Engineering of Mesoporous Silica Coated Carbon-Based Materials Optimized for an Ultrahigh Doxorubicin Payload and a Drug Release Activated by pH, T , and NIR-light

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Materials Optimized for an Ultrahigh Doxorubicin

Payload and a Drug Release Activated by pH, T , and

NIR-light

Connor Wells, Ophélie Vollin-Bringel, Vincent Fiegel, Sebastien Harlepp,

Benoit van der Schueren, Sylvie Bégin-Colin, Dominique Begin, Damien Mertz

To cite this version:

Connor Wells, Ophélie Vollin-Bringel, Vincent Fiegel, Sebastien Harlepp, Benoit van der Schueren, et al.. Engineering of Mesoporous Silica Coated Carbon-Based Materials Optimized for an Ultrahigh Doxorubicin Payload and a Drug Release Activated by pH, T , and NIR-light. Advanced Functional Materials, Wiley, 2018, 28 (17), pp.1706996. �10.1002/adfm.201706996�. �hal-02413449�

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Article type: Full Paper

Engineering of mesoporous silica coated carbon based materials

optimized for an ultra-high doxorubicin payload and a drug

release activated by pH, T and NIR-light

Connor Wells, Ophélie Bringel, Vincent Fiegel, Sébastien Harlepp, Benoit Van der Schueren, Sylvie Bégin-Colin, Dominique Bégin, Damien Mertz*

C. Wells, V. Fiegel, Dr. S. Harlepp, Prof. S. Bégin-Colin, Dr. D. Mertz,

Institut de Physique et Chimie des Matériaux de Strasbourg (IPCMS), UMR-7504 CNRS-Université de Strasbourg, 23 rue du Loess, BP 34 67034, Strasbourg Cedex 2, France

E-mail: damien.mertz@ipcms.unistra.fr

O. Bringel, Dr. B. Van der Schueren, Dr D. Bégin.

Institut de Chimie et Procédés pour l'Energie, l'Environnement et la Santé (ICPEES), UMR-7515 CNRS-Université de Strasbourg, 25 rue Becquerel, 67087 Strasbourg, Cedex 2, France. Keywords: Carbon nanotubes, Graphene, Mesoporous silica, Nanocomposites, Drug loading/release, Stimuli responsive materials

Abstract

Among the nanomedecine challenges, engineering nanomaterials able to combine imaging and multi-therapies is hugely needed to address issues of a personalized treatment. In that context, a novel class of drug releasing and remotely activated nanocomposites based on carbon-based materials coated with mesoporous silica and loaded with an outstanding level of the anti-tumoral drug doxorubicin (DOX) has been designed. Such nanocomposites are shown able thus to combine drug delivery, phototherapy and imaging, thanks to the carbon based materials. First, carbon nanotubes (CNTs) and graphene sheets (called “few layer graphene” FLGs) are processed to afford a distribution size that is more suitable for nanomedicine applications. Then, the controlled coating of mesoporous silica (MS) shell having a thickness tailored with the sol-gel parameters (amount of precursor, sol-gel time) around the sliced CNTs and exfoliated FLGs are reported. Furthermore, the drug loading in such mesoporous nanocomposites is investigated in full and the surface modification with an aminopropyltriethoxysilane (APTS) coating leading to a controlled polysiloxane layer provides an ultra-high payload of DOX (up to 3 folds the mass of the composites). Such new

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CNTs based-nanocomposites are demonstrated to release DOX at low acidic pH, high temperature (T) and remotely when they are excited by NIR light. Such nanoconstructs may find applications as components of innovative biomedical devices (scaffolds implants, etc..) for dual therapy displaying photo-thermal properties combined with drug delivery.

1. Introduction

Carbon-based materials such as carbon nanotubes (CNTs) or graphene have attracted much attention over the past two decades due to their unique physical, chemical and mechanical properties.[1–4] Both carbon-based materials have already been reported in nanomedecine as innovative theranostics carriers[5–8] for biomedical applications. Hence, they were used as drug delivery carriers,[9–13] or as contrast agents for imaging (NIR fluorescence, photo-acoustic and Raman.)[14] They were also loaded with magnetic nanoparticles inside their cavity to combine MRI, magnetic hyperthermia and phototherapy.[15] Additionally they can be used in tissue engineering for their mechanical strength[16] or to ensure cell differentiation in bone tissue regeneration[17] and they were also reported to be degraded by macrophage cells.[18] Besides, among all these features, the property of CNTs and graphene to convert near infrared (NIR) light into local heat is highly attractive for photothermal applications[15,19] to kill cancer cells. Indeed, CNTs and graphene have a very broad absorbance spectrum which covers the NIR optical window where biological tissues are transparent.5 After exposure to NIR light (in the range of 750 to 1400 nm), CNTs (and graphene) enter into an excited state and release vibrational energy converted into local heat. Furthermore, the NIR light conversion into heat by graphene/CNTs could also be beneficial for remotely releasing drugs for instance within the cells by brownian activation or by triggering the cleavage of thermosensitive non covalent or covalent bonds.[11,20–22]

However, there are various issues currently raised by using CNTs or graphene as nano-objects for phototherapy combined with drug delivery. Indeed, the aspect ratio for CNTs

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and the dimension of the graphene sheets remain still too large to ensure suitable interactions with cells such as cell internalization and this effect is reported to promote cell toxicity. Moreover, for biomedical applications, CNTs or graphene were mainly coated with polymers and this may lead to polymer desorption with time, loss in the control of the colloidal stability and to uncontrolled heat dissipation after NIR light application.

In terms of shortening CNTs, one technique as shown by Chew et al., is to compress CNTs, aided by sonic booms, which results in the buckling of CNTs and their structures fragmenting at certain points[23].Another approach is to treat CNTs in acidic conditions, as developed by Liu et al.[24] who based their experiments on similar procedures used for the exfoliation of graphite. They succeeded in shortening single-walled CNTs to have a length range of 100 – 300 nm. For graphene exfoliation, several techniques have been developed to form thin layers including the mechanical pulling of graphite using adhesive tape[25] or the CVD[26] (chemical vapor deposition) which however lack respectively in the control of the number of graphene layers and the yield for the first one and in an expensive cost for the second one. Probably, one of the most common technique is few layer graphene production assisted by sonication[27], either from "graphite oxide", or from graphitic materials in the presence of surfactants in aqueous media.

The coating of mesoporous silica (MS) shells has recently been reported on graphene or CNTs as a way to form a highly stable and hydrophilic coating for such C-based materials.[28,29] As for IO cores, the silica thickness is controlled by the reagents stoichiometry and the reaction time. All types of CNTs, single or multi walled, big or small, can be coated with MS, and can be used as platforms for further functionalization. Coating of MS induces an important rise of the surface area of the material from several tens (CNT alone) up to 500-1000 m².g-1. The created mesopores have an average diameter of ca. 2-3 nm. Because of the facile and high level of mass transportation through the pores, this important surface area makes this material interesting for various applications including heterogeneous catalysis,

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removal of pollutants or as addressed in this work, for drug delivery. Indeed, in the last few years, MS coated graphene and CNTs, loaded with DOX as a model drug were shown as highly efficient for drug delivery combined with phototherapy.[30–32] The carriers were then injected in cancer cells and after exposition to NIR light, the combination of the drug release and of the photothermal activity of graphitic materials improved efficacy of the treatment compared with NIR light and drug release alone.

Herein, these challenges described above are addressed. A new class of carbon-based materials having features well suited for nanomedecine applications have been designed. First, with the aim to afford CNTs or graphene layers having distribution size more suitable for biomedical applications, the slicing of CNTs and exfoliation of graphene are investigated. Then, MS coatings strategies are applied around these two C-based materials. The tuning of the MS shell thickness around the CNTs is reported by playing on parameters such as the time of sol-gel reaction or the amount of TEOS precursor Then, with the aim to optimize the drug payload in such composites, a drug loading study is performed in full with varying parameters such as drug concentration and the influence of the surface modification. For this latter point, the surface modification with the aminosilane aminopropyltriethoxysilane (APTS) in comparison to unmodified surface is investigated deeply for various MS coated C-based systems: CNTs, FLG and MCM-41 (MS spheres without carbon) and for various silica shell thicknesses on CNTs. The release of the drug is then investigated with different local or remote stimuli : under low acidic conditions (pH ca. 4), in high T (80°C) and by applications of a NIR light on such systems with the aim to use such materials as smart drug releasing carriers (Figure 1).

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Figure 1. Scheme showing the principle of synthesis of CNT@MS or FLF@MS grafted with

APTS, the loading with doxorubicin and the drug release actuated by T, pH or NIR light.

2. Results and discussion

2.1 Processes for the cleavage of carbon nanotubes and exfoliation of graphene

The carbon-based materials CNTs and FLGs, were first reduced in size to afford adapted distributions for cellular interactions. First, a process to cleave CNTs was applied to ensure a better-suited length, typically from a range of 2-100 microns to a few hundred nanometers. The cleavage was achieved by dispersing CNTs in a mixture of nitric and sulfuric acids, which enhances the oxidation of the nanotubes causing slicing at surface defects. The now-acidic CNTs were neutralised with NaOH before further modification. After washing and centrifugation, and removing the salts formed during neutralisation (sodium sulphate and sodium nitrate), the CNTs were heated under argon, to remove the oxygenated functions from their surface. Following transmission electron microscopy (TEM) imaging and using the software ImageJ, the length was found in a range of 100 – 1000 nm (see top line TEM images in Figure 2, and S1 for additional TEM images).

Regarding the synthesis of few-layer graphene (FLG), expanded graphite was combined with CTAB, which acted as a surfactant, and ensured graphite sheets exfoliation by dispersion in water via ultra-sonication. The solution was then decanted 36 h and the sediment was removed (Figure 2. SEM images on the bottom line).

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Figure 2. Processes showing the cleavage of CNTs and exfoliation of graphite into FLGs

which were followed by TEM and SEM before and after the process.

With the aim to determine the number of FLG layers after decantation, Raman spectrum (Figure 3.A) of the FLG was acquired. Analysis of the spectrum indicated that the exfoliated FLG was composed of 3 to 4 layers of graphene. Moreover, the "quasi" absence of the D band on the Raman spectrum attested to the purity of the as synthesized graphene.

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Regarding the process to control the size of the graphene sheets, the longer the time of sonication, the smaller the size of FLG was (Figure 3.B). Thus, the size decreased from ca. 2.8 to 1.1 µm. The time of decantation was also a parameter to isolate the FLG, the heavier particles falling to the bottom of the beaker. After 36 hours, the sediment was removed and the supernatant was made of FLG with ca. 3-4 layers.

2.2. Coating CNTs and FLG with MS: parameters controlling the MS shell thickness

A sol-gel procedure adapted from Bian et al.[28] was used to cover the CNTs and FLGs with a homogenous MS shell with pore size of ca. 2.5 nm. CNTs or FLGs were added to CTAB, in water and ethanol and thoroughly dispersed by ultra-sonication (see Materials and Methods section). As CTAB is a cationic surfactant, it forms a lipid-like layer around the hydrophobic carbon surface and positively charges micelles in solution outside the carbon materials. After addition of tetraethylorthosilicate (TEOS) and NaOH, the sol-gel process was initiated from the CNTs or FLG surface. NaOH acts as a catalyst which kick-starts the hydrolysis of TEOS, which condenses into silicate polyanions around the positive micelles, and then forms Si-O-Si network of MS around the CNTs or FLG. After the sol-gel reaction, the CTAB micelles were removed by extractions with NH4NO3. The removal of CTAB was followed by measuring Zeta potential (ZP) decreasing from positive to negative values after several extractions, upon which a plateau was reached (ca. -25 mV) assuming a CTAB free MS shell as described before.[33] At this point, the composites were denoted CNT@MS or FLG@MS. This micelle templated sol-gel process was also used for the synthesis of MCM-41, except without any carbon materials: CNTs/FLG. Figure 4 shows the TEM and SEM images of these three composites (CNT@MS or FLG@MS and MCM-41, top figure) and their associated zoomed TEM images (bottom of the figure 4) highlighting their similar meso-porosity.

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Figure 4. TEM and SEM images of the carbon-based MS composites obtained by applying a

sol-gel procedures adapted by Bian et al.[28] (A) around CNTs to form CNT@MS, (B) around FLG to form FLG@MS, (C) in the absence of carbon materials to form MCM-41 sub-micron spheres. D, E, F images correspond to TEM zoomed images showing the mesoporosity of the composites.

The thickness of the MS layer was determined via TEM images. The conditions applied by following the process of Bian et al (24 mg CNTs or FLG, 124 mg of CTAB, and 240 µL of TEOS) afforded an average MS thickness of ca. 65 nm of CNT@MS, ca. 50 nm for FLG@MS and an average diameter of ca. 610 nm for MCM-41 sub-micron MS spheres. With the aim to finely tune the thickness of the MS shell, we investigated the influence of two key parameters of the sol-gel process during the MS coating: the reaction time (hours) and the amount of TEOS added (µL). Thus in a first set of experiments, by applying the same process used above as a reference, the sol-gel reaction was stopped after 1 h, 2 h and 4 h, allowing the thickness of the MS coating to be controlled with MS shells obtained at ca. 38±5, 50±6 and 60±6 nm, respectively (Figure 5.A-C, respectively). A shell thickness of ca. 60 nm was also

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found for a sample that underwent 19 h of reaction time (usual conditions of the Figure 4.A) thus we deduced that after 4 h of reaction, the silica polymerisation reached a plateau and did not continue further. However, let us note that when the aging time increased, a moderate amount of MCM-41 spheres formed along with CNT@MS and increased regularly during the sol-gel process (S2).

Figure 5. TEM images showing the evolution of the MS coating thickness by increasing the

reaction time: A, B, C respectively at 1, 2 and 4 h.

In a second set of experiments, the amount of TEOS was varied by decreasing its volume compared to the above initial procedure (using 240 µL TEOS) respectively by a factor of 8, 4 and 2. (Figure 6. A, B, C respectively). The aging time was maintained at 16 hours. The results show that by increasing the amount of TEOS at volumes of 30, 60 and 120 µL, MS shells of ca. 8.5 ± 3, 30 ± 7 and 60 ± 6 nm thicknesses were respectively obtained ensuring a rational control of the thickness over the parameters of the sol-gel process. Moreover, in these conditions, the formation of free MCM-41 was very negligible which allowed validating these conditions as the most optimized for the control of the MS shell thickness and its specific formation around the CNTs .

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Figure 6. TEM images showing the evolution of the MS coating thickness by increasing silica

precursor amount respectively at 30, 60, 120 µL of TEOS added during the sol-gel process.

These composites described above having various MS shell thicknesses, were thus characterized by thermogravimetric analysis (TGA) to evaluate their composition ratio C/silica. TGA curves were traced for CNT@MS 30, 50 and 65 (respective to their MS shell thickness), FLG@MS and MCM-41 (Figure 7.A). TGA was performed in air (heating rate 10°C/min) and displayed for the CNTs@MS a rapid weight loss around 500°C while the weight loss for FLG@MS was rather continuous from 450 to 700°C. More in details, the masses losses at T lower than ca. 200°C correspond to the evaporation of solvents, (EtOH and then H2O, respectively) while the weight losses at ca. 550-600°C corresponds to the T of max conversion of carbon into carbon dioxide. By taking into account the limited contribution of solvents, this allowed us to calculate the C/SiO2 mass ratio for each CNT@MS nanocomposite.

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Figure 7. A. TGA analyses for CNT@MS30, CNT@MS50, CNT@MS65, FLG@MS and

MCM-41. B. representative N2 isotherm adsorption of CNTs@MS65 associated with the pore size (2.5 nm) distribution measured by BJH model.

The TGA data are summarized in Table 1 and show that the evolution of the C/SiO2 ratio in CNTs@MS is consistent with the MS thickness (i.e.) the SiO2 mass fraction was increased regularly from 35 to 55 % when the MS thickness was increased from 30 to 65 nm. FLG@MS with 50 nm thickness had a very high amount of silica (81%) which was attributed to the coating of both sides of the graphene sheets contrary to the CNTs surface, but mainly because of the number of graphene layers which is very higher in the case of CNTs. The mesoporous composites were also characterized by nitrogen adsorption-desorption Brunauer-Emmett-Teller (BET) technique. Overall, for the 5 systems, type IV adsorption-desorption isotherms were found, with a surface area ranging from 777 to 829 m2.g-1. Furthermore, for the 5 systems, the pore size was found in the range 2.3-2.8 nm with a unimodal pore size distribution. A typical isotherm adsorption for instance for CNT@MS65 is shown in Figure

7.B. As for TGA analyses, a summary of the BET analyses is given for CNT@MS30,

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Table 1. Summary of the BET and TGA analyses for CNT@MS30, CNT@MS50,

CNT@MS65, FLG@MS50 and MCM-41 CNT@MS30 CNT@MS50 CNT@MS65 FLG@MS50 MCM-41 % C (TGA) 65 57 45 19 0 % Si02 (TGA) 35 43 55 81 100 Pore size (nm) 2.8 2.3 2.5 2.6 2.3 Surface area BET ( m².g-1) 810 829 800 777 804

2.3. Parameters influencing the drug loading in CNT@MS, FLG@MS and MCM-41

In this section, we investigated various parameters influencing the drug loading within the different composites previously synthesized (CNT@MS, FLG@MS and MCM-41) including the concentration of the drug solution, the effect of the surface modification and the thickness of the porous silica shell. The principle behind the loading technique is a simple drug impregnation in water within the porous materials, with the drug payload parameters (DLC and DLE) being determined by the UV-Vis technique. The two parameters used to characterize the drug loading are the drug loading capacity (DLC) that transcribes as the mass of DOX loaded versus the mass of composite, and the drug loading efficacy (DLE) which is the mass of DOX loaded versus the initial mass of DOX introduced. To quantify these parameters of drug loading, a calibration curve (S3) obtained by UV-Vis spectrophotometry was traced to measure the absorbance (480 nm) of DOX in water at various concentrations. For the loading procedure, DOX was incubated with the composite with continuous stirring for 16 h, after which the mixture was centrifuged. A volume of the supernatant (ca. 400 μL) was diluted with water (10 times) and the UV/Vis absorbance was measured. Using the

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calibration curve, the mass of DOX from the supernatant, (i.e.) the DOX that has not entered the silica shell, could be deduced.

First, we investigated the surface modification of the different composites with an aminosilane (aminopropyltriethoxsilane, APTS) compared with unmodified (bare) systems. For both surface chemistries, we assessed and compared the drug loading for various drug concentrations by tracing DLC and DLE profiles with drug concentration. Thus, for each composite synthesized above: CNT@MS30, CNT@MS50, CNT@MS65, FLG@MS and MCM-41, APTS was condensed into the silanols of the MS shell in EtOH:NH4OH(aq) (ca. 5:1 in vol.) media to afford an amino-functionalized coating (cf Materials and Methods section). Then, the DOX loading was assessed in comparison with the unmodified MS shell in the concentration range of DOX of ca. 0.25-2 mg.mL-1. Figure 8 displays the resultant DLC profiles with [DOX] for all the various composites (Figures 8.A for CNT@MS30, CNT@MS50 and CNT@MS65, 8.B for FLG@MS, 8.C for MCM-41) whereas the DLE curves can be found in the SI (S4). A first observation is that for all the composites used, there is a consistent increase in DLC as the concentration of DOX rises, which is consistent with the DOX isotherm adsorption on the porous materials. Regarding the effect of the surface modification, it can be clearly stated that for each system CNTs@MS, FLG@MS and MCM-41, the modification with APTS has an obvious enhancing effect on the amount of drugs entering the MS layer compared to the bare surfaces. For instance, Figure 8.A, shows that at 2 mg.mL-1, CNT@MS30@APTS had a higher DLC of ca. 80%, whereas the bare counterpart has reached a value of 18%, corresponding to a 4-fold increase. Furthermore,

Figures 8.B and 8.C show also a DLC enhancement with APTS modified-FLG@MS and

MCM41 which had, at 2 mg.mL-1 DOX solution impregnation, DLC values at ca. 45% and 75%, compared with unmodified FLG@MS and MCM-41 which had a DLC of ca. 9 and 17% corresponding both to a ca. 5 fold increase.

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Hence, these results obtained with APTS-modified CNTs@MS, FLG@MS and MCM41 systems indicate an exceptional enhancement of drug payloads compared to the bare surfaces. To the best of our knowledge, this article is the first report investigating the DOX loading on such MS coated carbon-based composites modified with APTS and comparing it with a bare surface. Studies on the drug loading of DOX were reported previously on bare MS (only composed of silica) and the DLC was found in the range of 15-30% depending on the sizes of the MS and on the DOX impregnation conditions.[34,35] These results are in agreement with our DLC found with our bare CNTs@MS which have DLC close to 20% (at 2 mg.ml-1 DOX impregnated).

Besides, another effect which was investigated for the drug loading study was the influence of the MS shell thickness for CNTs@MS systems. Figure 8.A show clearly that for APTS modified systems, when the silica shell thickness decreased from 65 nm to 30 nm, the DLC values (at 2 mg.mL-1) increased from 30 to 75%. This result was reproduced by additional experiments where DLC increased from 24 to 78% by decreasing the thickness of the MS. Furthermore, the corresponding bare composites have a DLC no greater than 20% for the CNTs, regardless of the thickness of the silica shell. To explain these results, we assume that the APTS organic matrix is so importantly grafted at the inorganic CNT@MS surface that it decreases importantly the global density of the resulting nanocomposite when the MS shell thickness is reduced from 65 to 30 nm. Thus, by decreasing the MS shell thickness, and thus the silica proportion, the DLC value which is reported per gram of APTS-modified composite, may thus increase. Another effect would be also a cooperative effect of the APTS surface with the attractive effect of the CNTs substrate underneath the MS which was reported to bind importantly aromatic cycles of drugs through pi-pi stacking and hydrophobic interactions.

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Figure 8. DLC curves as a function of [DOX] in water and effect of APTS modification

compared to non-modified surface (bare) for A. CNT@MS, B. FLG@MS, C. MCM-41, in the range 0.25-2 mg.mL-1. For A. CNTs@MS, 30, 50 and 65 correspond to the thicknesses of the porous silica shell (nm).

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Higher concentrations of DOX for impregnations were also used in the range of 2-8 mg.mL-1 applied on all these composites modified with APTS. Different assays performed in this range of [DOX] are listed in the Table 2. As it was found above, the DLC increased by increasing the [DOX] for all systems, and by decreasing the MS thickness for the different CNT@MS systems. However as it can be seen in this table, some samples displayed a greater error bars in the DLC measurements compared to those achieved in the range 0.25-2 mg.mL-1 Nevertheless, in these conditions ultra-high payloads of DOX into the APTS-modified composites achieved at 8 mg.mL-1 DOX, were obtained. For instance, CNT@MS30@APTS, FLG@MS@APTS and MCM-41@APTS achieved loading capacities of up to 318%, 266%, and 105%, respectively. Worthy to note, is that the DLC of a system can indeed be above 100%. This is because if the mass of some substances, such as carbon nanotubes for example, is lower than that of the drug, then it becomes possible to charge more amounts of drug into the carriers than the carriers itself.

Table 2. Summary of DLC for CNT@MS30, CNT@MS50, CNT@MS65, FLG@MS and

MCM-41 at higher [DOX] obtained in the range of 2-8 mg.mL-1. [DOX] during impregnation CNT@MS30 @APTS CNT@MS50 @APTS CNT@MS65 @APTS FLG@MS50 @APTS MCM-41 @APTS DLC at 2 mg.mL-1 79 % 79 % 42 % 58% 45% 30 % 25 % 45 % 74 % 60 % 75% DLC at 4 mg.mL-1 159 % N/A 38 % 134 % 146 % 64% DLC at 8 mg.mL-1 318 % 144 % 110 % N/A 51 % 92 % 266 % 297 % 105%

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Regarding the reported results about the drug loading of DOX on pure silica MS modified with APTS, the values differ greatly depending on the grafting conditions of APTS silanes and there is no clear trend. Wang et al.[35] reported that DOX payloads were in the range of [5-15%] when the APTS silane condensation was made in water and at an APTS organosilane added at ca. 0.1% in vol. Meng et al. reported a DLC(DOX) of only 0.1%[36] when APTS was condensed in situ during the sol-gel process of MS nanoparticles when the APTS silane condensation was made at ca. 0.1% vol. In our system the APTS grafting is made after the MS shell coating on CNTs, after CTAB extraction, and the condensation of APTS is made according to a previously used strategy in Stöber-like conditions (EtOH: NH4OHaq25%) at a highy APTS concentration at ca. 15 % in vol. Hence, the surface chemistry and the surface density of the silanes grafts should not be the same as the different APTS modified systems.

The APTS coating procedure in ethanol reported here was adapted from our previous works which involved the formation of a primer APTS layer at the surface of silica microparticles used as sacrificial templates to prepare self-supported polymer microcapsules.[37,38] In these works, the polysiloxane layer was preferred to the APTS monolayer built in toluene or in ethanol at low siloxane amount as it ensured a better primer layer for the further polymer adsorption on the silica to build membranes of capsules. This APTS coating strategy was also used to coat and disperse iron oxide NPs synthesized by the thermal decomposition method[39] The formation of a dense polysiloxane layer of several nm thickness at the surface of magnetic nanoparticles was shown essential to ensure their colloidal stability which could not be obtained with a thin siloxane shell.

To provide insights on the drug loading mechanism, the APTS coating onto the CNTs@MS surface was investigated by different methods (TEM, zeta potential, BET N2 isotherms and TGA). All these results are gathered in the Figure 9. First, we performed TEM imaging after APTS coating of different CNT@MS having different MS shell

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thicknesses to visualize the effect of the APTS coating process conducted in this basic ethanol media. The effect of the APTS process was imaged on three typical systems built in the study: CNTs@MS30 synthesized with a sol-gel performed during 1h with one equivalent TEOS; CNTs@MS30 made with a sol-gel performed during 16h with 0.25 equivalent TEOS; CNTs@MS65 with a sol-gel performed during 16h with one equivalent TEOS. Interestingly, TEM images before and after APTS coating indicated that the first system had a change in its porous structure with a pore enhancement and a better transmission of electron beam through the MS thickness (Figure 9.A). Here, the process of APTS silane condensation to the silica surface could be related with an effect of dissolution in basic media. Indeed, the etching process occurring upon this basic media across the MS layer could explain the change in the porous structure. Various works showed these last years that a polymer deposited at MS surface could protect the MS surface and ensure internal etching of the silica by forming a lighter etched MS shell.[40,41] Oppositely, the second and third systems built in longer times of sol-gel were found unchanged in their MS structure after APTS coating. (Figure 9.B and C). To explain these differences, it was assumed that CNT@MS made at longer condensation times have a higher chemical stability due to a better Si-O-Si cross-linking compared to the one at short condensation times. This may explain why the 30 nm MS shell made at 1h was etched whereas the one at 16h was not.

The surface charge as a function of the pH was also characterized by zeta potential (ZP) measurements for non-modified and APTS modified surfaces CNTs@MS30 (16h condensation) (Figure 9.D). ZP values showed that the grafted APTS systems had an isoelectric point (IEP) around ca. 8, which is consistent with an amine grafting at silica surface whereas the IEP of bare silica is close to ca. 3 consistently to a silica surface. These data clearly demonstrated the charge reversal and the effective APTS surface functionalization.

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Next, the pore accessibility was investigated in CNT@MS systems by BET-N2 isotherm adsorption analysis (Figure 9.E). Compared to bare CNT@MS counterparts which had ca. 800 m2.g-1 surface area as seen above, it was found here that after the APTS coating, the systems CNT@MS30@APTS (1h condensation) and CNT@MS65@APTS (16h condensation) displayed both an important decrease of the surface area with respectively 67 ± 3 and 113 ± 7 m2.g-1. This clearly showed the effect of the APTS surface modification on the CNTS@MS surface, which had almost closed the pores by the formation of the polysiloxane layer. A first conclusion that can be drawn here is that the mechanism of drug loading between the bare MS and MS@APTS systems must be very different. Hence, contrary to the bare systems, the drug payloads after APTS coating are probably achieved more via a surface mechanism than via a volume loading (i.e. the drug is loaded by interaction with the APTS layer). With the aim to quantify the amount of grafted APTS onto the CNT@MS systems, the CNTS@MS30@APTS (1h sol-gel condensation) was analyzed by thermogravimetric analysis (TGA) (Figure 9.F). The data showed as expected that the calcination of APTS coating occurred before the CNT calcination. This allowed to estimate a mass loss of APTS at ca. 15% mass of the total composite CNTs@MS@APTS. A similar result was found with CNTs@MS65@APTS where APTS contributed to ca. 10% in mass. This clearly demonstrated that a non-negligible amount of polysiloxane layer is formed at the surface of CNTs@MS.

Then, to confirm this mechanism, the drug loading at a very high DLC (318%) on the CNT@MS@APTS surface was imaged by TEM before and after the drug loading .(Figure

10). TEM images at low (Figure 10.A and B) and higher magnification (Figure 10.C)

showed that a dense and sticky organic matrix is formed around the CNTs@MS@APTS. Thus, the formed APTS polysiloxane layer acts probably as a kind of organic sponge attracting DOX at the CNT@MS@APTS surface confirming the assumption of a surface- mediated drug loading mechanism on this matrix. Combination of interactions through H

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bonding and pi-pi stacking are probably involved to ensure accumulation of DOX at this surface. [9,10,30]

Figure 9. TEM images of CNT@MS30@APTS (1h sol gel condensation) (A).

CNT@MS30@APTS (16h condensation) (B) and CNT@MS65@APTS (16h condensation) (C) Zeta potential measurements of CNTS@MS30 and CNTS@MS30@APTS (D), and N2 isotherm adsorption (E) and TGA analysis (F) of CNTS@MS30@APTS.

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Figure 10. TEM images showing CNTs@MS30@APTS@DOX at DLC =318% which

provide a view of the CNT@MS inorganic composite and the APTS-DOX matrix at low (A, B) and high magnification (C).

2.4. Drug release study : influence of pH, T, and NIR light

With the aim to demonstrate the potential of drug release from such composites, we first investigated the effect of two different stimuli: i) a low pH (pH ca. 4) mimicking acidic conditions in cellular endo-lysosomes and ii) heating to temperatures of 80°C to assess the possibility to trigger the drug by local photothermal effect. For that, two suspensions of CNT@MS50@APTS loaded with DOX at DLC=45% were respectively placed in an aqueous solution buffered at pH ca. 4 or in water at pH=7 at 80°C and were compared to similar aqueous suspension at pH=7 at ambient T used as a control. The results of released DOX obtained after 3 and 6 h incubation time are shown in Figure 11. Firstly, these results indicate that the DOX level is constant (ca. 20-25 µg) in the control samples signifying the limited spontaneous release of DOX in the absence of any stimulus. Furthermore, when the composite’s suspension was lowered to pH ca. 4, there was a significant release of DOX after 3h that reached a plateau after 6h (ca. 50-60 µg.). Similar results were observed when the T was set to 80°C in water at pH=7 where the DOX released was also of the order of ca. 75-80 µg.mL-1 which is about 3 times the dose released compared with the control sample. Pictures at the right of Figure 11 show distinctively the difference of red contrast in the different supernatants. The drug releases triggered by low acidic pH and T effects are attributed respectively to the weakening of electrostatic or H bonds interactions involved in DOX binding with the APTS-modified surface.

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Figure 11. Drug release after 3 and 6 hours achieved on CNTS@MS50@APTS samples

loaded with DOX at DLC=45% triggered by low pH (pH ca. 4) or by increasing T. Pictures of the associated supernatants of the three suspensions are provided at the top of the figures.

In a following study, the possibility to induce the drug release by NIR light application, converting the photonic wave into localized heating was investigated from these carbon-based materials nanocomposites. The laser was set to 1064 nm at 6 W/cm2 for various times and the surface area of the sample container was 1 cm2 for 1 mL of suspsension. A wavelength of 1064 nm is well suited to this study because it is within the NIR optical window where biological tissues are transparent and it ensures the excitation of carbon-based materials to transform NIR-light into localized heat which has not been measured in this study. For this study, samples of CNT@MS30@APTS were chosen, with two high DLCs of 160% and 318% (hereafter referred to as samples 160NIR and 318NIR), each with their own control, which was kept at ambient temperature (160C and 318C). An initial trial was carried out with a 1 hour NIR light exposure (Figure 12.A). Results show for the “160 samples” a release of

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ca. 188 μg of DOX for 160NIR, compared with 12 μg for 160C which is over 15 times more amount released. Regarding the “samples 318”, 318NIR liberated ca. 44 μg of DOX via NIR irradiation, compared with 13 μg released for 318C which is over 3 times more amount released. Such differences might be attributed to strong stacking which might appear when samples are highly loaded preventing a facile diffusion out from the composite. Moreover, 1 hour NIR application may raise issues for preclinical applications because of potential overheating of tissues (we have previously shown that temperature can raise very fast with such composites6 ). Investigating the possibility of a pulsatile release may hence be of a higher interest for controlled drug release in space and location. Then, the time frame of NIR irradiation was adjusted to 2 x 15 min NIR pulses with an hour’s break in between, once a day over four days. The results for this study can be found in Figure 12.C for samples at DLC =160 % and 12.D for the samples DLC=318%. Hence, both graphs show a substantial sequential increase of the DOX released after each consecutive day of NIR light. Worthy to note that, for sample 160NIR on day four, there is an abrupt decline of DOX released. This can be explained by the observed precipitation that was observed on the side of the tube after supernatant separation by centrifugation (S5). Nonetheless, sample 160NIR by day 3 and 318NIR by day 4 released respectively ca. 3 and 4 times as much DOX (i.e. 44 and 68 μg of DOX) after the NIR irradiation, compared to the controls at ambient T (15 and 16 μg of DOX released). Photographs of the DOX released from both samples are shown in Figure 12.B for DLC=160% (after 1h NIR irradiation, top picture) and for DLC=318% (after 4 consecutive 15 min NIR irradiations separated by 1 h break, bottom picture) and illustrate the results of this study.

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Figure 12. Drug release study upon NIR irradiation on CNT@MS30@APTS at DLCs of

160% and 318% (hereafter referred to as samples 160NIR and 318NIR), each with their own control :160C and 318C. A. A first initial trial was carried out with a 1 hour NIR light exposure. B. Photographs of the DOX released after NIR exposure. C. and D. NIR study when the time frame was adjusted to 2 x 15 min NIR pulses with an hour’s break in between, for two applications per day, over four days for samples 160 and 318%, respectively.

Similar experiments were also carried out on FLG@MS@APTS with DLCs of 134%. After the first trial, with an hour of NIR irradiation at 1064 nm, 134NIR liberated 16 μg of DOX, 1.6 times more than without NIR treatment (134C, 10 µg DOX) (Figure 13.A). A second trial, with the 2 x 15 min pulse sequence, was carried out over 4 days for this sample 134 (Figure

25

was of 27 μg for 134NIR compared with 18 μg for 134C, respectively. In this study, the two first NIR pulses allowed releasing the drug however from day two to day four, for both 134NIR and 134C, the drug released level did not change anymore. Figure 13.C illustrates a slight difference in contrast of the supernatants between the samples submitted to NIR light and the control.

Figure 13. Drug release study upon NIR irradiation on FLG@MS50@APTS at DLCs of

134% A. A first initial trial was carried out with a 1 hour NIR light exposure. B. NIR study when the time frame was adjusted to 2 x 15 min NIR pulses with an hour’s break in between, for two applications per day, over four days. C. picture after 2 irradiations 15 min separated by 1 hour break showing a slight difference between 134C at the left and 134 NIR at the right.

3. Conclusion

A novel class of C-based nanocomposites has been designed and optimized for drug loading / release applications. First, C-based materials such as long CNTs or expanded graphite were processed to afford sliced CNTs and few layer graphene (FLG). Then, the coating of mesoporous silica shell around CNTs or FLG was performed and the fine tuning of MS shell thickness was shown around CNTs by adjusting sol-gel reaction parameters (time or the amount of silica precursor). Furthermore, we showed that an ultrahigh drug loading was obtained when the MS shells were grafted with APTS aminosilanes with drug payloads reaching several times the mass of the composites (DLC up to 300%) in comparison with bare

26

surface (DLC up to 20%). The DOX release from these composites was efficiently obtained when the DOX loaded on APTS-coated CNT@MS and FLG@MS were submitted to external stimuli such as high T (80°C), acidic pH (ca. 4) or remotely applied NIR-light (1064 nm). Such systems present a huge interest for releasing drugs in living systems and may be used as components of smart implantable systems. Future works will thus address important issues on the fundamental or the biomedical point of view such as elucidation of the driving force for ultra-high DOX loading, or the coating with a biocompatible polymer or a protein to ensure a suitable biointerface .

4. Experimental Section

Chemicals. Carbon nanotubes PR-19XT-HHT (CNT) were provided by Pyrograf-III, while

Mersen provided expanded graphite. Tetraethyl orthosilicate (TEOS), 3-aminopropyltriethoxysilane (APTS), sodium hydroxide (NaOH) and nitric acid (HNO3) were obtained from Sigma-Aldrich (France). Doxorubicin HCl, 99% (DOX) was acquired from OChem Incorporation (France). Sulfuric acid (H2SO4) and ethanol (EtOH) were obtained from Carlo Erba Reagents (France). Ammonium hydroxide (25% assay) came from Fluka. Cetyltrimethylammonium bromide used for the synthesis of CNT@MS and FLG@MS (CTAB) was obtained from Roth (France), whereas CTAB* used for the synthesis of MCM-41, was obtained from Alfa Aesar.

Procedures

Synthesis of cleaved carbon nanotube (CNTs). Typically, 500mg of CNTs were added to a

mixture of H2SO4 (54 mL) and HNO3 (18 mL), and sonicated for 24 h at 0°C. Then the acidic CNTs were pipetted dropwise into 100 mL NaOH and the solution was monitored until neutral. The CNTs were then washed and centrifuged with H2O (2 x 25 mL, 5000g, 10 min). After washing, the CNTs were heated under argon for 5 h at 900°C.

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Synthesis of few layer graphene (FLG). Firstly, the graphene was prepared by exfoliation. 1

g of CTAB and 100 mg of expanded graphite were dispersed in 100 mL of H2O by ultrasonication (4 h, power = 700 W, amplitude = 20%, temperature = 25°C). The composite was decanted for 36 h. After this time, the sediment was removed.

Synthesis of CNT@MS. This method was adapted from Bian et al.[28] In a typical procedure, 124 mg of CTAB was dissolved in a mixture of H2O (30 mL) and EtOH (20 mL) and stirred at 60°C for 2 h. 24 mg of CNTs was added to this solution and dispersed by ultrasonication (2 x 10 min, power = 750 W, amplitude = 40%, temperature = 30°C, runs: 50” ON, 50” OFF, Vibracell 75043 from Bioblock Scientific). Upon addition of TEOS (240 μL) and 1M NaOH (60 μL), the sol gel process had begun. The mixture was stirred for 15 h at room temperature on a mechanical wheel (STUART Rotator SB3 at 40 rpm). After this time, the composite was washed and centrifuged with EtOH (2 x 25 mL, 12000g, 12 min) and re-dispersed in EtOH.

Synthesis of FLG@MS. 12 ml of FLG (2 mg/mL) was added in a mixture of H2O (18mL),

EtOH (20 mL), TEOS (1.2 mL) and 1M NaOH (60 μL) and stirred for 2 h at room temperature on the mechanical wheel. The composite was sonicated in an ultrasonication bath for 2 min then stirred for a further 15 h at room temperature on the mechanical wheel. After 15 h, the composite was washed and centrifuged with EtOH (25mL, 5000 g, 7 min) and dispersed again in EtOH.

Synthesis of MCM-41. This method was adapted from Bian et al. 124 mg of CTAB was

dissolved in a mixture of H2O (30mL) and EtOH (20mL) and stirred at 60°C for 2 h. After this time, 60µL of NaOH (1M) and 240µL of TEOS were added and the solution was stirred for 15 h at room temperature on the mechanical wheel. After this time, the composite was washed and centrifuged with EtOH (25mL, 5000g, 7 min) and dispersed again in EtOH.

Surfactant extraction from CNT@MS, FLG@MS, MCM-41 composites. CTAB removal

from the silica pores was achieved by dispersing the composite in NH4NO3 (25 mL at 20 mg/mL in EtOH), followed by magnetic stirring at 60°C for 1 h. To determine when all the

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CTAB had been removed, the surface charge of the silica was measured by Zeta potential. The procedure was repeated until all the CTAB had been extracted (approximately 5 extractions were required). At this point, the composite was denoted CNT@MS, FLG@MS or MCM-41, respective to the original composite.

Surface modification with aminopropylsilane APTS. This protocol was adapted from our

previous works on silica and iron oxide nanoparticles. [38,39,42–44]. In a standard procedure, 50 mg of CNT@MS was dispersed in 27 mL EtOH. To this solution, NH4OH (1.2 mL, 25 % in water) and APTS (5 mL) were added and the mixture was stirred for 2 h on the mechanical wheel at room temperature. Afterwards, the amino-modified composites were washed and centrifuged with EtOH (2 x 20 mL, 13000g, 14 min). Further washing with water was done (2 x 20 mL). The composites at this point were denoted CNT@MS@APTS. This procedure was also used for the surface modification of FLG@MS and MCM-41.

Measurement of dried composites for mass determination. Each composite was stored in

EtOH to allow a rapid solvent removal. 1 mL of each composite was left on a weighed petri glass under the fumehood until all the liquid EtOH had evaporated. Then, the petri dish was transferred to an oven set between 80 and 100°C. Initially, the sample was measured every two hours, up to 6 hours to determine when the solvent was completely removed. An accurate mass of the composites could then be determined for the DOX-loading stages.

Drug Loading procedures in CNT@MS, FLG@MS and MCM-41: Impregnation of Doxorubicin. 2.5 mg of composite was dispersed in 1 mL of DOX(aq) at a given concentration and stirred on the mechanical wheel overnight. After ~16 h, the solution was centrifuged (14000 g, 14 min) and 400 μL of the supernatant was removed. 3.6 mL H2O was added (to give a dilution factor of 10) and the solution was analysed by UV/vis spectrometry (Lambda 950 UV/VIS Spectrometer by Perkin Elmer). The absorption measurements of the supernatant allowed the mass of DOX loaded into the various composites to be determined.

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NIR activated drug release from CNT@MS@APTS and FLG@MS@APTS. After

centrifuging to remove the impregnation solution, the samples were washed with H2O (x 1 mL) then put into fresh 1 mL H2O. Samples were placed in a 1 mL plastic cuvette, which was positioned underneath the NIR laser. The laser was adjusted to have a diverging lens that spanned 1 cm2 with a wavelength of 1064 nm, at 6 W/ cm2. For trial 1, the sample was irradiated for 1 h; for trial 2, the sample was exposed to 15 min of NIR light, followed by a break lasting 1 h, then a further 15 min of irradiation. After the activation, the samples were centrifuged and the supernatant removed for UV/vis absorbance to be measured. Using this measurement and the calibration curve, the mass of DOX released from the samples was calculated.

Characterization Methods

TEM microscopy. Morphology of the different nanocomposites: CNTs, FLG, CNTs@MS

FLG@MS and MCM-41 were characterized by transmission electron microscopy (TEM) with a TOPCON 002B ultra high resolution microscope operating at 200 kV. TEM samples were prepared by depositing one drop of the nanocomposite in solution on a carbon-coated copper grid, then letting the solvent evaporate a few hours before observation. The thickness of the silica was determined using ImageJ software on the TEM pictures and the results are indicated as mean layer thickness (nm) ± standard deviation (nm).

SEM microscopy The size of graphene particles and the morphology of silica on FLG were

characterized by scanning electron microscopy (Gemini500 - ZEISS - 1nm resolution). One drop of the sample, in ethanol or water solution, was deposited on a carbon-coated copper grid and then the solvent was evaporated. To measure the MS thickness on FLG@MS, it was necessary to find several FLG@MS at the vertical of the detector. The FLG is very small compared with the MS shell the silica so the FLG’s thickness was assumed negligible. The image J software allowed to measure the thickness of FLG@MS with the SEM images.

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Zeta potential. Zeta potential measurements of the CNTs@MS and FLG@MS

nanocomposites at different stages of the synthesis was measured by using a Zetasizer nano ZS by Malvern Instruments. The measurements were performed using DTS1070 folded capillary cells in which 10 µL of suspension was diluted in 1 mL of water. The pH of the measured solution was set at pH ca. 7 , adjusted by using HCl and NaOH 100mM.

Nitrogen adsorption / BET analysis. Mesoporous silica characteristics were determined by

nitrogen adsorption-desorption analysis through Brunauer-Emmett-Teller (BET) method to determine surface area and Barrett-Joyner-Halenda (BJH) method to determine pore size and pore volume. All the analyses were done on a Tristar 3000 Gas Adsorption Analyzer by Micromeritics Instruments. Before the tests, the samples were outgassed under vacuum at 150°C for about 4h.

Thermal gravimetric analysis (TGA). TGA was carried out on a Q5000 Automatic Sample

Processor by TA Instruments. Before the analyses, the samples in solution were dried over night under the hood, then 48h in a sterilizer at 80°C. For the runs, the samples were heated from 25 to 1000°C at a rate of 40°C / min.

UV/Vis spectroscopy. UV-vis spectroscopy was used to determine the amount of drug loaded

into the composite through the supernatant titration after drug impregnation. The UV-vis spectra, were recorded with a Lambda 950 UV/vis Spectrometer by Perkin Elmer. For all the measurements, the samples were diluted in a UV/vis quartz cell and into a final volume of 3 mL of solution. The solutions and the quartz cell were always protected from light using aluminum foil until the measurement was done.

NIR light irradiation set-up. A Navigator laser from SpectraPhysics J40-BL6-106Q was

used to shine 1064nm irradiation onthe samples. It was set to 6W lasing power and the beam was expended trough an IR treated lens to reach a beam diameter of about 1cm. Then the beam was directed to a spectroscopy cuvette to trigger the drug release through photothermal activation.

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Supporting Information.

Supporting Information is available from the Wiley Online Library or from the author.

Acknowledgements

D.M. thanks The University of Strasbourg for funding through the IDEX-Attractivité framework.

Received: ((will be filled in by the editorial staff))

Revised: ((will be filled in by the editorial staff))

Published online: ((will be filled in by the editorial staff))

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The table of contents

A novel class of drug releasing and remotely activated nanocomposites based on carbon-based materials coated with mesoporous silica and loaded with an outstanding level of the anti-tumoral drug doxorubicin (DOX) has been designed. Such new CNTs

based-nanocomposites are demonstrated to release DOX at low acidic pH, high temperature (T) and remotely when they are excited by NIR light.

Keywords : Carbon nanotubes, Graphene, Mesoporous silica, Nanocomposites, Drug

loading/release, Stimuli responsive materials

Connor Wells, Ophélie Bringel, Vincent Fiegel, Sébastien Harlepp, Benoit Van der Schueren, Sylvie Bégin-Colin, Dominique Bégin, Damien Mertz*

Engineering of mesoporous silica coated carbon based materials optimized for an ultra-high doxorubicin payload and a drug release activated by pH, T and NIR-light

34

Supporting Information

Engineering of mesoporous silica coated carbon based materials

optimized for an ultra-high doxorubicin payload and a drug

release activated by pH, T and NIR-light

Connor Wells1_{, Ophélie Bringel}2_{, Vincent Fiegel}1_{, ,Sébastien Harlepp}1 _{Benoit Van der}

35

S1.Size and isotherm adsorptions properties of CNTs before and after their cleavage S1.A CNTs before cleavage

S1.B CNTs after cleavage

S1.C Typical N2 BET isotherm

Isotherm adsorption of CNTs before cleavage having a surfacea area of ca. 30 m2.g-1. A similar graph was found after cleavage with a surface Area = ca. 34.5 m2.g-1.

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S2. TEM images showing the progressive formation of MCM-41 submicron spheres with the aging time by applying the conditions of Figure 5

37

38

S4. DLE curves corresponding to the DLC curves of Figure 8