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.) 2 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 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.) Academic Editors: Ahmad Mehdi and Sébastien Clément   Received: 29 January 2020; Accepted: 20 February 2020; Published: 21 February 2020 Abstract: 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. 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 [1–3]. Hollow Mesoporous Organosilica Nanoparticles have been more recently described and reviewed as well [4]. PMONPs are synthesized from trialkoxypolysilylated organic precursors and usually 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 [5–12]. Indeed, PMONPs can be loaded with hydrophobic or hydrophilic molecules and present better hemolytic properties than MSN [13,14]. 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]. Molecules 2020, 25, 974; doi:10.3390/molecules25040974 www.mdpi.com/journal/molecules degradability can be triggered by glutathione [16]. 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 Molecules 2020, 25, 974 2 of 16 HIFU2020, Molecules [25–27]. 25, 974Furthermore, recent efforts have been dedicated to the preparation of carriers able2to of 16 generate CO 2 bubbles [28]. In the course of our studies on PMONPs, we were interested in molecules and present better hemolytic properties than MSN [13,14]. PMONPs are more stable than nanomaterials MSN in medical possessing relevanttheconditions tert‐butoxycarbonyl [15], but, (BOC) group. Indeed, with disulfide bridgesBOC,in as an amine their protective structure, their Functionalized group, can be can MSN quickly [17,18] removedby and PMONPs inglutathione water at 90–100 [19–22] were combined with high-intensity focused degradability be triggered [16]. °C, generating CO2 [29,30]. As HIFU can heat up ultrasound to 70–80 (HIFU) for MSN °C [31,32], Functionalized theranostic we [17,18] thoughtapplications andofPMONPs of [19–22] synthesizingcancer. Indeed, PMONPs were HIFUpolytrialkoxysilylated from combined has withbeen used for the high‐intensity ablation BOC‐ focused of pathological functionalized lesions and molecules intumors order [23,24]. to develop Bubbles are PMONP‐based usually the contrast most agents developed for ultrasound (HIFU) for theranostic applications of cancer. Indeed, HIFU has been used for the ablation HIFU. contrast agent for of HIFU We present [25–27]. here our Furthermore, studies recent of the efforts syntheses have been of three dedicated bis(triethoxysilylated) to the pathological lesions and tumors [23,24]. Bubbles are usually the most developed contrast agent for preparation P1 of and carriers abletetra(triethoxysilylated) to generate HIFU [25–27]. COFurthermore, molecules 2 bubbles [28]. In recent P2, P3course the efforts (Scheme have 1)dedicated of our been possessing studiestoon a PMONPs, theBOC group,we preparation and their ofwere reaction interested carriers able to in through nanomaterials the sol‐gel generate COpossessing procedure 2 bubbles the with bis(triethoxysilyl)ethylene [28].tert-butoxycarbonyl In the course of our (BOC) group. studies E. The on Indeed, PMONPs, materials BOC, called weaswere an amine E‐Pn 90/10 protective interested in PMONPs group, can beor nanomaterials E‐Pn quickly 75/25 removed possessing PMONPsin watercorrespond at 90–100to (BOC) the tert‐butoxycarbonyl ◦ a sol‐gel C, reaction generating group. CO2with Indeed, 90%as [29,30]. BOC, Asmolecule anHIFU amine Eprotective can and heat10%up to molecule 70–80 canPn, beorwe ◦ C [31,32], group, 75% quickly molecule removed thought E and 25% molecule in water of synthesizing 90–100Pn, atPMONPs respectively. °C,from generating COE PMONPs wasBOC-functionalized 2 [29,30]. As HIFU polytrialkoxysilylated also synthesized can heat up and corresponds to 70–80in °C molecules order to 100% [31,32], of molecule we thought to develop E. The materials of synthesizing PMONP-based were contrast PMONPs analyzed agents forfrom with different techniques HIFU.polytrialkoxysilylated BOC‐ and showed mesoporosity functionalized at a 10% proportion of P1–P3 molecules. When the proportion increased, no We presentmolecules here our in order to develop studies of the PMONP‐based syntheses contrast of threeagents for HIFU. bis(triethoxysilylated) P1 moreWe porosity present was observed. here our Pure studies P3 PMONPs of the with syntheses the tetra(triethoxysilylated) of three molecule bis(triethoxysilylated) P1P3 are and and tetra(triethoxysilylated) molecules P2, P3 (Scheme 1) possessing a BOC group, and their reaction also described and showed tetra(triethoxysilylated) mesopores molecules of 5.6 nm. PMONPs are, therefore, promising materials for through the sol-gel procedure with P2, P3 (Scheme 1) possessing bis(triethoxysilyl)ethylene E.a The BOCmaterials group, and their called reaction E-Pn 90/10 theranostic through theapplications. sol‐gel procedure with bis(triethoxysilyl)ethylene E. The materials called E‐Pn 90/10 PMONPs or E-Pn 75/25 PMONPs correspond to a sol-gel reaction with 90% molecule E and 10% PMONPs or E‐Pn 75/25 PMONPs correspond to a sol‐gel reaction with 90% molecule E and 10% molecule 2. or 75% molecule E and 25% molecule Pn, respectively. E PMONPs was also synthesized Pn, and Results molecule Pn, orDiscussion 75% molecule E and 25% molecule Pn, respectively. E PMONPs was also synthesized and corresponds to 100% of molecule E. The materials were analyzed with different techniques and corresponds We envisaged to to 100% of molecule synthesize E. The materials were analyzed the bis(triethoxysilylated) with different techniques and tetra(triethoxysilylated) and molecules and showed mesoporosity at a 10% proportion of P1–P3 molecules. When the proportion increased, showed mesoporosity P1–P3 possessing at a group a BOC 10% proportion through aofcopper‐catalyzed P1–P3 molecules.azide‐alkyne When the proportion increased, cycloaddition no reaction no more more porosity was porosity was observed. observed.PurePureP3 P3PMONPs PMONPswith withthe thetetra(triethoxysilylated) tetra(triethoxysilylated) molecule molecule P3 P3 are are (CuAAC) under anhydrous conditions [33], by treatment of the appropriate dialkyne with 3‐ alsoalso described and showed mesopores described and showed mesopores of 5.6 nm. PMONPs are, therefore, promising of 5.6 nm. PMONPs are, therefore, promising materials forfor materials azidopropyltriethoxysilane, 3‐azido‐N,N‐bis(triethoxysilylpropyl)propanamine or 2‐azido‐N,N‐ theranostic applications. theranostic applications. bis(triethoxysilylpropyl)ethanamine[34], respectively (Scheme 1). 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‐ n azidopropyltriethoxysilane, 3‐azido‐N,N‐bis(triethoxysilylpropyl)propanamine or 2‐azido‐N,N‐ bis(triethoxysilylpropyl)ethanamine[34], respectively (Scheme 1). Scheme Scheme 1. 1. Proposed Proposed bis(triethoxysilylated) bis(triethoxysilylated)and tetra(triethoxysilylated) and molecules tetra(triethoxysilylated) P1–P3 P1–P3 molecules for thefor preparation of PMONPs. the preparation of PMONPs. n 2. Results andprotection N‐Boc Discussionof propargyl amine 1 furnished carbamate 2 in 91% yield, which was reacted with propargyl bromide We envisaged in thethe to synthesize presence of NaH in anhydrous bis(triethoxysilylated) THF to afford dialkyne and tetra(triethoxysilylated) 3 in molecules quantitative P1–P3 yield (Scheme possessing a BOC2)group [35]. through a copper-catalyzed azide-alkyne cycloaddition reaction (CuAAC) under anhydrous conditions [33], by treatment of the appropriate dialkyneScheme 1. Proposed bis(triethoxysilylated) 3-azido-N,N-bis(triethoxysilylpropyl)propanamine with 3-azidopropyltriethoxysilane, and tetra(triethoxysilylated) molecules P1–P3 for the or preparation of PMONPs. 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 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 with propargyl bromide in the presence of NaH in anhydrous THF to afford dialkyne 3 in yield (Scheme 2) [35]. Scheme 2. Synthesis of tert‐butyl di(prop‐2‐yn‐1‐yl)carbamate. quantitative yield (Scheme 2) [35]. Scheme2.2.Synthesis Scheme Synthesisof oftert-butyl tert‐butyl di(prop‐2‐yn‐1‐yl)carbamate. 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 Molecules Molecules 2020, 2020, 25,25, 974974 3 of 316of 16 Molecules 2020, 25, 974 3 of 16 The bis‐silylated precursor P1 was obtained by the reaction of dialkyne 3 with (3‐ The bis‐silylated precursor P1 was obtained by the reaction of dialkyne 3 with (3‐ azidopropyl)triethoxysilane catalyst [CuBr(PPh3 )3 ] was used, under butcopper we found(I) catalysis under that it was anhydrous difficult conditions. to remove it fromFirst, the copper the crude mixture azidopropyl)triethoxysilane under copper (I) catalysis under anhydrous conditions. First, the copper by catalyst washing [CuBr(PPh with 3)3] was used, but we found that it was difficult to remove it from the crude different solvents after the reaction. Then, CuI was chosen catalyst [CuBr(PPh3)3] was used, but we found that it was difficult to remove it from the crude as a copper source mixture andmixture by washing with different solvents tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine after the reaction. Then, CuI was chosen as a copper by washing with different solvents after the (TBTA) reaction.asThen, a ligand CuIthat wasstabilizes chosen asCu(I) ions in a copper source and tris[(1‐benzyl‐1H‐1,2,3‐triazol‐4‐yl)methyl]amine (TBTA) as a ligand that stabilizes Cu(I) ◦ C overnight a 1:1 mixture of anhydrous Et N/anhydrous THF at source and tris[(1‐benzyl‐1H‐1,2,3‐triazol‐4‐yl)methyl]amine 3 50 (Scheme 3). After the work-up (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 andions purification, the bis(silylated) in a 1:1 mixture of anhydrousprecursor P1 wasTHF Et3N/anhydrous obtained at 50 °Cat overnight 73% yield.(Scheme The purification 3). After thewas work‐up and purification, the bis(silylated) precursor P1 was obtained at 73% yield. The purification performed work‐up by andwashing withthe purification, hotbis(silylated) anhydrous precursor pentane several P1 was times. obtainedAsatthe 73%product remains yield. The partially purification was performed by washing with hot anhydrous pentane several times. As the product remains dissolved in pentane, was performed the isolated by washing withyield hot is lower. pentane several times. As the product remains anhydrous partially dissolved in pentane, the isolated yield is lower. partially dissolved in pentane, the isolated yield is lower. Scheme3.3. Synthesis Scheme of bis(silylated) Synthesis of bis(silylated)precursor precursorP1. P1. Scheme 3. Synthesis of bis(silylated) precursor P1. Similarly, Similarly,the theother othertwo twosilylated silylated precursors andP3 P2 and precursors P2 wereobtained, P3were obtained,both both at at 96%96% yield, yield, by by Similarly, the other two silylated precursors P2 and P3 were obtained, both at 96% yield, by reacting thethe reacting corresponding correspondingbis(silylated bis(silylated azide) with dialkyne azide) with dialkyne33under underthe thesame same reaction reaction conditions conditions reacting the corresponding bis(silylated azide) with dialkyne 3 under the same reaction conditions (Scheme (Scheme 4).4). (Scheme 4). Scheme 4. Synthesis of silylated precursors P2 and P3. Scheme4.4.Synthesis Scheme Synthesis of silylated silylatedprecursors precursorsP2 P2and andP3. P3. With those precursors in hands, we next performed the sol‐gel procedure in highly diluted WithWith those those precursorsininhands, precursors hands, wewe next next performed performed the thesol‐gel sol-gelprocedure procedureinin highly highly diluted diluted conditions with 1,2‐bis(triethoxysilyl)ethene (E) as the major reagent. First, the synthesis was conditions conditions with with 1,2‐bis(triethoxysilyl)ethene (E) as the major reagent. First, the synthesis was performed in 1,2-bis(triethoxysilyl)ethene (E) as the major reagent. Mili‐Q water with cetyltrimethylammonium bromideFirst, (CTAB)the synthesis was performed micellar template and performed in sodium in Mili‐Q Mili-Q hydroxide water with water with cetyltrimethylammonium cetyltrimethylammonium bromide (CTAB) micellar templatesodium and catalysis at 80 °C. The template bromide was prepared (CTAB) at thismicellar template temperature for 50and min with sodium hydroxide hydroxide catalysis ◦ at 80 °C. The template was prepared at this temperature for 50 min with a stirringcatalysis at 80 C. speed adjusted atThe 1400template rpm. Then,was1,2‐bis(triethoxysilyl)ethene prepared at this temperature (in afor 50 min with proportion a stirring of 90% or a stirring speed speed atadjusted at 1400 rpm. Then, 1,2‐bis(triethoxysilyl)ethene a (in a proportion of 90% or or 75%)adjusted was added 1400P1–P3 with rpm. Then, (in 1,2-bis(triethoxysilyl)ethene a proportion of 10% or 25%) and the(in proportion mixture of two reacted for 90% 75%) hours. was75%) addedwas with addedP1–P3 with P1–P3 (in a (in a proportion proportion of of 10% 10% or or 25%)and 25%) and the the mixture mixture reacted reacted forfor twotwohours. hours. Nanoparticles were collected by centrifugation and the template was removed by washing with an Nanoparticles were collected by centrifugation and the template was removed by washing with an Nanoparticles were collected ethanolic solution of ammonium by centrifugation and the material nitrate; the resulting templatewas waswashed removed by washing successively with with ethanolic solution of ammonium nitrate; the resulting material was washed successively with an ethanol, ethanolicMili‐Q water solution and ethanol.nitrate; of ammonium Herein,the two differentmaterial resulting ratios ofwas E‐Pn were tested—90/10, washed successively with 75/25—in ethanol, ethanol, Mili‐Q water and ethanol. Herein, two different ratios of E‐Pn were tested—90/10, 75/25—in orderwater Mili-Q to study andtheir influence ethanol. on the Herein, twoNPs’ size and different morphology. ratios of E-Pn were tested—90/10, 75/25—in order to order to study their influence on the NPs’ size and morphology. All the mixed periodic mesoporous study their influence on the NPs’ size and morphology. nanoparticles were characterized by TEM, organosilica All the mixed periodic mesoporous organosilica nanoparticles were characterized by TEM, nitrogen‐sorption measurements All the mixed periodic (BET), p‐XRD, mesoporous FTIR, 13nanoparticles organosilica C‐CP MAS solid‐state NMR, DLS, by were characterized zeta‐ TEM, nitrogen‐sorption measurements (BET), p‐XRD, FTIR, 13C‐CP MAS solid‐state NMR, DLS, zeta‐ potential, TGA nitrogen-sorption and DSC. measurementsSome physical data p-XRD, are given 13 in Table 1. potential, TGA and DSC. Some(BET),physical FTIR, data are givenC-CP MAS1.solid-state NMR, DLS, zeta-potential, in Table TGA and DSC. Some physical data are given in Table 1. Molecules2020, Molecules 2020,25, 25,974 974 4 of 164 of 16 Table 1. 1. Some Somephysical physicaldata dataofof E‐Pn PMONPs E-Pn andand PMONPs P3 PMONPs. P3 PMONPs. N2‐Sorption Measurements N2 -Sorption Measurements Zêta ZêtaPotential Potential Material SBET Vpore Particles Particles Size (nm) c TGA d Material S V pore∅ pore (nm) b pH (mV) TGA d (m2∙g2−1BET ) −1 (cm3∙g−1) 3a −1 a ∅pore (nm) b Size (nm) c pH (mV) (m ·g ) (cm ·g ) E‐P1 90/10 PMONPs 683 0.48 2.8 447 6.96 −28 78% E-P1 90/10 PMONPs 683 0.48 2.8 447 6.96 −28 78% E‐P2 E-P290/10 PMONPs 90/10 PMONPs 509509 0.34 0.34 2.7 2.7 800 800 5.92 5.92 3232 75% 75% E‐P3 90/10 E-P3 90/10PMONPs PMONPs 505505 0.36 0.36 2.8 2.8 479 479 5.44 5.44 3636 72% 72% E‐P1 E-P175/25 PMONPs 75/25 PMONPs lowlow nd nd nd nd 507 507 7.34 7.34 −22 −22 67% 67% E-P2 E‐P2 75/25 75/25 PMONPs PMONPs lowlow nd nd nd nd 513 513 6.25 6.25 2020 58% 58% E-P3 E‐P3 75/25 75/25 PMONPs PMONPs lowlow nd nd nd nd 321 321 4.86 4.86 2424 57% 57% P3 PMONPs 186 0.26 5.6 269 8.94 11 nd e P3 PMONPs 186 0.26 5.6 269 8.94 11 nd e a Determined from the uptake at saturation at p/p◦ = 0.98. b Diameter determined by BJH. c Hydrodynamic a Determined from the uptake at saturation at p/p° = 0.98. b Diameter determined by BJH. c diameters from DLS. d Residual mass, TGA with a heating rate of 10 ◦ C/min from 30 to 1000 ◦ C. e Not determined. 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 The morphologies nanoparticles of E‐Pn were obtained were first analyzed by TEM ranging (Figurefrom 200for 1), and nmthe formajority (E-P1, E-P3 of the90/10) tomaterials 300 nm long for E-P2. For E-P3 75/25, nanoparticles of 150 nm were observed. The rodlike‐shape nanoparticles were obtained ranging from 200 nm for (E‐P1, E‐P3 90/10) to hydrodynamic diameters 300 nm longof the nanorods for E‐P2. were75/25, For E‐P3 investigated by DLS, nanoparticles (Table of 150 1) and nm were the obtained observed. diameters were The hydrodynamic indiameters agreement with of the the sizes nanorods weredetermined investigated bybyTEM. (Tableshowed DLS, E-P2 higher 1) and the hydrodynamic obtained diameters diameters were in agreement than E-P1 andwithE-P3. the sizes Thedetermined hydrodynamic by TEM. E‐P2 showed diameter of E-P3higher 75/25hydrodynamic was lower than diameters (E-P1,than E‐E-P3 E-P2, P1 and 90/10) inE‐P3. The hydrodynamic agreement with a smaller diameter of E‐P3 size and 75/25 particle was lower than morphology (E‐P1, E‐P2, compared E‐P3 90/10) to nanorods. in zeta The 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 potentials were the determined presence of Si-Oin− water at the (Table surface1).ofE‐P1 showed negative the nanorods. The zetazeta potential, potential in agreement of E-P2 and E-P3 with the was positive 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. 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). Figure 1. TEM images of PMONPs (E-P1; E-P2; E-P3, proportion 90/10 and 75/25). Molecules 2020, 25, 974 5 of 16 Molecules 2020, 25, 974 5 of 16 The N2 adsorption-desorption isotherms were next examined (Figure 2). All the materials 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. showed an isotherm between type I and type IV, and between microporous and mesoporous systems. 2 ·g−1 Nanorods from E (already synthesized) [36] showed a very high specific surface area of 800 m Nanorods from E (already synthesized) [36] showed a very high specific surface area of 800 m2∙g−1 with an average small pore size of 2.5 nm. When incorporating P1, E-P1 90/10 showed a decrease in with an average small pore 2size of 2.5 nm. When incorporating P1, E‐P1 90/10 showed a decrease in specific surface area to 683 m ·g−1 and a small increase in pore size to 2.8 nm. The incorporation of P2 specific surface area to 683 m2∙g−1 and a small increase in pore size to 2.8 nm. The incorporation of P2 2 −1 with an increase andand P3P3at at a proportion a proportionofof90/10 90/10ledledtotoaadecrease decrease in in specific surfacearea specific surface areatoto500 500mm2∙g·g −1 with an increase of pore of pore size to 2.7 and 2.8 nm, respectively. Note that when increasing the proportion of of size to 2.7 and 2.8 nm, respectively. Note that when increasing the proportion precursors precursors P1-3 to to P1‐3 75/25, 75/25,materials E‐P1‐3,showed materialsE-P1-3, showed no no adsorption ofN adsorption of N2 2any anymore moreatat7878K,K, which which suggests suggests thatthat thethe porosity porosity is is not accessible not accessibletotoNN22at at this this temperature. temperature. Figure 2. N22.adsorption-desorption Figure experiments N2 adsorption‐desorption (BET) experiments of of (BET) E, E, E-P1, E-P2, E‐P1, E‐P3,atataaproportion E‐P2,E-P3, proportion of of 90/10. 90/10. Small Angle X-rays diffraction of nanorods from E [36] (Figure 3) showed the d(1,0,0) Bragg peak at 2.2 θ,Small with Angle the satellites X‐rays at d(1,1,0) 4.0 diffraction θ and d(2,0,0) of nanorods from E4.6 characteristics θ, (Figure [36] 3) showed of athe well-ordered d(1,0,0) Bragg hexagonal peak at 2.2 θ, with mesoporosity. Thetheother satellites at d(1,1,0) materials are more4.0 θdisordered and d(2,0,0) 4.6aθ, with characteristics wormlike of a well‐ordered mesoporosity and a broad hexagonal mesoporosity. non-structured band (see XRD Theofother E-P1materials 90/10 and areE-P1 more disordered 75/25 with a wormlike as representative examples).mesoporosity and a broad non‐structured band (see XRD of E‐P1 90/10 and E‐P1 75/25 FTIR were recorded for all the precursors and materials. A representative example as representative examples). is given in Figure FTIR 4 were with recorded P2, E-P2for90/10 all the andprecursors and materials. E-P2 75/25. P2 showed A representative the presence example of theisBOC givengroup in Figure 4 with −1 P2, E‐P2 90/10 and E‐P2 75/25. P2 showed the presence −1 of the at 2973 cm (νCH3 ), the carbon chains at 2927 and 2883 cm (νCH2 ), the carbonyl of the carbamate BOC group at 2973 cm −1 (νCH3), the carbon chains at 2927 and 2883 cm (νCH2), the the triazole νCH at 1457–1386 cm−1 , carbonyl of the carbamate at 1696 cm−1 −1 −1 at 1696 cm−1 (νC=O), the silicon–oxygen bonds at 1073 cm (νC=O), the triazole νCH at 1457–1386 cm−1, the silicon–oxygen bonds at 1073 cm−1 (νSi‐O). All the (νSi-O). All the bands were essentially the same in the materials, with a shift in the carbonyl to bands were essentially the same in the materials, with a shift in the carbonyl to 1675–1672 cm−1 1675–1672 cm−1 showing a different environment of the BOC group in the material. A broad band showing a different environment of the BOC group in the material. A broad band between 1200–1000 between 1200–1000 cm−1 ((νSi-O-Si), characteristic of a well-condensed material, was observed. cm13 ((νSi‐O‐Si), characteristic of a well‐condensed material, was observed. −1 C-CPMAS 13C‐CPMASsolid-state, solid‐state, NMR NMR were were also also recorded. Spectrum(E‐P2 recorded. Spectrum (E-P2 is is 75/25) 75/25) representative representative (Figure 5) and showed the characteristic signals (in ppm) at 155.9 (C=O), (Figure 5) and showed the characteristic signals (in ppm) at 155.9 (C=O), 145.3 (C=C, Cq 145.3 (C=C, Cq triazole), 124.0 triazole), (CH triazole), 80.4 (Cq, tBu), 55.2 124.0 (CH triazole), 80.4 (Cq, tBu), 55.2(CH -N, CH 2 (CH2‐N, -triazole), 28.6 (CH tBu) 21.6(-CH 2 CH2‐triazole), 28.6 3(CH3 tBu) 21.6(‐CH 2 -), 11.2 (CH 2‐), 11.2 (CH -Si). 2 2‐ Si). Molecules 2020, 25, 974 6 of 16 Molecules 2020, 25, 974 6 of 16 Molecules 2020, 25, 974 6 of 16 Figure 3. Small angle XRD diagram of E (blue line), E‐P1 90/10 (red line), E‐P1 75/25 (green line). Figure 3. Small angle XRD diagram of E (blue line), E-P1 90/10 (red line), E-P1 75/25 (green line). 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. Figure4.4. FTIR Figure FTIR of (A) P2, of (A) P2,(B) (B)E‐P2 E-P290/10 and(C) 90/10and (C) E-P2 E‐P2 75/25. 75/25. TGA analyses 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. Molecules 2020, 25, 974 7 of 16 Molecules 2020, 25, 974 7 of 16 Molecules 2020, 25, 974 7 of 16 Figure 5. 13C‐CP‐MAS solid‐state NMR of E‐P2 75/25. TGA analyses 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 Figure 13C‐CP‐MAS 5. 13 C-CP-MASsolid‐state NMR solid-state of of NMR E‐P2 75/25. of the organic part of theFigure materials was shown. E-P2 75/25. TGA analyses 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. 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. 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. WeWe then then performedthe performed thesynthesis synthesis of of P3 P3 PMONPs. PMONPs. For Forthis, this,P3P3was wasused usedasas thethe only precursor only precursor (no silica source). CTAB was dissolved in aqueous NaOH at 80 °C ◦in highly diluted conditions for 50 (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 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. stirred at 80 ◦ C for 24 h, then extraction of CTAB and drying of the sample was performed as above. The nanoparticles were analyzed We then performed usingof the synthesis different techniques. P3 PMONPs. For this, P3 was used as the only precursor First, TEM images (Figure 7) showed spherical (no silica source). CTAB was dissolved in aqueous NaOH monodisperse nanoparticles at 80 °C in highly (100 nm diameter) diluted conditions for 50 which seem to be composed of sheets of organosilica. The nanoparticles are clearly porous on TEM Molecules 2020, 25, 974 8 of 16 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. Molecules 2020, 25, 974 were analyzed using different techniques. The nanoparticles 8 of 16 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 images. The porosity was confirmed with N2 sorption experiments (BET). Indeed, a type IV isotherm images. The porosity was confirmed with N22 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 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 was observed at 269 nm and zeta potential was positive, in agreement with the protonation of tertiary nitrogen nitrogen atoms atoms(Table (Table1).1). The The precursor precursor P3 was not P3 was not damaged damagedafter afterreaction, reaction,asas shown shown by by FTIR FTIR spectroscopy (Figure 9) with the carbonyl group observed at 1682 cm −1 . −1 spectroscopy (Figure 9) with the carbonyl group observed at 1682 cm−1 . FigureFigure 7. 7. TEM TEMof P3of Periodic Mesoporous P3 Periodic OrganosilicaOrganosilica Mesoporous Nanoparticles (PMONPs) at different Nanoparticles (PMONPs) at different magnifications. magnifications. Molecules 2020, 25, 974 Figure 8.8.NN22adsorption–desorption Figure experiments(BET) adsorption–desorption experiments (BET)ofofP3P3 PMONPs. PMONPs. 9 of 16 2 Figure 9. Figure 9. FTIR FTIR of of P3 P3PMONPs. PMONPs. 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 Molecules 2020, 25, 974 9 of 16 Figure Figure 9. FTIR 9. FTIR of P3 of P3 PMONPs. PMONPs. E-P3 75/25 PMONPs were selected for the release of CO2 , first under water conditions at 100 ◦ C. E‐P3E‐P3 75/25 75/25 PMONPs PMONPs were were selected selected forfor thethe release release of of COCO 2, first 2, first under under water water conditions conditions at at 100100 °C.°C. A total of 50 mg of E-P3 75/25 PMONPs were added to 10 mL of distilled water, then the mixture was A total A total of of 50 50 mgmg of of E‐P3 E‐P3 75/25 75/25 PMONPs PMONPs were were added added to to 10 10 mL mLof of distilled distilled water, water, then then ◦ C and stirred at this temperature for 30 min. The experiment was performed the the mixture mixture was was quickly quickly heated heated to 100 quickly heated to to 100100°C°C andand stirred stirred at at this this temperature temperature forfor 30 30 min. min. TheThe experiment experiment waswas performed performed at two at at two two different different different pH: pH:pH:7 and 7 and 7 and 1.1.At 1. At At pH7,7,we pHpH 7,wewe did diddid not notnot observe observe observe any any any bubbles; bubbles; bubbles; at at at 1pH pHpH 1could 1 we we we could could clearly clearly clearly seesee see aalot lot of a lot bubbles of of bubbles bubbles in the in in thethe mixture mixture mixture (Figure (Figure (Figure 10). 10).10). Then, Then, Then, E-P3 E‐P3 E‐P3 75/25 75/25 75/25 PMONPs PMONPs PMONPs were were were collected collected collected byby by centrifugation centrifugation centrifugation and and washed with EtOH. Finally, E‐P3 75/25 PMONPs were dried for FTIR analyses. At pH 7,pH washed and washed withwithEtOH. EtOH. Finally, Finally,E-P3 E‐P3 75/25 75/25 PMONPs PMONPs were were dried dried for for FTIR FTIR analyses. analyses. At At pH 7,BOC 7, the the the BOC BOC group was still present, but at pH 1 the absorption of tBu (2987 cm −1 ) and C=O (1680 cm −1 ) disappeared group group waswas still still present, present, butbut at pH at pH 1 the 1 the absorption absorption of of tButBu (2987 (2987 cmcm) and −1 −1) and C=O C=O (1680 (1680 cmcm) disappeared −1 −1 ) disappeared in in the in spectrum thethe spectrum spectrum (Figure (Figure (Figure 11). 11). 11). These These These results results results confirmed confirmed confirmed the thethe successful successful successful release release release of of COCO 2of in2CO in pHpH2 1inwater 1pH water1atwater at at 100 °C.◦°C. 100 100 C. Figure Figure Figure 10.10. 10. Images Images Images ofE-P3 E‐P3 ofofE‐P3 75/25 75/25 in water inin 75/25 water water at 100 at 100 at °C °C 100 Cand ◦and pHpH and 11(A) 1 (A) pH (A)5atat at 5 5min min and minand (B)(B) and at 30 at(B) 30 min. min. at 30 min. Figure Figure FTIR 11. FTIR Figure 11.11. FTIRofofE-P3 E‐P3 of E‐P3 75/25 75/25 75/25 (A) (A)(A) before before before treatment, treatment, treatment, (B) (B)(B) after after after heating heating heating inin distilled distilled in distilled water, water, water, (C) (C)(C) after after after heating heating heating in water at in water in water pH = at pH at pH 1. = 1.= 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 1 H- and 13 C-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 Ressonància Magnètica Nuclear of the Universitat Autònoma de Barcelona (UAB). The 13 C-CP-MAS solid state NMR spectra were obtained from a Varian VNMRS 300 MHz instrument (Les Ulis, France) which belongs to the University of Molecules 2020, 25, 974 10 of 16 Montpellier; the repetition time was 5 s with contact times of five milliseconds. From the Servei d’Anàlisi Química of the UAB, the following experimental data were acquired: infra-red spectra (IR), 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, CH3 CN and pyridine were dried by molecular sieves, THF, CH2 Cl2 and pentane were from solvent processing equipment (PureSolv, Innovative Technology, Newburyport, MA, USA). Cetyltrimethylammonium bromide (CTAB), sodium hydroxide, ammonium nitrate (NH4 NO3 ), 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 Et3 N (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, 73% yield). 1 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); 13 C-NMR (91 MHz, CDCl ) 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, 3 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 C29 H57 N7 O8 Si2 Na: 688.3880, found: 688.3877. Molecules 2020, 25, 974 11 of 16 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 Et3 N (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 evaporated at reduced pressure to afford the final product as colorless oil (2.27 g, 96% yield). 1 H-NMR (360 MHz, 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); 13 C-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 C51 H107 N9 O14 Si4 Na: 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 Et3 N (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 g, 96% yield). 1 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); 13 C-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 C53 H111 N9 O14 Si4 Na: 1232.7220, found: 1232.7195. 3.5. Synthesis of E-BTSE Grubbs Catalyst™ 1st Generation C823 (PCy3 )2 Cl2 Ru=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 g, 71% yield, 4 mbar, 124 ◦ C). E-BTSE was identified by 1 H-NMR as a diastereoisomerically pure product (100% E). 1 H-NMR (360 MHz, CDCl3 ) δ 6.66 (s, 2 H; E-isomer), 3.83 (q, J = 7.2 Hz, 12 H), 1.23 (t, J = 7.2 Hz, 18 H). 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 µL, 2.29 mmol) was added rapidly under stirring. The condensation process was conducted 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 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 [NH4 NO3 , 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 NH4 NO3 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 NH4 NO3 (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: 13 C-CP-MAS NMR (75 MHz) δ: 155.6, 146.1, 123.8, 81.1, 57.9, 52.5, 28.2, 24.9, 17.4, 10.4. IR ν (ATR) (cm−1 ): 3337.7, 1671.3, 1416.6, 1187.7, 1036.9, 927.3. BET: SBET = 683 m2 ·g−1 , Vpore = 0.48 cm3 ·g−1 , ∅pore = 2.8 nm. TGA (air, 5 ◦ C/min, 20–1000 ◦ C) residual mass 78%. Zeta Potential: ζ = −28.7 mV, pH = 6.99. DLS: 447 nm. E-P2 90/10 PMONPs: 13 C-CP-MAS NMR (75 MHz) δ: 155.4, 146.0, 125.2, 81.0, 58.1, 52.8, 48.2, 28.5, 20.6, 10.8. IR ν (ATR) (cm−1 ): 3322.9, 1673.0, 1412.0, 1187.7, 1036.2, 923.7, 187.9. BET: SBET = 509 m2 ·g−1 , Vpore = 0.34 cm3 ·g−1 , ∅pore = 2.7 nm. TGA (air, 5 ◦ C/min, 20–1000 ◦ C) residual mass 75%. Zeta Potential: ζ = 32.6 mV, pH = 5.92. DLS: 800 nm. E-P3 90/10 PMONPs: 13 C-CP-MAS NMR (75 MHz) δ: 156.1, 146.1, 124.7, 81.0, 58.0, 48.0, 28.4, 18.5, 10.6. IR ν (ATR) (cm−1 ): 3361.9, 1655.6, 1370.1, 1188.0, 1033.4, 924.1, 788.8. BET: SBET = 505 m2 ·g−1 , Vpore = 0.66 cm3 ·g−1 , ∅pore = 2.8 nm. TGA (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 NH4 NO3 (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: 13 C-CP-MAS NMR (75 MHz) δ: 156.1, 145.5, 123.6, 81.9, 57.7, 52.8, 28.3, 24.8, 17.9, 10.4. IR ν (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: ζ = −22.9 mV, pH = 7.34. DLS: 507 nm. E-P2 75/25 PMONPs: 13 C-CP-MAS 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: 13 C-CP-MAS NMR (75 MHz) δ: 1561.2, 146.1, 124.8, 81.7, 58.2, 48.8, 28.6, 19.8, 10.3. IR ν (ATR) (cm−1 ): 3294.2, 1676.8, 1388.2, 1012.7, 923.4, 786.5. TGA (air, 5 ◦ C/min, 20–1000 ◦ C) residual mass 57%. Zeta Potential: 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 µL of 2 M NaOH was added. The mixture 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 NH4 NO3 (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, 1367.4, 1103.1, 1023.6, 783.8, 688.4. BET: SBET = 186 m2 ·g−1 , Vpore = 0.26 cm3 ·g−1 , ∅pore = 5.64 nm. Zeta Potential: ζ = 10.9 mV, pH = 8.94. DLS: 269 nm. 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 13 C 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 14 of 16 Author Contributions: Conceptualization, R.P., P.M., C.P. and J.-O.D.; methodology, H.L., C.G. and A.D.; formal analysis, 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 Economía, Industria y Competitividad (MINECO) of Spain (Projects CTQ2014-53662-P and CTQ2016-81797-REDC), Ministerio de Ciencia, Innovación 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. References 1. Du, X.; Li, X.; Xiong, L.; Zhang, X.; Kleitz, F.; Qiao, S.Z. Mesoporous silica nanoparticles with organo-bridged silsesquioxane framework as innovative platforms for bioimaging and therapeutic agent delivery. Biomaterials 2016, 91, 90–127. [CrossRef] [PubMed] 2. Chen, Y.; Shi, J. 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