Periodic mesoporous organosilica nanoparticles with BOC group, towards HIFU responsive agents

Article abstract of DOI:10.3390/molecules25040974

Periodic Mesoporous Organosilica Nanoparticles (PMONPs) are nanoparticles of high interest for nanomedicine applications. These nanoparticles are not composed of silica (SiO₂). They belong to hybrid organic-inorganic systems. We considered using these nanoparticles for CO₂ release as a contrast agent for High Intensity Focused Ultrasounds (HIFU). Three molecules (P1-P3) possessing two to four triethoxysilyl groups were synthesized through click chemistry. These molecules possess a tert-butoxycarbonyl (BOC) group whose cleavage in water at 90-100 °C releases CO₂. Bis(triethoxysilyl)ethylene E was mixed with the molecules Pn (or not for P3) at a proportion of 90/10 to 75/25, and the polymerization triggered by the sol-gel procedure led to PMONPs. PMONPs were characterized by different techniques, and nanorods of 200-300 nm were obtained. These nanorods were porous at a proportion of 90/10, but non-porous at 75/25. Alternatively, molecules P3 alone led to mesoporous nanoparticles of 100 nm diameter. The BOC group was stable, but it was cleaved at pH 1 in boiling water. Molecules possessing a BOC group were successfully used for the preparation of nanoparticles for CO₂ release. The BOC group was stable and we did not observe release of CO₂ under HIFU at lysosomal pH of 5.5. The pH needed to be adjusted to 1 in boiling water to cleave the BOC group. Nevertheless, the concept is interesting for HIFU theranostic agents.

Full text of DOI:10.3390/molecules25040974

molecules
Article
Periodic Mesoporous Organosilica Nanoparticles
with BOC Group, towards HIFU Responsive Agents
Hao Li 1,2, Carolina Gascó 2, Anthony Delalande 3, Clarence Charnay 1 , Laurence Raehm 1,
Patrick Midoux 3, Chantal Pichon 3, Roser Pleixats 2,* and Jean-Olivier Durand 1,
*
1
ICGM, Univ Montpellier, CNRS, case 1701, Place Eugène Bataillon, CEDEX 05, 34095 Montpellier, France;
Hao.Li@uab.cat (H.L.); clarence.charnay@umontpellier.fr (C.C.); laurence.raehm@umontpellier.fr (L.R.)
Departament de Química and Centro de Innovación en Química Avanzada (ORFEO-CINQA),
Facultat de Ciències, Universitat Autònoma de Barcelona, UAB Campus, C/dels Til.lers,
08193 Cerdanyola del Vallès (Barcelona), Spain; Carolina.Gasco@uab.cat
2
3
Center for Molecular Biophysics, (CBM, UPR 4301), Rue Charles Sadron, 45071 Orléans, France;
anthony.delalande@cnrs-orleans.fr (A.D.); patrick.midoux@cnrs-orleans.fr (P.M.);
chantal.pichon@cnrs-orleans.fr (C.P.)
*
Correspondence: roser.pleixats@uab.cat (R.P.); jean-olivier.durand@umontpellier.fr (J.O.-D.);
Tel.: +33-4-67-14-45-01 (J.O.-D.)
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
Academic Editors: Ahmad Mehdi and Sébastien Clément
Received: 29 January 2020; Accepted: 20 February 2020; Published: 21 February 2020
ꢀꢁꢂꢃꢄꢅꢆ
Abstract:PeriodicMesoporousOrganosilicaNanoparticles(PMONPs)arenanoparticlesofhighinterestfor
nanomedicine applications. These nanoparticles are not composed of silica (SiO2). They belong to hybrid
organic–inorganic systems. We considered using these nanoparticles for CO2 release as a contrast agent for
High Intensity Focused Ultrasounds (HIFU). Three molecules (P1
groups were synthesized through click chemistry. These molecules possess a tert-butoxycarbonyl (BOC)
group whose cleavage in water at 90–100 ◦C releases CO2. Bis(triethoxysilyl)ethylene
was mixed with
–P3) possessing two to four triethoxysilyl
E
the molecules Pn (or not for P3) at a proportion of 90/10 to 75/25, and the polymerization triggered by
the sol-gel procedure led to PMONPs. PMONPs were characterized by different techniques, and nanorods
of 200–300 nm were obtained. These nanorods were porous at a proportion of 90/10, but non-porous
at 75/25. Alternatively, molecules P3 alone led to mesoporous nanoparticles of 100 nm diameter. The BOC
group was stable, but it was cleaved at pH 1 in boiling water. Molecules possessing a BOC group
were successfully used for the preparation of nanoparticles for CO2 release. The BOC group was stable
and we did not observe release of CO2 under HIFU at lysosomal pH of 5.5. The pH needed to be
adjusted to 1 in boiling water to cleave the BOC group. Nevertheless, the concept is interesting for HIFU
theranostic agents.
Keywords: HIFU; periodic mesoporous organosilica nanoparticles; cancer; CO2
1. Introduction
The use of Periodic Mesoporous Organosilica Nanoparticles (PMONPs) has grown a lot in
the last decade, particularly for biological applications, and the field has been recently reviewed [
Hollow Mesoporous Organosilica Nanoparticles have been more recently described and reviewed
as well [ ]. PMONPs are synthesized from trialkoxypolysilylated organic precursors and usually
1–3].
4
silica sources are not used for their preparation. PMONPs possess improved properties compared to
Mesoporous Silica Nanoparticles (MSN), which have been considerably reported for their nanomedicine
applications [
and present better hemolytic properties than MSN [13
5
–
12]. Indeed, PMONPs can be loaded with hydrophobic or hydrophilic molecules
14]. PMONPs are more stable than MSN in
,
medical relevant conditions [15], but, with disulfide bridges in their structure, their degradability can
be triggered by glutathione [16].
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Functionalized MSN [17
,
18] and PMONPs [19
–
22] were combined with high-intensity focused
ultrasound (HIFU) for theranostic applications of cancer. Indeed, HIFU has been used for the ablation
of pathological lesions and tumors [23 24]. Bubbles are usually the most developed contrast agent
for HIFU [25 27]. Furthermore, recent efforts have been dedicated to the preparation of carriers
,
–
able to generate CO2 bubbles [28]. In the course of our studies on PMONPs, we were interested in
nanomaterials possessing the tert-butoxycarbonyl (BOC) group. Indeed, BOC, as an amine protective
group, can be quickly removed in water at 90–100 ◦C, generating CO2 [29
,
30]. As HIFU can heat up to
32], we thought of synthesizing PMONPs from polytrialkoxysilylated BOC-functionalized
molecules in order to develop PMONP-based contrast agents for HIFU.
We present here our studies of the syntheses of three bis(triethoxysilylated) P1
and tetra(triethoxysilylated) molecules P2 P3 (Scheme 1) possessing a BOC group, and their reaction
through the sol-gel procedure with bis(triethoxysilyl)ethylene . The materials called E-Pn 90/10
PMONPs or E-Pn 75/25 PMONPs correspond to a sol-gel reaction with 90% molecule and 10%
molecule Pn, or 75% molecule and 25% molecule Pn, respectively. E PMONPs was also synthesized
and corresponds to 100% of molecule . The materials were analyzed with different techniques
and showed mesoporosity at a 10% proportion of P1 P3 molecules. When the proportion increased,
70–80 ◦C [31
,
,
E
E
E
E
–
no more porosity was observed. Pure P3 PMONPs with the tetra(triethoxysilylated) molecule P3 are
also described and showed mesopores of 5.6 nm. PMONPs are, therefore, promising materials for
theranostic applications.
Scheme 1. Proposed bis(triethoxysilylated) and tetra(triethoxysilylated) molecules P1
–P3 for
the preparation of PMONPs.
2. Results and Discussion
We envisaged to synthesize the bis(triethoxysilylated) and tetra(triethoxysilylated) molecules
P1
–
P3 possessing a BOC group through a copper-catalyzed azide-alkyne cycloaddition
reaction (CuAAC) under anhydrous conditions [33], by treatment of the appropriate
dialkyne with 3-azidopropyltriethoxysilane, 3-azido-N,N-bis(triethoxysilylpropyl)propanamine or
2-azido-N,N-bis(triethoxysilylpropyl)ethanamine [34], respectively (Scheme 1).
N-Boc protection of propargyl amine 1 furnished carbamate 2 in 91% yield, which was reacted
with propargyl bromide in the presence of NaH in anhydrous THF to afford dialkyne
3 in quantitative
yield (Scheme 2) [35].
Scheme 2. Synthesis of tert-butyl di(prop-2-yn-1-yl)carbamate.
The bis-silylated precursor P1 was obtained by the reaction of dialkyne
3 with
(3-azidopropyl)triethoxysilane under copper (I) catalysis under anhydrous conditions. First, the copper

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catalyst [CuBr(PPh3)3] was used, but we found that it was difficult to remove it from the crude mixture
by washing with different solvents after the reaction. Then, CuI was chosen as a copper source
and tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine (TBTA) as a ligand that stabilizes Cu(I) ions in
a 1:1 mixture of anhydrous Et3N/anhydrous THF at 50 ◦C overnight (Scheme 3). After the work-up
and purification, the bis(silylated) precursor P1 was obtained at 73% yield. The purification was
performed by washing with hot anhydrous pentane several times. As the product remains partially
dissolved in pentane, the isolated yield is lower.
Scheme 3. Synthesis of bis(silylated) precursor P1.
Similarly, the other two silylated precursors P2 and P3 were obtained, both at 96% yield, by
reacting the corresponding bis(silylated azide) with dialkyne
(Scheme 4).
3 under the same reaction conditions
Scheme 4. Synthesis of silylated precursors P2 and P3.
With those precursors in hands, we next performed the sol-gel procedure in highly diluted
conditions with 1,2-bis(triethoxysilyl)ethene (E) as the major reagent. First, the synthesis was performed
in Mili-Q water with cetyltrimethylammonium bromide (CTAB) micellar template and sodium
hydroxide catalysis at 80 ◦C. The template was prepared at this temperature for 50 min with a stirring
speed adjusted at 1400 rpm. Then, 1,2-bis(triethoxysilyl)ethene (in a proportion of 90% or 75%)
was added with P1–P3 (in a proportion of 10% or 25%) and the mixture reacted for two hours.
Nanoparticles were collected by centrifugation and the template was removed by washing with
an ethanolic solution of ammonium nitrate; the resulting material was washed successively with ethanol,
Mili-Q water and ethanol. Herein, two different ratios of E-Pn were tested—90/10, 75/25—in order to
study their influence on the NPs’ size and morphology.
All the mixed periodic mesoporous organosilica nanoparticles were characterized by TEM,
nitrogen-sorption measurements (BET), p-XRD, FTIR, 13C-CP MAS solid-state NMR, DLS, zeta-potential,
TGA and DSC. Some physical data are given in Table 1.

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Table 1. Some physical data of E-Pn PMONPs and P3 PMONPs.
N2-Sorption Measurements
Zêta Potential
Particles
Material
TGA d
Vpore
Size (nm) c
SBET
(nm) b
pH
(mV)
∅
pore
(m2·g−1
)
a
(cm3·g−1
)
E-P1 90/10 PMONPs
E-P2 90/10 PMONPs
E-P3 90/10 PMONPs
E-P1 75/25 PMONPs
E-P2 75/25 PMONPs
E-P3 75/25 PMONPs
P3 PMONPs
683
509
505
low
low
low
186
0.48
0.34
0.36
nd
2.8
447
800
479
507
513
321
269
6.96
5.92
5.44
7.34
6.25
4.86
8.94
−28
32
36
78%
75%
72%
67%
58%
57%
nd e
2.7
2.8
nd
nd
nd
5.6
−22
nd
nd
0.26
20
24
11
a
b
c
Determined from the uptake at saturation at p/p◦ = 0.98. Diameter determined by BJH. Hydrodynamic
diameters from DLS. d Residual mass, TGA with a heating rate of 10 ◦C/min from 30 to 1000 ◦C. e Not determined.
The morphologies of E-Pn were first analyzed by TEM (Figure 1), and for the majority of
the materials rodlike-shape nanoparticles were obtained ranging from 200 nm for (E-P1, E-P3 90/10)
to 300 nm long for E-P2. For E-P3 75/25, nanoparticles of 150 nm were observed. The hydrodynamic
diameters of the nanorods were investigated by DLS, (Table 1) and the obtained diameters were
in agreement with the sizes determined by TEM. E-P2 showed higher hydrodynamic diameters
than E-P1 and E-P3. The hydrodynamic diameter of E-P3 75/25 was lower than (E-P1, E-P2, E-P3
90/10) in agreement with a smaller size and particle morphology compared to nanorods. The zeta
potentials were determined in water (Table 1). E-P1 showed negative zeta potential, in agreement with
the presence of Si-O− at the surface of the nanorods. The zeta potential of E-P2 and E-P3 was positive
because the amino groups of P2 and P3 were protonated below pH 7.
Figure 1. TEM images of PMONPs (E-P1; E-P2; E-P3, proportion 90/10 and 75/25).

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The N2 adsorption-desorption isotherms were next examined (Figure 2). All the materials
showed an isotherm between type I and type IV, and between microporous and mesoporous systems.
Nanorods from
with an average small pore size of 2.5 nm. When incorporating P1
specific surface area to 683 m2 g−1 and a small increase in pore size to 2.8 nm. The incorporation of P2
and P3 at a proportion of 90/10 led to a decrease in specific surface area to 500 m2 g−1 with an increase
E
(already synthesized) [36] showed a very high specific surface area of 800 m2
·
g−1
,
E-P1 90/10 showed a decrease in
·
·
of pore size to 2.7 and 2.8 nm, respectively. Note that when increasing the proportion of precursors
P1-3 to 75/25, materials E-P1-3, showed no adsorption of N2 any more at 78 K, which suggests that
the porosity is not accessible to N2 at this temperature.
Figure 2. N2 adsorption-desorption experiments (BET) of E, E-P1, E-P2, E-P3, at a proportion of 90/10.
Small Angle X-rays diffraction of nanorods from
E
[
36] (Figure 3) showed the d(1,0,0) Bragg peak
, characteristics of a well-ordered hexagonal
at 2.2 , with the satellites at d(1,1,0) 4.0 and d(2,0,0) 4.6 θ
θ
θ
mesoporosity. The other materials are more disordered with a wormlike mesoporosity and a broad
non-structured band (see XRD of E-P1 90/10 and E-P1 75/25 as representative examples).
FTIR were recorded for all the precursors and materials. A representative example is given
in Figure 4 with P2
,
E-P2 90/10 and E-P2 75/25
CH3), the carbon chains at 2927 and 2883 cm−1
C=O), the triazole
CH at 1457–1386 cm−1, the silicon–oxygen bonds at 1073 cm−1
.
P2 showed the presence of the BOC group
at 2973 cm−1
at 1696 cm−1
(ν
(ν
CH2), the carbonyl of the carbamate
(
ν
ν
(νSi-O). All the bands were essentially the same in the materials, with a shift in the carbonyl to
1675–1672 cm−1 showing a different environment of the BOC group in the material. A broad band
between 1200–1000 cm−1 ((νSi-O-Si), characteristic of a well-condensed material, was observed.
13C-CPMAS solid-state, NMR were also recorded. Spectrum (E-P2 75/25) is representative
(Figure 5) and showed the characteristic signals (in ppm) at 155.9 (C=O), 145.3 (C=C, Cq triazole), 124.0
(CH triazole), 80.4 (Cq, tBu), 55.2 (CH2-N, CH2-triazole), 28.6 (CH3tBu) 21.6(-CH2-), 11.2 (CH2-Si).

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Figure 3. Small angle XRD diagram of E (blue line), E-P1 90/10 (red line), E-P1 75/25 (green line).
Figure 4. FTIR of (A) P2, (B) E-P2 90/10 and (C) E-P2 75/25.
TGA analyse◦s were performed (Figure 6). The first loss of mass corresponded to water (up to 120 ◦C).
From 150 to 220 C, the loss of the BOC group was observed. The BOC group was very stable in
the materials in dry state, as no loss of mass was observed before 120 ◦C. Then, the decomposition of
the organic part of the materials was shown.

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Figure 5. 13C-CP-MAS solid-state NMR of E-P2 75/25.
Figure 6. TGA-DSC of (A) E-P1 90/10. (B) E-P1 75/25. (C) E-P2 75/25, (D) E-P3 75/25.
We then performed the synthesis of P3 PMONPs. For this, P3 was used as the only precursor
(no silica source). CTAB was dissolved in aqueous NaOH at 80 ◦C in highly diluted conditions for
50 min, then the precursor P3, dissolved in EtOH, was rapidly added. The condensation mixture was
stirred at 80 ◦C for 24 h, then extraction of CTAB and drying of the sample was performed as above.
The nanoparticles were analyzed using different techniques.
First, TEM images (Figure 7) showed spherical monodisperse nanoparticles (100 nm diameter)
which seem to be composed of sheets of organosilica. The nanoparticles are clearly porous on TEM

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images. The porosity was confirmed with N2 sorption experiments (BET). Indeed, a type IV isotherm
was obtained (Figure 8) and the porosity was determined to be 5.6 nm. The hydrodynamic diameter
was observed at 269 nm and zeta potential was positive, in agreement with the protonation of tertiary
nitrogen atoms (Table 1). The precursor P3 was not damaged after reaction, as shown by FTIR
spectroscopy (Figure 9) with the carbonyl group observed at 1682 cm−1
.
Figure 7.
TEM of P3 Periodic Mesoporous Organosilica Nanoparticles (PMONPs)
at different magnifications.
Figure 8. N2 adsorption–desorption experiments (BET) of P3 PMONPs.
Figure 9. FTIR of P3 PMONPs.

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E-P3 75/25 PMONPs were selected for the release of CO2, first under water conditions at 100 ◦C.
A total of 50 mg of E-P3 75/25 PMONPs were added to 10 mL of distilled water, then the mixture was
quickly heated to 100 ◦C and stirred at this temperature for 30 min. The experiment was performed
at two different pH: 7 and 1. At pH 7, we did not observe any bubbles; at pH 1 we could clearly see
a lot of bubbles in the mixture (Figure 10). Then, E-P3 75/25 PMONPs were collected by centrifugation
and washed with EtOH. Finally, E-P3 75/25 PMONPs were dried for FTIR analyses. At pH 7, the BOC
group was still present, but at pH 1 the absorption of tBu (2987 cm−1) and C=O (1680 cm−1) disappeared
in the spectrum (Figure 11). These results confirmed the successful release of CO2 in pH 1 water
at 100 ◦C.
Figure 10. Images of E-P3 75/25 in water at 100 ◦C and pH 1 (A) at 5 min and (B) at 30 min.
Figure 11. FTIR of E-P3 75/25 (A) before treatment, (B) after heating in distilled water, (C) after heating
in water at pH = 1.
We then tried to carry out BOC removal under HIFU conditions. The different nanoparticles were
suspended in water at a concentration of 10 mg/mL and submitted to HIFU, but due to the stability of
BOC group in the materials, we did not observe any cleavage of the BOC group (data not shown).
3. Materials and Methods
3.1. General Remarks
The 1H- and 13C-NMR spectra in solution were recorded on Bruker DPX-360 MHz or Avance-II
600 MHz (Bruker Biospin, Rheinstetten, Germany) and are referenced to solvent signals (CDCl3:
δ
= 7.26 ppm). All the spectra were calibrated using the residual solvent signal (CHCl3, δH, 7.26 and δC,
77.16 ppm). Chemical shift data are expressed in ppm and coupling constant (J) values in Hz.
The multiplicity of peaks is abbreviated as s (singlet), d (doublet), t (triplet), q (quartet) and dd
(doublet of doublets). These NMR instruments belong to the Servei de Resson
the Universitat Aut
noma de Barcelona (UAB). The 13C-CP-MAS solid state NMR spectra were obtained
from a Varian VNMRS 300 MHz instrument (Les Ulis, France) which belongs to the University of
ància Magnètica Nuclear of
ò

Molecules 2020, 25, 974
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Montpellier; the repetition time was 5 s with contact times of five milliseconds. From the Servei
d’An lisi Qu mica of the UAB, the following experimental data were acquired: infra-red spectra (IR),
à
í
mass-spectrometry (MS) and elemental analysis. IR spectroscopy was recorded with a Bruker Tensor
27 spectrometer (Bruker Biosciences Española, Madrid, Spain) using a Golden Gate ATR module
with a diamond window. Low- and high-resolution mass spectra were obtained by direct injection of
the sample with electrospray techniques in a Hewlett-Packard 5989A and microTOF-Q instruments
(Bruker Biosciences Española, Madrid, Spain), respectively. Elemental analysis of C, N and H were
performed using Flash 2000 Organic Elemental analyser (Barcelona, Spain) of Thermo Fisher Scientific
with BBOT as an internal standard. Transmission Electron Microscopy (TEM) was performed on
JEM-2011 Electron Microscope 200 Kv (JEOL Ltd., Akishima, Tokyo, Japan), which belongs to the Servei
de Microscòpia of UAB. Powder X-ray diffraction (P-XRD) was performed in the Servei de Difracció
de Raigs X of UAB with X’Pert Power from PANalytical (Almelo, The Netherlands) 45 Kv/40 mA,
Kα 1.5419 Å with a cooper anode. Dynamic light scattering (DLS) and Zeta potential were obtained
from University of Montpellier. DLS analyses were performed using a Cordouan Technologies DL
135 (Pessac, France) particle size analyzer instrument at University of Montpellier. Zeta potential
measurements were performed on MALVERN Instruments ZETA SIZER Nano series (Orsay, France)
with 1–3 mg samples dispersed in 5 mL of aqueous solution (distilled water or 0.01 M NaCl solution)
between pH 4 and 9. The surface areas were determined by the Brunauer-Emmet-Teller (BET) method
from N2 adsorption–desorption isotherms obtained with a Micromeritics ASAP2020 analyzer (Mérignac,
France) which belongs to the University of Montpellier after degassing samples for 12 h at 80 ◦C under
vacuum. The total pore volumes were evaluated by converting the volume adsorbed at p/po 0.98 to
the volume of liquid adsorbed (single point adsorption total pore volume of pores less than 4000 Å
at p/p◦
≈
0.98). The pore size distributions for PMONPs were determined from the desorption branch
using the Barrett–Joyner–Halenda (BJH) method, which relies on the Kelvin equation to relate the width
of the pores to the condensation pressure. When required, experiments were carried out with standard
high vacuum and Schlenk techniques. Chromatographic purifications were performed under N2
pressure using 230–400 mesh silica gel (flash chromatography). Dry solvents and reagents were obtained
following standard procedures: triethylamine was distilled over CaH2; DMF, CH3CN and pyridine
were dried by molecular sieves, THF, CH2Cl2 and pentane were from solvent processing equipment
(PureSolv, Innovative Technology, Newburyport, MA, USA). Cetyltrimethylammonium bromide
(CTAB), sodium hydroxide, ammonium nitrate (NH4NO3), and potassium bromide were purchased
from Sigma-Aldrich. Absolute ethanol was purchased from Fisher Chemicals and hydrochloric acid
from VWR PROLABO.
3.2. Synthesis of P1
To a dry 100 mL Schlenk flask equipped with a stir bar and under Argon atmosphere, CuI (7.60 mg,
0.04 mmol), TBTA (21.20 mg, 0.04 mmol) and anhydrous THF (20 mL) were added. The resulting
mixture was stirred for 30 min, then (3-azidopropyl)triethoxysilane (988.5 mg, 4.0 mmol), tert-butyl
di(prop-2-yn-1-yl)carbamate (463.46 mg, 2.4 mmol) and anhydrous Et3N (1.0 mL, 0.73 g/mL 7.2 mmol)
◦
were added by using a syringe. The resulting mixture was stirred at 50 C (Argon atmosphere)
until tert-butyl di(prop-2-yn-1-yl)carbamate was fully consumed (16 h, reaction monitored by TLC). Then,
the solvent was evaporated under reduced pressure and the residue was washed with cold anhydrous
pentane to remove some remaining azide. Hot anhydrous pentane was added to the insoluble fraction to
extract the disilylated product; this digestion with hot pentane was repeated several times and the pentane
extracts were concentrated under reduced pressure to provide the pure product as a colorless oil (999 mg,
1
73% yield). H-NMR (360 MHz, CDCl3)
δ
7.53 (s, 1H), 7.42 (s, 1H), 4.51 (s, 4H), 4.29 (t, J = 7.2 Hz, 4H),
3.78 (q, J = 7.2 Hz, 12H), 2.00–1.96 (m, 4H), 1.42 (s, 9H), 1.17 (t, J = 7.2 Hz, 18H), 0.56 (t, J = 7.2 Hz, 4H);
13C-NMR (91 MHz, CDCl3)
δ 155.2, 122.7, 121.9, 80.3, 58.5, 58.4, 52.4, 28.4, 24.2, 18.2, 7.4. IR (film) 3132.4,
2973.6, 2927.2, 1692.6, 1390.2, 1163.5, 1073.1, 954.5, 783.2 cm−1. MS (ESI) m/z: 688.4, 632.3, 588.3, 558.3;
HRMS (ESI) m/z [M + Na]+ calcd. for C29H57N7O8Si2Na: 688.3880, found: 688.3877.

Molecules 2020, 25, 974
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3.3. Synthesis of P2
To a dry, 100 mL Schlenk flask equipped with a stir bar and under Argon atmosphere,
CuI (7.60 mg, 0.04 mmol), TBTA (21.20 mg, 0.04 mmol) and anhydrous THF (20 mL) were
added. The resulting mixture was stirred for 30 min, and then N-(2-azidoethyl)-3-(triethoxysilyl)
-N-(3-(triethoxysilyl)propyl)propan-1-amine (1.98 g, 4.0 mmol), tert-butyl di(prop-2-yn-1-yl)carbamate
(463.46 mg, 2.4 mmol) and anhydrous Et3N (1.0 mL, 0.73 g/mL, 7.2 mmol) were added by
◦
using a syringe. The resulting mixture was stirred at 50 C (Argon atmosphere) until tert-butyl
di(prop-2-yn-1-yl)carbamate was fully consumed (20 h, reaction monitored by TLC). Then, the solvent
was partially evaporated under reduced pressure, the remaining solution was filtered through
Whatman® membrane filters PTFE (pore size 0.2
at reduced pressure to afford the final product as colorless oil (2.27 g, 96% yield). H-NMR (360 MHz,
µ
m, diam. 47 mm) and the filtrate evaporated
1
CDCl3)
δ 7.61 (s, 1H), 7.57 (s, 1H), 4.51 (s, 4H), 4.36 (t, J = 7.2 Hz, 4H), 3.80 (q, J = 7.2 Hz, 24H), 2.88
(t, J = 7.2 Hz, 4H), 2.47 (t, J = 7.2 Hz, 8H), 1.53–1.45 (m, 17H), 1.20 (t, J = 7.2 Hz, 36H), 0.54 (t, J = 7.2 Hz,
8H); 13C-NMR (91 MHz, CDCl3)
δ 155.1, 128.9, 127.9, 80.1, 58.2, 57.0, 54.1, 48.8, 28.3, 28.1, 20.2, 18.1, 7.7.
IR (film): 2972.4, 2927.2, 2882.9, 1695.7, 1457.0, 1389.6, 1164.1, 1073.0, 952.5, 770.1 cm−1. MS (ESI) m/z:
1204.7, 1182.7, 688.4, 591.9, 413.3; HRMS (ESI) m/z [M + Na]+ calcd. for C51H107N9O14Si4Na: 1204.6906,
found: 1204.6894.
3.4. Synthesis of P3
To a dry, 100 mL Schlenk flask equipped with a stir bar and under Argon atmosphere, CuI (7.60 mg,
0.04 mmol), TBTA (21.20 mg, 0.04 mmol) and anhydrous THF (20 mL) were added. The resulting
mixture was stirred for 30 min, then 3-azido-N,N-bis(3-(triethoxysilyl)propyl)propan-1-amine (2.03 g,
4.0 mmol), tert-butyl di(prop-2-yn-1-yl)carbamate (463.46 mg, 2.4 mmol) and anhydrous Et3N (1.0 mL,
◦
0.73 g/mL, 7.2 mmol) were added by using a syringe. The resulting mixture was stirred at 50 C
(Argon atmosphere) until tert-butyl di(prop-2-yn-1-yl)carbamate was fully consumed (20 h, reaction
monitored by TLC). Then, the solvent was partially evaporated under reduced pressure, the remaining
solution was filtered through Whatman® membrane filters PTFE (pore size 0.2
µm, diam. 47 mm)
and the filtrate was evaporated at reduced pressure to afford the final product as colorless oil (2.33
1
g, 96% yield). H-NMR (360 MHz, CDCl3)
δ
7.55 (s, 1H), 7.47 (s, 1H), 4.53 (s, 4H), 4.36 (t, J = 7.2 Hz,
4H), 3.81 (q, J = 7.2 Hz, 24H), 2.48–2.36 (m, 12H), 2.01 (t, J = 7.2 Hz, 4H), 1.52–1.46 (m, 17H), 1.21
(t, J = 7.2 Hz, 36H), 0.57 (t, J = 7.2 Hz, 8H); 13C-NMR (91 MHz, CDCl3)
δ
155.2, 129.1, 128.0, 80.3, 58.3,
57.1, 56.8, 50.9, 48.5, 28.4, 26.8, 20.1, 18.3, 7.9. IR (film) 2972.7, 2926.8, 2883.2, 1695.4, 1455.6, 1389.7,
1164.0, 1072.8, 952.5, 770.3 cm−1. MS (ESI) m/z: 1232.7, 1210.7, 816.5, 702.4, 605.9, 509.3, 438.3; HRMS
(ESI) m/z [M + Na]+ calcd. for C53H111N9O14Si4Na: 1232.7220, found: 1232.7195.
3.5. Synthesis of E-BTSE
Grubbs Catalyst™ 1st Generation C823 (PCy3)2Cl2Ru=CHPh (82.30 mg, 0.1 mmol)
and vinyltriethoxysilane (VTES) (19.02 g, 100 mmol) were added to a Schlenk flask under argon.
After the solution was stirred for one hour and the mixture was refluxed for 24 h, unreacted VTES
was distilled off. Subsequently, BTSE was vacuum distilled to give a clear colorless liquid (12.53
◦
1
g, 71% yield, 4 mbar, 124 C). E-BTSE was identified by H-NMR as a diastereoisomerically pure
product (100% E). 1H-NMR (360 MHz, CDCl3)
(t, J = 7.2 Hz, 18 H).
δ 6.66 (s, 2 H; E-isomer), 3.83 (q, J = 7.2 Hz, 12 H), 1.23
3.6. Preparation of E-BTSE PMONPs
In an open 250 mL round-bottom flask without reflux condenser, a solution of
cetyltrimethylammonium bromide (CTAB, 250 mg, 0.686 mmol) was placed in Mili-Q water (120 mL)
and then 875
µL of 2 M NaOH was added (1.75 mmol of NaOH). The mixture was stirred at 1000 rpm
at 80 ◦C for 50 min. Then, the stirring speed was enhanced to 1400 rpm and 1,2-bis(triethoxysilyl)ethene

Molecules 2020, 25, 974
12 of 16
(BTSE, 800
for two hours at 80 ◦C. The suspension was cooled to room temperature while stirring and the NPs
were collected by centrifugation (13,500 rpm in 6 25 mL flasks tubes for 45 min). The supernatant was
µL, 2.29 mmol) was added rapidly under stirring. The condensation process was conducted
×
removed and an aliquot of the solid was taken and dried for FTIR. In order to remove the surfactant,
10 mL of an alcoholic solution of ammonium nitrate [NH4NO3, 6 g/L in 96% EtOH] was added to
◦
each tube. The six tubes were sonicated for 30 min at 50 C, then cooled and centrifuged (30 min
at 13,500 rpm at 25 ◦C), the supernatant was discarded. This NH4NO3 washing was performed three
times. Each solid in the tubes was washed successively with 96% ethanol, Mili-Q water, 96% ethanol
using the same protocol (30 min at 50 ◦C sonication, centrifugation). The final product was dried
for few hours under vacuum at room temperature. The E PMONPs were obtained as a white solid.
At the same time, another aliquot of the solid was taken and dried for FTIR. It was compared to the IR
of the sample before removal of the surfactant and after removal to check whether the CH2 bands of
CTAB had completely disappeared.
3.7. Preparation of E-Pn 90/10 PMONPs
In an open 250 mL round-bottom flask without reflux condenser, a solution of CTAB (250 mg,
0.686 mmol) was placed in Mili-Q water (120 mL) and then 875 µL of 2 M NaOH was added (1.75 mmol
of NaOH). The mixture was stirred at 1000 rpm at 80 ◦C for 50 min. Then, the stirring speed was
enhanced to 1400 rpm and a mixture of 100% E-BTSE (704.34 mg, 2.00 mmol) with Pn (0.2 mmol)
were added rapidly under stirring. The condensation process was conducted for two hours at 80 ◦C.
Afterwards, the suspension was cooled to room temperature while stirring and the NPs were collected
by centrifugation during 45 min at 13,500 rpm. The samples were then extracted three times with
a solution of NH4NO3 (6 g/L in 96% EtOH), and washed three times with 96% ethanol, Mili-Q
water, 96% ethanol, respectively. Extraction and the following steps were identical to those described
for E PMONPs. The E-Pn 90/10 PMONPs were obtained as a white solid. E-P1 90/10 PMONPs
13C-CP-MAS NMR (75 MHz)
: 155.6, 146.1, 123.8, 81.1, 57.9, 52.5, 28.2, 24.9, 17.4, 10.4. IR (ATR)
(cm−1): 3337.7, 1671.3, 1416.6, 1187.7, 1036.9, 927.3. BET: SBET = 683 m2 −1, Vpore = 0.48 cm3 g−1
= 2.8 nm. TGA (air, 5 ◦C/min, 20–1000 ◦C) residual mass 78%. Zeta Potential:
28.7 mV,
: 155.4, 146.0, 125.2, 81.0,
: 3322.9, 1673.0, 1412.0, 1187.7, 1036.2, 923.7, 187.9.
:
δ
ν
·
g
·
,
∅
ζ
=
−
pore
pH = 6.99. DLS: 447 nm. E-P2 90/10 PMONPs
58.1, 52.8, 48.2, 28.5, 20.6, 10.8. IR ν (ATR) (cm−1
: δ
13C-CP-MAS NMR (75 MHz)
)
BET: SBET = 509 m2
mass 75%. Zeta Potential:
NMR (75 MHz) : 156.1, 146.1, 124.7, 81.0, 58.0, 48.0, 28.4, 18.5, 10.6. IR
·
g
−1, Vpore = 0.34 cm3
·
g−1
,
∅
= 2.7 nm. TGA (air, 5 ◦C/min, 20–1000 ◦C) residual
pore
ζ
= 32.6 mV, pH = 5.92. DLS: 800 nm. E-P3 90/10 PMONPs
(ATR) (cm−1): 3361.9, 1655.6,
g−1
= 2.8 nm. TGA
:
13C-CP-MAS
δ
ν
1370.1, 1188.0, 1033.4, 924.1, 788.8. BET: SBET = 505 m2
·
g
−1, Vpore = 0.66 cm3
·
,
∅
pore
(air, 5 ◦C/min, 20–1000 ◦C) residual mass 72%. Zeta Potential: ζ = 36.0 mV, pH = 5.44. DLS: 479 nm.
3.8. Preparation of E-Pn 75/25 PMONPs
In an open 250 mL round-bottom flask without reflux condenser a solution of CTAB (250 mg,
0.686 mmol) was placed in Mili-Q water (120 mL) and then 875 µL of 2 M NaOH was added (1.75 mmol
of NaOH). The mixture was stirred at 1000 rpm at 80 ◦C for 50 min. Then, the stirring speed was
enhanced to 1400 rpm and a mixture of 100% E-BTSE (581.09 mg, 1.65 mmol) with Pn (0.55 mmol)
were added rapidly under stirring. The condensation process was conducted for two hours at 80 ◦C.
Afterwards, the suspension was cooled to room temperature while stirring and the NPs were collected
by centrifugation during 45 min at 13,500 rpm. The samples were then extracted three times with
a solution of NH4NO3 (6 g/L in 96% EtOH), and washed three times with 96% ethanol, Mili-Q
water, 96% ethanol, respectively. Extraction and the following steps were identical to those described
for E PMONPs. The E-Pn 75/25 PMONPs were obtained as a white solid. E-P1 75/25 PMONPs
13C-CP-MAS NMR (75 MHz)
: 156.1, 145.5, 123.6, 81.9, 57.7, 52.8, 28.3, 24.8, 17.9, 10.4. IR (ATR)
(cm−1): 3302.3, 1670.8, 1414.8, 1186.9, 1012.0, 926.6, 784.0. TGA (air, 5 ◦C/min, 20–1000 ◦C) residual
mass 67%. Zeta Potential: 22.9 mV, pH = 7.34. DLS: 507 nm. E-P2 75/25 PMONPs
13C-CP-MAS
:
δ
ν
ζ
=
−
:

Molecules 2020, 25, 974
13 of 16
NMR (75 MHz)
δ
: 155.7, 145.5, 125.3, 80.2, 57.8, 28.4, 21.7, 11.1. IR
ν
(ATR) (cm−1): 3331.9, 1672.4,
1412.3, 1186.7, 1011.4, 923.1, 785.6. TGA (air, 5 ◦C/min, 20–1000 ◦C) residual mass 58%. Zeta Potential:
ζ
= 19.9 mV, pH = 6.25. DLS: 513 nm. E-P3 75/25 PMONPs: δ: 1561.2,
13C-CP-MAS NMR (75 MHz)
146.1, 124.8, 81.7, 58.2, 48.8, 28.6, 19.8, 10.3. IR
786.5. TGA (air, 5 ◦C/min, 20–1000 ◦C) residual mass 57%. Zeta Potential:
ν
(ATR) (cm−1): 3294.2, 1676.8, 1388.2, 1012.7, 923.4,
ζ = 24.2 mV, pH = 4.85.
DLS: 321 nm.
3.9. Synthesis of P3 PMONPs
In an open 100 mL round-bottom flask without reflux condenser a solution of CTAB (125 mg,
0.34 mmol) was placed in Mili-Q water (60 mL) and then 437 L of 2 M NaOH was added. The mixture
µ
was stirred at 1000 rpm at 80 ◦C for 50 min. Then, the stirring speed was enhanced to 1400 rpm and P3
(0.1 mmol) in absolute ethanol (1.0 mL) was added rapidly under stirring. The condensation process
was conducted for 24 h at 80 ◦C. Afterwards, the suspension was cooled to room temperature while
stirring and the NPs were collected by centrifugation during 15 min at 20,000 rpm. The samples were
then extracted twice with a solution of NH4NO3 (6 g/L in EtOH), and washed three times with ethanol,
Mili-Q water, ethanol, respectively. Extraction and the following steps were identical to those described
for E PMONPs
.
P3 PMONPs were obtained as a white solid. IR
ν
(ATR) (cm−1): 3395.7, 2936.4, 1682.3,
−1, Vpore = 0.26 cm3 g−1
= 5.64 nm.
1367.4, 1103.1, 1023.6, 783.8, 688.4. BET: SBET = 186 m2
Zeta Potential: ζ = 10.9 mV, pH = 8.94. DLS: 269 nm.
·
g
·
,
∅
pore
3.10. HIFU Stimulation
Nanoparticles were resuspended in PBS at a concentration of 10 mg/mL in an eppendorf tube.
The tube was submitted to an ultrasound field from a focused 1 MHz probe (Precision Acoustics, UK)
driven by a signal generator (Agilent 33220a, Les Ulis, France) and RF power amplifier
(ADECE, Artannes sur Indre, France). The transducer was placed in a custom-made three-axis
motorized positioning system to send ultrasound accurately on the eppendorf tube at the focus of
the probe. Degased water was applied between the transducer and the eppendorf tube. Peak negative
pressures of 1.0, 1.4 and 1.6 MPa in continuous mode were used in this study. Ultrasound stimulation
time was set to 15 s. The transducer was calibrated using an HGL-200 PVDF bullet type hydrophone
(Onda, Sunnyvale, CA, USA), at the focus distance of the transducer.
4. Conclusions
In conclusion, we studied the preparation of nanomaterials possessing a BOC group. Molecules with
BOC and two to four triethoxysilyl groups were successfully synthesized through click chemistry. Then,
the molecules were reacted through the sol gel procedure with bis(triethoxysilyl)ethylene, at a proportion
of 10/90 to 25/75, and the materials were analyzed with different techniques. The nanorods E-P1
,
E-P2 E-P3, at a proportion of 90/10, possessed small wormlike mesopores, while, at a proportion of
,
75/25, the nanomaterials were non-porous at 78 K. The BOC group was not damaged during the sol
gel-procedure, as shown by FTIR and 13C CPMAS solid-state NMR. TGA showed the stability of BOC
group inside the materials, as no decomposition occurred before 120 ◦C. Alternatively, P3 PMONPs
were synthesized from molecule P3 without any other source of silicate. The obtained nanoparticles
were of 100 nm diameter, and were mesoporous with a porosity of 5.6 nm. The BOC was very stable
as we needed to adjust the pH to 1 to cleave the BOC at 100 ◦C. We were not able to observe bubbles
at pH 5.5 under our conditions of HIFU, but the concept is nevertheless promising for future contrast
agents for HIFU. Indeed, the electrophilicity of BOC has been shown to strongly influence the reaction
rate and reactivity of the cleavage. [37] Therefore, the synthesis of precursor molecules possessing more
electrophilic BOC groups can be considered. Another possibility is to introduce carboxylic acid functions
in the PMONPs using triethoxysilanes possessing this function [38] during the sol-gel procedure, in
order to facilitate BOC cleavage in the materials.

Molecules 2020, 25, 974
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Author Contributions:Conceptualization, R.P., P.M., C.P. andJ.-O.D.; methodology, H.L., C.G. andA.D.; formalanalysis,
C.C., R.P. and J.-O.D.; writing—original draft preparation, H.L., J.-O.D. and R.P., writing—review and editing, L.R.;
supervision, R.P., J.-O.D. and C.P. All authors have read and agreed to the published version of the manuscript.
Funding: This research was funded by CNRS (France), Ministerio de Econom
ía, Industria y Competitividad
(MINECO) of Spain (Projects CTQ2014-53662-P and CTQ2016-81797-REDC), Ministerio de Ciencia, Innovaci
ó
n
y Universidades (MCIU) of Spain (Project RTI2018-097853-B-I00 and RED2018-102387-T), DURSI-Generalitat
de Catalunya (Project SGR2017-0465) and China Scholarship Council (CSC) (predoctoral scholarship to Hao Li,
CSC No. 201606890025).
Acknowledgments: The authors are grateful to China Scholarship Council (CSC) for predoctoral scholarships
to H.L.
Conflicts of Interest: The authors declare no conflict of interest.
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2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
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