained in the resulting hybrid materials.
1
pletely disappeared and the liquid H NMR spectrum of the
recovered solution confirms the nature of 3. Moreover, the
IR spectra of BS3A and BS0 are identical and can be com-
pletely superimposed. All these observations demonstrate
the complete elimination of 3 from BS3 after acid treat-
ment.
The hybrid material BS3 was also characterized by using
usual techniques (see the Supporting Information, S3 and
S4). The nitrogen adsorption isotherms of the different ma-
terials (BS3 and BS3A) were realized. Essentially weak
sorption was observed for both samples meaning that both
materials are poorly porous. After the acid treatment of
BS3 to remove 3, the specific surface did not increase signif-
icantly.
Material BS2 and BS3 using precursor P1 and 2 and 3: BS2
and BS3 were prepared under the same conditions as above.
In these cases, due to the multiple units of 5-FU, ratios of
1:2 and 1:3 were used for 2 and 3, respectively. In both cases
the gels were formed in a much shorter time than for BS1
(2 h vs. 1 day). This different behavior may result from an
easy organization of bridged silsesquioxane due to the exis-
tence of the complex. To confirm the presence of 5-FU de-
rivatives in the hybrid materials, FTIR spectroscopy studies
were performed.
1
As already reported, solid-state H NMR spectroscopy is
an appropriate technique to probe the existence of complex
through hydrogen bonding.[18a,20] Similarly to the solution
1H NMR studies on P1, it was possible to check the exis-
ꢀ
BS2 behaves similarly to BS1, with no retention of 2 de-
spite the presence of two ADA patterns most probably be-
cause the complex was still not strong enough to resist to
several aqueous washings (see the Supporting Information,
S2).
tence of the N3 H bonding of 3 with the silylated triazine
fragment in BS3. The observations in the solid state were
similar to those found in the liquid phase with P1. Indeed,
ꢀ
the N3 H proton signal at d=11.1 ppm of 3 alone nearly
disappeared and a weaker band was found downfield at d=
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C. Carcel et al.
15.3 ppm for the hybrid BS3 indicating that the hydrogen-
bonding interactions between the anchored triazine deriva-
tive and the 5-FU fragments were really preserved in the
hybrid material (Figure 10).
Figure 11. Release of 3 from BS3 in PBS conditions at different pH
~
&
^
values ( pH 7, pH 5, pH 3).
compared with 5-FU have been synthesized. They were all
able to form complexes in solution by molecular recognition
with a triazine-based organosilane precursor, as demonstrat-
1
1
Figure 10. CP-MAS H NMR spectra of BS3 and 3.
ed by liquid H NMR studies. Attempts to retain these com-
pounds within the resulting sol–gel-made materials (keeping
the ADA pattern of the drug linked to the DAD pattern of
the anchored triazine unit to the silica matrix) to produce
pH-responsive bridged silsesquioxanes failed in the case of
5-FU and 2, but was successful with 3. This clearly indicates
that at least three ADA patterns are necessary to strongly
hold the prodrug in the BS. Moreover, no release of 3 was
observed from the BS3 at neutral pH but it can be stimulat-
ed under mild acid pH. This study demonstrates the interest
of developing well-designed bridged silsesquioxanes, despite
being non-porous, as new functional hybrid materials for bi-
omedical applications. We envision the use of these BS as
carriers for drug-delivery systems since they obviously
comply with the required conditions for therapeutic applica-
tions, namely a non-premature release (the drug remains in
the material under neutral pH) and a controlled-release trig-
gered by an acid stimulus (pH close to weak acidic media of
the cell compartments). Work is in progress to synthesize
the corresponding nano-BS with the appropriate size to
evaluate the acid-triggered self-release of the prodrug inside
the cancer cells.
It thus appears clearly that the 9 H-bonds between P1
and 3 were strong enough to maintain 3 in BS3 under the
sol–gel conditions and during washing process. Treating BS3
with an acid breaks all the H-bonds to release 3 in solution,
which was recovered by filtration and washing of the materi-
al. BS3 is therefore appropriate for an evaluation as a drug
carrier.
Prodrug release: For application as a drug-delivery system,
the hybrid material should meet moderate pH conditions
like those found in lysosomal compartments. We thus envis-
age three experiments at different pH: pH 7 to confirm that
the material meets the zero premature-release criterion,
pH 3 to favor a complete release, and pH 5 to mimic the ly-
sosomal pH. The pH-release profile was determined by
quantitative HPLC analysis. A suspension of BS3 was stir-
red at 378C in a phosphate buffer solution (PBS) at the
three selected pH (7, 5, and 3). Aliquots of the solution
were taken at defined time intervals and replaced by fresh
PBS at the same pH to maintain the total volume.
The results are given in Figure 11. The release rate de-
pends directly on the pH value with higher rates at higher
concentrations of H+ ions as expected. At pH 3, the release
is complete after one day, whereas 25% of 3 were released
for endosomal pH 5. After two days at pH 5, the cleavage of
3 occurred inducing wrong HPLC data. Interestingly, in neu-
tral condition no release was observed. These observations
confirm that the system bridged silsesquioxane/3 (BS3) can
be appropriate as an autonomous drug delivery system with
zero premature-release.
Experimental Section
General: All reactions requiring anhydrous conditions were performed
under N2 atmosphere by using vacuum line and standard Schlenk techni-
ques. Chemicals and solvents were either A.R. grade or purified by
standard techniques. Thin layer chromatographies (TLC) were realized
with silica gel plates Merck 60F254 and compounds were visualized under
UV light. Column chromatography was performed using flash chroma-
tographer Grace Reveleris, equipped with prepack silica columns (Inter-
chim, 30 mm) with eluent given in parentheses. 5-fluorouracil, chloroace-
tic acid and 1,1,1-tri(hydroxymethyl)propane, 2,2-dimethylpropane-1,3-
diol were purchased from Aldrich, USA. All other solvents and chemi-
cals were obtained from commercial sources.
Conclusion
Compound 1 (5-FUA): Compounds 2 and 3 were synthesized from 5-
FUA, which was prepared according to a previously reported method.[23a]
In this work, two new 5FU-based prodrugs (2 and 3), bear-
ing additional ADA patterns (two and three, respectively)
1
Yield: 73%; m.p. 274–2768C; H NMR (400 MHz, [D6]DMSO): d=11.93
12812
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Chem. Eur. J. 2013, 19, 12806 – 12814

A Designed 5-Fluorouracil-Based Bridged Silsesquioxane
FULL PAPER
(s, 1H), 8.09 (d, 1H), 4.37 ppm (s, 2H); HRMS (ESI+): calcd for
C6H6N2O4F: 189.0312 [M+H+]; found: 189.0313.
Characterizations: Discontinuous nitrogen sorption isotherms were meas-
ured at 77.15 K using a Micromeritics ASAP 2010. The samples were de-
gassed at 608C under vacuum for 12 h before the measurements. The
Brunauer–Emmet–Teller (BET) method was used to calculate the specif-
ic surface area. Apparatus ESI high-resolution mass spectra (Q-TOF
ES+) were obtained by infusing samples into a JEOL MS-DX 300 mass
spectrometer. FTIR spectroscopy was carried out using a Perkin–Elmer
FTIR system Spectrum BX spectrophotometer. Solid-state NMR spectra
were obtained on a Bruker FT-AM 400 spectrometer by using magic-
angle spinning mode (MAS) for 1H and CP(cross-polarization)-MAS for
13C and 29Si. Liquid NMR spectra were recorded on a Bruker AC-400
spectrometer. 1H NMR and 13C NMR analysis was performed using
CDCl3 or [D6]DMSO as solvent at room temperature. The chemical
shifts are expressed in ppm relative to TMS (0 ppm). 1H liquid NMR in-
vestigations of the H-bonding were performed in [D6]DMSO with similar
concentration of P1 as used for the hybrids BS1–3 synthesis. The melting
points were determined on Bꢃchi Melting Point B-540 and are uncorrect-
ed.
Compound 2: Compound 1 (0.5 g, 2.660 mmol) was suspended in freshly
distilled SOCl2 (7 mL) at 708C under N2. After 10 h, the excess of SOCl2
was removed under vacuum and the residue was dissolved in dry THF
(10 mL). After cooling to 08C, 2,2-dimethylpropane-1,3-diol (87.5 mg,
0.841 mmol) was added. The mixture was stirred at room temperature for
10 h and was then filtered on Celite. The solvent was removed under
vacuum and the crude product was recrystallized (cyclohexane/dichloro-
methane: 25:75). Yield: 75%; M.p. decomposition at 1808C; 1H NMR
(400 MHz, [D6]DMSO): d=11.99 (s, 2H), 8.07 (d, 2H), 4.50 (s, 4H), 3.92
(s, 4H), 0.88 ppm (s, 6H); 13C NMR (400 MHz, [D6]DMSO): d=20.9,
34.7, 48.7, 69.4, 130.4, 138.2, 140.5, 149.6, 157.2, 167.6 ppm; HRMS
(ESI+): calcd for C17H19N4O8F2; 445.1171 [M+H+]; found: 445.1189.
Compound 3: Compound 1 (0.5 g, 2.660 mmol) was suspended in freshly
distilled SOCl2 (7 mL) at 708C under N2. After 10 h, excess of SOCl2 was
removed under vacuum and the residue was dissolved in dry THF
(10 mL). After cooling to 08C, 1,1,1-tri(hydroxymethyl)propane (80 mg,
0.597 mmol) was added. The mixture was stirred at room temperature
overnight. The solvent was then removed under vacuum and the residue
was purified by column chromatography (dichloromethane/methanol:
98:2!80:20). Yield: 60%; m.p. decomposition after 1808C; 1H NMR:
(400 MHz, [D6]DMSO): d=12.03 (s, 3H), 8.03 (d, 3H), 4.49 (s, 6H), 4.04
(s, 6H), 1.36 (q, 2H), 0.79 ppm (t, 3H); 13C NMR (400 MHz,
[D6]DMSO): d=20.9, 34.7, 48.7, 63.4, 130.1, 138.1, 140.5, 149.6, 157.2,
167.6 ppm; HRMS (ESI+): calcd for C24H24N6O12F3: 645.1404 [M+H+];
found: 645.1412.
Prodrug release: The release profile was determined by suspending dried
samples of the prodrug hybrid carrier (50 mg) in 100 mL of a phosphate
buffer solution (PBS) at various pH (pH 7.4, 5, and 3). The solutions
were kept at 378C with constant stirring at 250 rpm. Aliquots (2 mL) of
the samples were taken at defined intervals, and filtered on 0.2 mm PTFE
membrane. 2 mL of fresh PBS was added for every aliquot taken at the
same pH to maintain the total volume. The release profile was deter-
mined by quantitative HPLC on a Shimadzu model LC 20A. The detec-
tor wavelength was set at 266 nm. Separation was achieved by isocratic
elution with a mobile phase (acetonitrile/ammonium acetate (5 mm aq,
80:20), delivered at a flow-rate of 0.5 mLminꢀ1 at ambient temperature
through a ZIC-HILIC analytical, MERCK column (150 mm lengthꢄ
4.6 mm i.d., 5 mm particle size).
Precursor P1: Compound P1 was prepared according to a reported pro-
cedure.[21] 1H NMR: (400 MHz, CDCl3): d=0.58 (t, 4H), 0.91 (t, 6H),
1.18 (t, 18H), 1.54 (m, 8H), 3.06 (q, 4H), 3.27 (m, 4H), 3.41 (m, 4H),
3.60 (m, 4H), 3.78 ppm (q, 12H); 13C NMR (400 MHz, CDCl3): d= 0.9,
7.5, 11.4, 18.2, 23.0, 23.6, 39.8, 42.5, 42.9, 58.3, 158.8, 165.6 ppm; HRMS
(ESI+): calcd for C33H71N10O8Si2: 791.4995 [M+H+]; found: 791.5006.
Cell culture conditions: Human breast cancer cells (MCF-7) were pur-
chased from ATCC (American Type Culture Collection, Manassas, VA).
MCF-7 cells were cultured in DMEM-F12 culture medium supplemented
with 10% foetal bovine serum and 50 mgmLꢀ1 gentamycin. These cells
were allowed to grow in humidified atmosphere at 378C under 5% CO2.
Hybrid BS0: Compound P1 (500 mg, 0.633 mmol) was completely dis-
solved in DMSO (2 mL) at 508C. After 30 min, water (140 mL) and
NH4F (150 mL, 0.25m solution) were added. A white gel was formed after
1 day and was left under static conditions at room temperature for 3
days. After washing successively with water (50 mL), EtOH (100 mL),
and acetone (50 mL), a white powder was obtained and dried under
vacuum. 29Si CP-MAS solid-state NMR: d=ꢀ57.5 (T2), ꢀ66.5 ppm (T3);
13C CP-MAS solid-state NMR: d=11.8, 23.8, 43.4, 160.4, 166.7 ppm.
Cytotoxicity evaluation: MCF-7 cells were seeded into 96-well plates at
2ꢄ104 cells per well in culture medium (200 mL) and allowed to grow for
24 h. Drug and prodrugs were freshly dissolved in DMSO at a concentra-
tion of 10ꢀ2 m and submitted to an ultrasonic bath until complete dissolu-
tion. Then, the cells were incubated for 72 h with compounds in a range
of concentrations from 5 to 50 mm. Next, a MTT assay was performed to
evaluate the toxicity.[28] Briefly, the cells were incubated for 4 h with
0.5 mgmLꢀ1 of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoli-
um bromide; Promega) in media. The MTT/media solution was then re-
moved and the precipitated crystals were dissolved in EtOH/DMSO
(1:1). The solution absorbance was read at 540 nm.
Hybrid BS1: Compound P1 (500 mg, 0.633 mmol) and 5-FU (82 mg,
0.631 mmol) were completely dissolved in DMSO (2 mL) at 508C. After
30 min, water (140 mL, 7.778 mmol) and NH4F (150 mL, 0.038 mmol,
0.25m solution) were added. A white gel was formed after 1 day and was
left under static conditions at room temperature for 3 days. After wash-
ing successively with water (50 mL), EtOH (100 mL), and acetone
(50 mL), BS1 was obtained as a white powder and dried under vacuum.
29Si CP-MAS solid-state NMR: d=ꢀ57.5 (T2), ꢀ66.5 ppm (T3); 13C CP-
MAS solid-state NMR: d=12.0, 23.8, 43.4, 160.5, 166.9 ppm.
Cell-cycle analysis: Flow cytometric analyses were performed on 350000
cells seeded in culture dishes (60 mm diameter) and allowed to grow for
24 h. The cells were then treated with compounds at 50 mm. 24 h after
treatment, the cells were harvested and fixed with 70% ethanol over-
night. The fixed cells were then incubated with RNase A (1 mgmLꢀ1
)
Hybrid BS2: The same procedure was applied to 2 (140 mg, 0.317 mmol),
P1 (500 mg, 0.633 mmol), water (140 mL, 7.778 mmol), DMSO (2 mL)
and NH4F (150 mL, 0.038 mmol, 0.25m solution). In this case a white gel
was formed after 2 h, yielding BS2 as a white powder.
and propidium iodide (40 mgmLꢀ1) for 48 h, in the dark at 48C. Finally,
the DNA content of the cells was analyzed by using a Cytomics FC 500
flow cytometer with CXP software (Beckman Coulter).
Hybrid BS3: The same procedure was applied to 3 (136 mg, 0.211 mmol),
P1 (500 mg, 0.633 mmol), water (140 mL, 7.778 mmol), DMSO (2 mL)
and NH4F (150 mL, 0.038 mmol, 0.25m solution). In this case a white gel
was formed after 2 h, yielding BS3 as a white powder. 29Si CP-MAS
solid-state NMR: d=ꢀ57.5 (T2), ꢀ66.5 ppm (T3); 13C CP-MAS solid-
state NMR: d=11.8, 24.0, 43.6, 120–150, 160.1, 166.3 ppm.
Acknowledgements
We are grateful to the CNRS and the French Higher Education and Re-
search Ministry. This work was funded with a young researcher grant
from the French National Research agency (ANR-2010-JCJC-1006–01).
Montpellier Rio Imaging platform was used for FACS analyses.
Hybrids BS1A–BS3A: Acidic treatments at pH 2 were realized on BS1–
BS3. BS1–BS3 (20 mg) was suspended in ethanol (50 mL) and concen-
trated HCl (42.5 mL 0.500 mmol) was added. The suspension was stirred
at room temperature for 24 h. After filtering and washing with ethanol
(20 mL), the solid was suspended in Et3N (5 mL) and stirred at room
temperature for 2 h. The suspension was filtered, washed with ethanol
(20 mL) and the hybrid products BS1A–BS3A were dried under vacuum.
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Received: March 20, 2013
Published online: August 8, 2013
12814
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Chem. Eur. J. 2013, 19, 12806 – 12814