Size-Controlled Functionalized Mesoporous Silica Nanoparticles for

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Size-Controlled Functionalized Mesoporous Silica Nanoparticles for Tunable Drug Release and Enhanced Anti-Tumoral Activity Meryem Bouchoucha, Marie-France Côté, Rene C.-Gaudreault, Marc-André Fortin, and Freddy Kleitz Chem. Mater., Just Accepted Manuscript • DOI: 10.1021/acs.chemmater.6b00877 • Publication Date (Web): 24 May 2016 Downloaded from http://pubs.acs.org on May 26, 2016

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Chemistry of Materials

Size-Controlled Functionalized Mesoporous Silica Nanoparticles for Tunable Drug Release and Enhanced Anti-Tumoral Activity Meryem Bouchoucha†,‡,§, Marie-France Côté‡, René C.-Gaudreault⊥, Marc-André Fortin* ‡,§, Freddy Kleitz* ,†,§ †

Department of Chemistry, Université Laval, Québec QC, G1V 0A6, Canada. Centre de recherche du centre hospitalier universitaire de Québec (CR-CHUQ), axe Médecine régénératrice, Québec QC, G1L 3L5, Canada. § Centre de recherche sur les matériaux avancés (CERMA), Université Laval, Québec QC, G1V 0A6, Canada. ⊥Centre de recherche du Centre hospitalier universitaire de Québec (CR-CHUQ), axe Oncologie, Québec QC, G1L 3L5, Canada. ‡

ABSTRACT: Mesoporous silica nanoparticles (MSNs) are considered as one of the most promising nanovectors to perform controlled drug delivery. For the design of ideal drug nanocarriers, several factors have to be taken into account, such as size and surface chemistry. Here, we report how MSNs surface functionalization and particle size critically affect the drug release performances and therapeutic capabilities. We illustrate the size effect of these functionalized MSNs on in vitro, intracellular and in vivo drug release efficiency, as well as on nanoparticle and drug diffusion into the targeted tissues (tumor). For this, dispersable MSNs with different particle sizes (from 500 nm to 45 nm), similar physiochemical properties (e.g., structural and textural properties) and high colloidal stability (even in saline conditions) were synthesized. Their surface was specifically functionalized with a phosphonatesilane according to a novel post-grafting strategy, for better control over loading and release of positively charged drugs. An efficient particles size-dependent and pH-dependent release of the loaded drug (i.e., doxorubicin) was achieved in physiological conditions with phosphonated-MSNs compared to pure-MSNs. The cellular uptake efficiency is revealed to be much higher with the smallest phosphonated-nanoparticles (45 nm). Furthermore, doxorubicin is efficiently released from the nanoparticles into the intracellular compartments and the drug reaches the nucleus in a time- and particle size-dependent manner. Intratumoral diffusion of the developed nanoparticles, as well as the drug release and its diffusion into the tumor matrix, are clearly enhanced with the smallest phosphonated-nanoparticles (45 nm), leading ultimately to a superior cell and tumor growth inhibition.

INTRODUCTION. Among the developed nanocarriers in the field of nanomedicine, mesoporous silica nanoparticles (MSNs) have emerged as one of the most promising nanomaterials for drug delivery applications owing to their unique characteristics such as high surface area and pore volume, high in vitro and in vivo biocompatibility, tunable particle size and versatile surface chemistry.1-6 MSNs have been explored for anti-inflammatory,7-9 antibiotic,10 growth control,11-12 gene delivery13 and anti-cancer therapeutic agents.14-21 Due to their large pore volume and surface-to-volume ratios, MSNs offer the possibility to transport large quantities of drugs into perfused organs via passive (e.g., enhanced permeability and retention (EPR) effect)22-23 and/or active targeting (e.g., specific receptor-mediated recognition).24-26 In addition, recent studies have shown the possibility to control drug elution from MSNs structures.7, 27-28 The main challenge of MSNs for targeted delivery remains the design of optimal carriers that could more specifically bind to and/or accumulate in the targeted organs or tissues (e.g., tumors). This strategy would provide higher and more selective drug doses, while minimizing nonspecific toxicity and side effects. To achieve this goal, size and surface chemistry, as well as surface charge, must be carefully balanced and controlled.

Particle size determines the cell uptake capacity of nanoparticles and their ability to penetrate through tissues.29-31 However, so far, ambiguous results have been found in the attempts made to correlate MSNs size with cell uptake. For instance, some studies have suggested that 50 nm was an optimum MSNs particle size for efficient cell uptake, by comparison to larger (≥ 100 nm: 105 – 120 – 170 – 280 nm) and smaller (30 – 25 nm) ones.32-33 In these studies, pure MSNs showing negative charge (≈ -20 mV at pH 7.4),32 as well as MSNs functionalized with a nuclear targeted peptide, and showing a positive charge (≈ 25 mV; in PBS)33 were used. In contrast, other studies involving amine-functionalized and polyethylene glycol (PEG)-coated MSNs, as well as pristine MSNs (≈ -15 mV; PBS pH 7.4), reported that 100 - 150 nm MSNs could be more efficiently taken-up by cells, even compared to 50 - 55 nm MSNs.34-35 However, high cellular uptake alone, does not guarantee the functionality of targeted drug delivery vectors: the compound must circulate in the blood long enough to provide enhanced and selective uptake by the targeted organs (e.g., tumors). Small particle size is assumed to correlate with long blood circulation times, and this is considered being beneficial to the general therapeutic outcome.36-37 Studies reporting on intrave-

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nous injections of pure and PEGylated MSNs, indicated that 80 nm – 120 nm particles were less prone to opsonization by the reticuloendothelial system, and therefore less accumulated in the liver and spleen compared to larger nanoparticles (≥ 200 nm)38. MSNs of sized ranging between 80 and 120 nm, also revealed enhanced accumulation around the leaky regions of the tumor vasculature.39 Such results suggested the enhancement of passive tumor accumulation of nanoparticles (due to EPR effect), with decreasing particle size. Furthermore, other reports have shown that MSNs of mean diameter close to 50 nm, could ensure the highest accumulation at the tumor site by passive accumulation (∼ 12%).22 This observation enabled the design of MSNs for active targeting (via acid folic grafting), that have reached high tumor accumulation (∼ 23% of the total initial injected MSNs dose).17 However, thus far, there is still a lack of more comprehensive and thorough studies which correlate particle size and in vitro and in vivo drug release efficiency, as well as substantiating particle size-dependent nanoparticle and drug diffusion into diseased tissues (e.g., tumor). Controlling the drug loading in MSNs, as well as the drug elution rate, are also major conditions to reach therapeutic efficiency. Indeed, total drug elution and the kinetics of drug release (elution rates) should ideally be closely matched with the type of disease (e.g., type of cancer), and if possible to its level of malignancy. The three factors that might have the highest impact on drug release from MSNs are 1) total pore volume, 2) particle size, and 3) surface physicochemical characteristics. The total pore volume dictates the total capacity of drug loading per nanoparticle unit. In typical MSNs nanoparticles (e.g., MCM-48), pores account for more than 60 - 70 % of the total volume of the particle.40 The particle size is expected to have a strong impact on drug elution rate, since the closer the drug is located from the MSN surface, in principle the higher chances it has of efficiently escaping from the pore network. Very few reports have until now discussed the impact of MSNs size on drug elution efficiency. Only one study performed in aqueous buffer solution (PBS pH 7.4), has revealed that large particle sizes (300 - 500 nm, pure MSNs) resulted in slower drug release rate (vitamine C and dexamentasone were used as drug models) and there was no significant differences on the drug release performances between 55 nm nanoparticles and 100 - 200 nm MSNs.34 This points to a possible limitation of small particles over large ones: indeed, there is a risk that by decreasing the size of MSNs, unacceptably fast drug release rates could be observed, with dramatic impact on the overall efficacy of the compound (i.e., drug release before the MSNs finds its target tissue). In order to ensure optimal drug retention and release control, it might be necessary to functionalize the pores with molecules that interact with the drugs and, by doing so, prolong their retention in the highly porous structure. However, to the best of our knowledge, there has been no report clearly substantiating the MSNs size effect on intracellular and in vivo drug release efficiency, as well as on nanoparticles and drug diffusion into targeted tissues (e.g., tumor). In this study, MSNs of various sizes (ranging from 500 to 45 nm) were synthesized, of equivalent surface and bulk physiochemical properties. Their structural and textural characteristics were studied, as well as their colloidal stability in different media. We also developed a novel post-grafting strategy using phosphonate silane, which helps to provide a better control over loading and release of doxorubicin, an anticancer

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drug widely used in cancer research and oncology. The biocompatibility, cell retention efficiency, as well as the drug release and cytotoxicity of drug-loaded nanoparticles were evaluated in vitro. Finally, the accumulation and diffusion of the nanoparticles, as well as the drug release kinetics, were evaluated in vivo using the chick embryo chorioallantoic tumor model. RESULTS AND DISCUSSION Synthesis and Characterization of Different-Sized MSNs. The ambiguous results reported in earlier studies,32-35 on the effect of nanoparticle size on cell uptake and drug release, are most likely related to a certain variability in the control of physicochemical properties (e.g., colloidal stability, textural and structural properties and controlled surface potential) of sub-100 nm MSNs. Indeed, it is a great challenge to ensure these requirements particularly for the synthesis of such small nanoparticles. Previous attempts to synthesize very small MSNs based on conventional approaches, mainly led to the synthesis of aggregates and/or fused nanoparticles.41 Although great efforts have been dedicated to prepare stable suspensions of sub-100 nm nanoparticles, only limited success was achieved. For example, stable colloids of pure and functionalized MSNs suspensions exhibiting diameters from 40 to 150 nm, 25 to 105 nm, 20 to 80 nm and 10 to 20 nm have been reported by Bein et al.,42-43 Shi et al.33, 44, Kuroda et al.45 and Wiesner et al.,46 respectively. However, most of these nanoparticles cannot be re-dispersed after drying process and usually large agglomerates of nanoparticles are obtained. Recently, Zhao and colleagues successfully synthesized MSNs that remain highly stable, once redispersible, even after drying process. The mean sizes of particles were 48, 72 and 100 nm. This was achieved through adjustments to Bein’s method and by grafting PEG.17 However, these nanoparticles lost the ordered mesostructure, one of the characteristics of larger conventional nanoparticles (120 – 150 nm). Yet, colloidal stability and nanoparticle mesostructure are critical parameters that impact on drug release profiles. In order to prepare redispersible mesostructured silica nanoparticles with different sizes and high colloidal stability, we modified the synthesis method of MCM-48 silica particles reported by Kim et al.47 MSNs were successfully prepared, with average particle diameters of 45 nm (MSN45), 90 nm (MSN90), 150 nm (MSN150), 300 nm (MSN300) and 500 nm (MSN500), as shown by the TEM images displayed in Figure 1. Conventional MCM-48 type MSNs with mean particle size of 150 nm were prepared according to a previously reported procedure with the molar composition of 1:0.16:0.017:605:84:9.16 TEOS:CTAB:F127:H2O:EtOH:NH3 (Figure 1-d).27, 47-48 Particle size control was achieved by tuning the concentration of the triblock copolymer Pluronic F127 as a nonionic surfactant.47, 49 Indeed, MSNs with an average particle diameter of 90 nm (MSN90, Figure 1-c) and 300 nm (MSN300, Figure 1-e), were successfully synthesized by increasing and decreasing the fraction of F127, respectively. Each sample was prepared at the optimal molar ratio of 1:0.16:y:605:84:9.16 TEOS:CTAB:F127:H2O:EtOH:NH3; "y" = 0.008 and 0.034 for MSN90 and MSN300, respectively. The optimal fraction of F127 should be kept between 0.008 and 0.034 to obtain well-dispersed spherical particles of size included in the range 90 - 300 nm. By increasing the molar ratio of F127 higher than (y = 0.07 – 0.14), very polydispersed

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MSNs are obtained with an average particle diameter of 80 nm ± 55 nm (Figure S1). On the contrary, a decrease in F127 concentration (y = 0 – 0.001) generates bigger aggregated particles with a mean particle size of about 490 – 530 nm (Figure S2), as observed in the literature.47 To achieve improved particle size control, triethanolamine (TEA) was added as a dispersion agent, as co-inhibitor of particle growth and/or as the base catalyzing the hydrolysis and condensation reactions of silicate species. Thus, easily redispersable small nanoparticles, exhibiting narrow size distribution and mean particle size of 45 nm (MSN45, Figure 1a,b), are successfully obtained by adding TEA instead of ammonia. In this case, TEA is used as a complexing agent for silicate species (from TEOS) and as a particle growth inhibitor for MSNs. The molar ratio of this composition was: 1:0.16:0.017:605:84:9.16 for TEOS:CTAB:F127:H2O:EtOH:TEA. Moreover, nonaggregated large particles (≈ 500 nm: MSN500; Figure 1-f) were prepared by substituting F127 with triethanolamine (TEA) as the dispersion agent, under an optimal molar composition of 1:0.16:9.15:605:84:9.16 for TEOS:CTAB:TEA:H2O:EtOH:NH3. All the obtained particles (from 45 to 500 nm) showed an ordered mesostructure as revealed by low-angle XRD patterns (Figure 2-a). Well-resolved typical peaks of highly ordered 3D cubic Ia3തd MCM-48 mesopore structure were found for MSNs of particle size ≥ 150 nm. For sub-100 nm MSNs, the XRD diffraction peaks appeared broadened and revealed MCM-like mesostructures. This broadening of the diffraction peaks could be attributed to the decrease in the reflection domains of the mesophase, occurring when the nanoparticle size decreases. This is observed especially when the particle size falls in the nanoscale range (sub-100 nm).50 Note that structural order of the sub-50 nm nanoparticles is rather unusual for MSNs of such small sizes. The mesostructure order for these nanoparticles (MSN45) is improved compared to that in previous reports, in which no peaks were usually obtained in the XRD pattern.17, 33 MCM-48 type nanoparticles are very interesting for drug delivery because of their 3D structure and their open interconnected network. This could improve the drug diffusion inside and from the pores, enhancing therefore drug loading and controlled drug release capacity. The nitrogen physisorption results (Figure 2-b) showed type IV isotherms for all the developed MSNx, which is characteristic of uniform mesoporous channels with narrow, cylindrical-like pores. The first capillary condensation occurred in the same relative pressure range of 0.15 – 0.3, regardless of the particle size. This indicates that these different-sized MSNs (from 45 to 500 nm) have almost the same mesopore size. Indeed, the average pore diameter was estimated at 3.4 nm for MSNx (x = 500; 300; 150 and 90) and 3.6 for sub-50 nm MSN: MSN45 (NLDFT method, Figure 2-c and Table 1). The isotherm of sub-50 nm MSNs (MSN45) showed slight differences in the heights of the first and the second capillary condensation steps: MSN45 showed the smallest step in the first capillary condensation, but exhibited the sharpest one in the second capillary condensation step, above the relative pressure of 0.93, which is related to interparticle voids. This fact is usually observed with the decrease of the size of very well-dispersed spherical particles.47 As determined from N2 sorption isotherms, all the MSNx samples possess high surface area, around 1200 m2 g-1 (BET method), and large total pore volume of 1 cm3 g-1 (Table 1). From all these results, we can conclude that we successfully

synthesized different-sized mesostructured nanoparticles without introducing major deformations or impairments to their structural and textural properties. This is rather unusual for such small MSNs sizes (especially sub-50 nm MSNs). The colloidal stability of these nanoparticles was investigated in aqueous and saline solutions (e.g., water and 154 mM NaCl) and monitored by dynamic light scattering (DLS). Hydrodynamic diameter distributions are shown in Figure 3-a,b. Number-weighted data showed a narrow hydrodynamic particle size distributions. Intensity-weighted data are also represented to facilitate the detection of signs of aggregation or flocculation. These data indicated that all the MSNx could be thoroughly suspended and well-dispersed in aqueous and saline solutions, even after drying and calcination, without any significant evidence of aggregation. The polydispersity index (PDI) and the hydrodynamic diameters reported in numberweighted data, intensity-weighted data and Z-average are summarized in Table S1 (Supporting Information). An acceptable PDI was recorded (between 0.02 - 0.14 in aqueous and saline solutions). The hydrodynamic diameters are slightly larger than the core particle sizes observed by TEM (Figure 1) which is usually the case taking into account the hydration corona around the particles. In addition, this colloidal stability was maintained at least for up to 1 week (Figure S2, Supporting Information): no evidence of aggregation or flocculation was found and no significant increase in hydrodynamic diameter was reported. This confirms the excellent colloidal stability of these systems. Nanoparticles with mean diameter ≤ 200 nm are widely used in today’s pre-clinical drug delivery systems because they have the advantage to: 1) be internalized by cells, based on non-specific cellular uptake mechanism, and 2) to ensure a passive tumor targeting, based on the EPR effect.23 In contrast, cellular uptake, tissue accumulation and blood circulation halflife of bigger particles (200 nm - 500 nm) are always limited due to their large sizes. In addition, when large particles are injected intravascularly, they are rapidly taken-up by the reticuloendothelial system and found in the liver and the spleen.38 From this point in the study, we focused our efforts in reaching ideal size diameters of optimal nanovectors for intravenous injections. Therefore, we focused our interest on MSN150, MSN90 and MSN45 nanoparticles. Drug Loading, in Vitro Drug Release Profiles and Phosphonate Grafting. To study the effect of MSNx size on the drug loading capacity and stimuli-triggered drug release, doxorubicin (Dox) was used as a model of anticancer agent. First, no significant dependence behavior of the drug loading efficiency was observed as a function of particle size. The loading capacities of pure MSN45, MSN90 and MSN150 were 2.1%, 2.3% and 2.4% (w/w), respectively. As these nanoparticles exhibit similar pore diameters and structural properties, the loaded amount of drug could be mainly determined by the surface area which is in correlation with the amount of the drug adsorption sites (i.e., silanol groups) at the surface of mesoporous nanoparticles. No significant difference was found on the surface area values of MSN45, MSN90 and MSN150 (Table 1). As a result, the drug loading capacity remains quite similar, as expected. The effect of MSNs size on drug release performances was also investigated in PBS pH 7.4 and in phosphonate buffer solution pH 5, which are conditions often used to mimic in vivo media (Figure 4-a). Dox release was pH-dependent: 15-



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16% at pH 5 vs. 9% at pH 7. However, a limited amount of drug was released (15-16% even after 6 days) and very slight size-dependence was observed. The low drug loading capacity of pure MSNx17, 51 and their limited drug release efficiency27 had also been observed in previous studies. We believe that these behaviors might be attributed to the nature and protonation state of the silanol groups on the nanoparticle surface and to the strong interactions taking place between Dox and the silica matrix. During the drug loading and release procedures, silanol groups present at the surface of MSNx, are mainly represented as Q3 and Q2 NMR species (Figure 5-a): Q3 at 110 ppm (one silanol attached to the silicon center) and Q2 at 101 ppm (two silanols attached to the silicon center). The pKa of Q3 silanols (deprotonation step for the reaction ≡SiOH ↔ ≡SiO− + H+) was reported to be around 2 to 4. The pKa of Q2 silanols (deprotonation step for the reaction =Si(OH)2 ↔ =Si(O−)2 + 2 H+) was reported to be around 7 to 8.52-53 According to 29Si MAS NMR data (Figure 5-a), a high (Q2+Q3)/Q4 ratio is observed and around 30% of silanols were assigned to Q2 species. It is assumed that this fraction of silanols is not quite effective for electrostatic binding during the Dox loading step and it contributes to a poor drug release. To improve the loading and the release efficiency of positively charged water-soluble drugs under physiological conditions (e. g. Dox, pKa = 8.3), we functionalized the surface of MSNx with phosphonate groups. For this, a phosphonate silane (THMP, trihydroxysilylpropyl methyphosphonate) was grafted onto MSNx according to a new post-grafting strategy. Phosphonate groups (pKa = 2) are expected to increase the surface electronegativity of MSNs and their stability in physiological conditions.51, 54 Furthermore, phosphonate grafting could improve blood compatibility of the silica nanoparticles. Indeed, it was shown that THMP grafting greatly reduced nonspecific protein adsorption on MSNs surface and exhibited similar performance as polyethylene glycol (PEG) grafting.55 In previous reports, MSNs functionalization with phosphonate groups had always been based on the co-condensation method (i.e., adding the organosilane during the synthesis as silica source in addition to TEOS).51, 54-55 In this way, a fraction of phosphonate groups are “inactive sites” for drug loading because they appear to be trapped and incorporated into the pore walls of the silica framework.56 In addition, the cocondensation method leads to highly hydroxylated nanoparticles. Consequently, a high fraction of silanol groups on the surface is present, including Q2 silanols species.56 For these reasons, we decided to develop a post-grafting method, which seemed more suitable to reach our goals (Dox adsorption and release). Conventional post-grafting strategies reported so far (e.g., in organic or alcohol media; under inert conditions and reflux), were impractical for the grafting of the phosphonate silane (as demonstrated in this study, see details in Supporting Information). To circumvent that impairment, we developed a particular post-grafting strategy, which is carried out in water, under reflux and in acidic conditions (see details in Materials and Methods section). The weight percentage (w/w) of grafted phosphonated silane molecules (THMP) was measured by TGA (Figure S3) and it equals to 8.8 %, 9.3 % and 9.6 % for PMSN45, PMSN90 and PMSN150, respectively. The successful grafting of the phosphonate silane on MSNx (PMSNx: PMSN45, PMSN90 and PMSN150) was confirmed by solid state NMR. Figure 5-b shows 31P MAS NMR profiles indicating the presence of phosphonate species (RPOCH3O−)

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with an intense single peak appearing at 25.8 ppm. The 13C CP/MAS NMR spectrum (Figure 5-c) revealed characteristic peaks of THMP (peak attributions are displayed in Figure 5). More interestingly, the 29Si MAS NMR spectrum of PMSNx (Figure 5-d) showed a sharp decrease in Q3 groups compared to pure MSNx (Figure 5-a) accompanied by a quasi-total elimination of Q2 silanol species after post-grafting of the phosphonate groups. This is due to the condensation between free silanols on the MSNx surface and silanol groups of THMP. Indeed, Figure 5-d shows the presence of Tn groups (CSi(OSi)n(OH)3-n) between [-50 to -70] ppm originating from the silicon of the covalently grafted phosphonate silane. Furthermore, a decrease in pore volume, specific surface area and pore size was observed after THMP post-grafting (Table 1). This is in good agreement with the introduction of phosphonate groups on the surface of MSNx. Nevertheless, a quite high porosity remained available for Dox loading: the surface area, the total pore volume and the mean pore size of PMSNx samples were around 900 m2 g-1, 0.7 cm3 g-1 and 3 nm, respectively (Table 1). In addition, the colloidal stability of MSNx was conserved after phosphonate grafting without any significant evidence of agglomeration or flocculation in aqueous and saline solutions (Figure 2-c,d and Table S2 in Supporting Information). Phosphonate grafting had a considerable impact on the electronegativity of nanoparticles as demonstrated by zeta potential measurements (Figure 5-e). This implies a more effective electrostatic binding of Dox into the MSNs pores, from where the drug could be subsequently easily released by acidification of the medium under abiotic and also biotic conditions. Indeed, the Dox loading efficiency of phosphonate-MSNx is highly improved (7× increase). PMSNx showed a remarkably high adsorption capacity reaching 14%, w/w (13.1%, 13.7% and 14.1% (w/w) for PMSN45, PMSN90 and PMSN150, respectively). Such enhancement in Dox adsorption capacities might be attributed to the electrostatic interactions taking place between positively charged Dox (DoxH+) molecules, the negative phosphonate groups (RPOCH3O−) as well as with the remaining deprotonated Q3 silanol groups (SiO−). Furthermore, this great loading capacity is twice higher than phosphonated MSNs prepared by co-condensation method.51 This could be attributed to the high efficiency of this developed post-grafting strategy, compared to co-condensation method. An efficient size-dependent and pH-dependent release of doxorubicin was achieved with the PMSNx systems, as demonstrated by the pharmacokinetics studies (Figure 4-b). The drug release was achieved in a time- and pH-dependent manner. Similar trend of Dox release profiles at pH 7.4 and pH 5 was observed. Figure 4-b shows also an increase in the Dox release rate with the decrease in the nanoparticle size. Under acidic conditions, a progressive high drug release was observed in the first 12 hours (inset Figure in Figure 4-b). It reached 93% when using small nanoparticles (PMSN45), 79% for PMSN90 and 60% with for larger particles (PMSN150). The highest burst effect was clearly observed for the smallest nanoparticles. Close to 30% of drug dose was released during the first hour vs 23% for PMSN90 and only 10% for larger particles (PMSN150). After 6 days, a subsequent slow release was observed and the total cumulative release of Dox reached 100%, 93% and 80% from PMSN45, PMSN90 and PMSN150, respectively. The cumulative releases of Dox under acid conditions are about five times higher than those observed in PBS at pH 7.4 (Figure 4-b). The low release level at pH 7.4 and the


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higher values achieved under more acidic condition, are related to the electrostatic interactions between Dox and adsorption sites at surface of nanoparticles (phosphonate and Q3 silanol groups). Indeed in neutral pH (pH 7.4), Dox (pKa = 8.2) is positively charged (DoxH+) and strongly bound to the predominant negatively charged phosphonate and silanol species (RPOCH3O− and SiO−, respectively). However, by lowering of the pH, Dox remains positively charged and the RPOCH3O− and SiO− groups become more and more protonated (RPOCH3OH and SiOH), inducing the release of Dox. This proton-sensitive mechanism indicates the possibility to trigger intracellular drug release once the particles are internalized in cells, enhancing therefore drug efficiency and reducing deleterious effects of free anti-cancer agents during chemotherapy. The size-dependent drug release was fitted by a simple mathematical model, in order to extract pharmacokinetic data. In vitro release data of Dox from PMSNx, at pH 5 and pH 7.4, were fitted using the semi-empirical power law equation below:57

Mt = k ×tn M∞ where Mt is the amount of the drug released at time t (in hours); M∞ is the total amount of the loaded drug; k is the kinetic release constant and n is a release factor The n value indicates the type of drug release mechanism and the k value provides information on the release rate.57 Both values (n and k) could be calculated from Higuchi’s model (cumulative % drug release vs. square root of time) or Korsmeyer-Peppas plot (log of cumulative % drug release vs. log time). Plots are presented in Figure S4 (Supporting Information) and the deduced results (n and k values) are compiled in Table 2. The release exponents (n) in the more acidic medium (phosphonate buffer at pH 5), were close to 0.5 for all PMSNx sizes (45, 90 and 150 nm), and thus presented pure Fickian diffusion. This means that the drug released from phosphonated nanoparticles mainly follows a model based on the diffusion from the pores. The kinetic release constant (k), which is directly correlated to the release rate, increased with the decrease in particle size (k = 38.9; 25.1 and 14.1 wt.% (hn)-1 for PMSN45, PMSN90 and PMSN150, respectively). The kinetic release constant for small phosphonated nanoparticles (45 nm) was ≈ 3 times higher than that for larger phosphonated nanoparticles (150 nm). This is in good agreement with the increase factor of the nanoparticle size (the size of PMSN45 is ≈ 3 times smaller than PMSN150). Consequently, this phenomenon might be attributed to mesoporous channel lengths and to the drug diffusion rate from the pores. Pore channels and diffusion distances appear shorter in the smaller nanoparticles compared with the bigger ones. This results in more rapid drug release kinetics. In addition, the external/inner surface ratio of the smaller spheres is higher for the smaller MSNs compared to the bigger ones, that increases the initial amount of drug released in the first minutes (burst effect), as has been observed for the release of Dox in the first hour. It also increases the k value. Meanwhile, a more progressive and prolonged release was achieved with larger particles (e. g., 150 nm). At pH 7.4, the release exponent (n value) shifted to 0.7 (Table 2), implying a non-Fickian release model, and the kinetic release constant (k value) was much lower than that at pH 5 (5 times

lower, Table 2). The kinetic release constant exhibited also a size-dependent behavior (k = 6.9; 4.6 and 3.3 wt.% (hn)-1 for PMSN45, PMSN90 and PMSN150, respectively). Those phenomena are most probably due to a combination of diffusion and erosion-controlled release. Such so-called "erosion" release is attributed to the degradation behavior of PMSNx nanoparticles at pH 7.4, a phenomenon that was also observed for MSNs submitted to simulated biological medium (pH 7.4).45 According to these in vitro drug release profiles and extracted pharmacokinetic data, sub-50 nm nanoparticles (45 nm) and conventional sized MSNs (150 nm) were chosen for an effective comparative intracellular and in vivo drug release studies. Cell Uptake Studies and Biocompatibility. The cellular uptake of PMSNx was investigated by TEM. For this, two human tumor cell lines (M21 and HT1080), a skin melanoma and a highly tumorigenic fibrosarcoma were used, respectively. Small and large phosphonated nanoparticles entered cancer cells within 5 h of incubation and were entrapped in intracellular compartments (vesicles: e.g., lysosomes), through a nonspecific adsorptive endocytosis (Figure 6). A size-dependence in cell uptake efficiency was also observed: it seemed much higher when using the smallest nanoparticles (PMSN45), compared to larger ones (PMSN150). This suggests that the physicochemical properties of phosphonated nanoparticles have significant impact on cell uptake. Higher apparent cell uptake might be attributed to the much higher external surface of the small-sizes MSNs, compared to larger ones. More nanoparticle-cell interactions lead to much stronger nanoparticle cell uptake through. In vitro cytotoxicity studies were also performed with PMSNx systems using the HT1080 and M21 cell lines, based on the resazurin (cell proliferation) assay. Small and large nanoparticles showed no significant cytotoxicity and antiproliferative effect for cells at concentrations up to 250 µg mL-1, and for incubation times up to 72 h (Figure 7). These results indicate desirable innocuousness of such nanocarriers (i.e., the phosphonated nanoparticles) for therapeutic drug delivery applications. Intracellular Drug Release and in Vitro Cytotoxicity. To investigate the size effect on the intracellular drug release, the phosphonated nanoparticles were labeled with a fluorescent agent (Alexa fluor 488 covalently grafted on silica nanoparticles), before Dox loading. These nanoparticles are referred to as Dox@PMSN45-F and Dox@PMSN150-F, (F: Alexa fluor 488). In this study, M21 and HT1080 cancer cells were incubated for 2 h and for 24 h with these nanoparticles. Merged fluorescent microscopic images of M21 cells and the corresponding cross-section image analysis (Figure 8) confirmed that small and large nanoparticles were internalized into the cells in a size-dependent manner. They suggest also that after endocytosis of the nanoparticles, doxorubicin was efficiently released from the nanoparticles and reached the nucleus in a time- and particle size-dependent manner. At 2 h, the orange/yellow fluorescence points (i.e., merge of the green fluorescence of PMSN45-F and the red fluorescence of Dox) were located into the cytoplasm of the cells (Figure 8-a,e). This indicates that small nanoparticles (Dox@PMSN45-F) were already efficiently endocytosed by the cells and a major fraction of Dox was not released yet from the nanoparticles after 2 h of incubation. In contrast, the orange/yellow fluorescence points, observed when using larger Dox-loaded nanoparticles



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(Dox@PMSN150-F), were particularly located at the interface cytoplasm/cell membrane (Figure 8-b, f). At 24 h, significantly improved uptake efficiency was observed (Figures 8-c, d, g, h). In addition, the uptake efficiency was closely related to the size of the nanoparticles. It was higher with 45 nm nanoparticles (Figure 8-c, g) than that observed with 150 nm nanoparticles (Figure 8-d, h), as observed in our TEM cell uptake study. These observations establish that small nanoparticles (45 nm) were internalized more easily and faster than larger nanoparticles (150 nm). Moreover, after 24 h of incubation, the red fluorescence, related to the released doxorubicin in merged fluorescent images, is mainly observed in the nucleus. This establishes that a significant fraction of Dox was already released from the nanoparticles and had diffused and transferred into the cell nuclei. Another fraction of Dox remained into the pores of silica nanoparticles, as some yellow/orange fluorescence points are also observed in the cytoplasm (Figure 8-c, d and Figure S5). In addition, green fluorescence points in the merged fluorescent images, related to nanoparticles once Dox is released, are localized into the cytoplasm. This suggests that after their endocytosis, the nanoparticles will be localized into lysosomes. Then, free doxorubicin is released from the nanoparticles into these acidic intracellular compartments. Finally, Dox is subsequently released from the lysosomes, probably through a proton pump mechanism,51 and diffuses into the nucleus. Similar results were obtained when incubating the nanoparticles with HT1080 cells (Figures S6 Supporting Information). To harmonize our findings with the improved cancer cell growth inhibition, in vitro cytotoxicity (antiproliferative activity) of Dox@PMSNx was evaluated with M21 and HT1080 cells (Figure 9). Cells were incubated with either small or large Dox-loaded nanoparticles (Dox@PMSN45 and Dox@PMSN150) or free Dox for 24 h, 48 h and 72 h. Figure 9 confirms that the longer the incubation time of Dox@PMSN45 and Dox@PMSN150 with the tumor cells, the higher the antiproliferative effect. Interestingly, the cell growth inhibition efficacy of both Dox-loaded nanoparticles was higher than that observed with free Dox, at equal concentrations. In addition, the antiproliferative activity of Dox-loaded nanoparticles was related to nanoparticle sizes. The half-maximal inhibitory concentration (IC50) values of Dox@PMSNx decreased in parallel with the increase in size of the phosphonated nanoparticles (Figure 9 and Table S3 where IC50 values were compiled, Supporting Information). The IC50 values of the small nanoparticles were approximately 2 times lower than that of larger nanoparticles, after 48 h and 72 h of incubation. The maximum difference between IC50 values were found at 72 h for M21 cells (IC50 = 0.17, 0.32 and 0.48 µg mL-1 for Dox@PMSN45, Dox@PMSN150 and free-Dox, respectively) and at 48 h for HT1080 cells (IC50 = 0.03, 0.07 and 0.13 µg mL-1 for Dox@PMSN45, Dox@PMSN150 and free Dox, respectively). On the basis of IC50 values, it is possible to rank the antiproliferative activity as follows: Dox@PMSN45 > Dox@PMSN150 > free Dox. This might be attributed to the efficient size-dependent cellular uptake of Dox-loaded nanoparticles and the efficient pH-responsive release of doxorubicine in a particle size-dependent manner. In Vivo Drug Release and Tumor Control. For in vivo studies, intratumoral and intravenous injections of Dox-loaded nanoparticles and free Dox were performed on M21 and HT1080 tumors grafted onto the chorioallantoic membrane of developing chick embryos. Dox-loaded nanoparticles

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(Dox@PMSN45 and Dox@PMSN150) were injected directly into the solid M21 tumors. Unloaded nanoparticles and a saline solution were also injected as controls. At 1 h and 18 h post-injection, tumors were collected and ex vivo fluorescence imaging was performed to investigate the kinetics of drug release into the tumor matrix. Ex vivo images of the tumors are displayed in Figure 10 after subtraction of the fluorescence of background (fluorescence of tumors obtained with the control). At 1 h, the fluorescence, coming from doxorubicin, was located in one single point. At 18 h, the fluorescence has diffused in a wider area that seems to be larger in tumors collected after injection of the smallest Dox-loaded nanoparticles (Dox@PMSN45). To strengthen these observations with semi-quantitative visual study, immunohistofluorescence analyses were performed on the M21 tumors (Figure 11). At 1 h post administration, the red fluorescence of Dox, was mainly located around the injection site, for both Dox@PMSN45 and Dox@PMSN150 (Figures 11-a,b,c). At 18 h, the red fluorescence had well diffused into the tumor matrix. The deep tumor penetration was clearly nanoparticle size-dependent and the highest intratumoral diffusion was observed for Dox@PMSN45 (Figures 11-d,e). This latter seems to be twice as deep as that of Dox@PMSN150 (Figure 11-f), in terms of diffusion depth. This diffusion could be related to the release of doxorubicin from the nanoparticles or to the diffusion of Dox-loaded nanoparticles (no Dox release) into the tumor matrix. To clarify these hypotheses, Dox@PMSN45-F and Dox@PMSN150-F were also injected into M21 tumors and the same study was performed. At 1 h, Dox@PMSNx-F nanoparticles were mainly localized around the injection site and doxorubicin was not yet released from the nanoparticles regardless the particle size. Indeed, a merging and co-localization of the green fluorescence of nanoparticles, the red fluorescence of doxorubicin and the blue fluorescence of labeled cancer cells were observed (Figures 11-g,h). In contrast, at 18 h, nanoparticles (green fluorescence) and doxorubicin (red fluorescence) were not co-localized and started diffusing into the intratumoral environment (Figures11j,k,l). The nanoparticles remained at the periphery of the tumor and doxorubicin had well diffused into the tumor matrix, suggesting that Dox was thus efficiently released from the nanoparticles. Once the nanoparticles diffused into the tumor matrix (e. g., acidic extracellular tumor fluid), doxorubicin was released based on a pH-sensitive mechanism and diffused then efficiently into the tumor tissue. Nanoparticle intratumoral diffusion as well as Dox release and diffusion into the dense collagen tumor matrix were size-dependent. They were more pronounced with the smallest nanoparticles, in term of diffusion depth (Figure 11-j, k vs Figure 11-l). The released Dox in the extracellular tumor environment might cross the plasma membrane of cancer cells by passive diffusion, increasing therefore the intracellular drug concentration and leading to tumor growth inhibition. To study the effect of nanoparticle size on in vivo drug delivery and tumor growth inhibition, Dox@PMSN45 and Dox@PMSN150 were administered as single intravenous injection into chicken embryos (100 µL of nanoparticles suspension, [NPs] = 3.5 mg mL-1 ; injected dose of Dox = 50 µg/egg). For this study, HT1080 fibrosarcoma tumors were grafted onto the chick embryo’s chorioallantoic membrane. We also included a saline-treated control as well as a group of embryos treated with unloaded nanoparticles (without doxorubicin). Seven days post-drug administration, tumor tissues were ex-


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cised for accurate weighing and comparative quantitative analysis of the tumor growth inhibition effect (Figure 12; n = 11 for each condition). Treatment with Dox-loaded nanoparticles clearly reduced tumor growth compared to the salinetreated control. No significant tumor growth inhibition was observed with unloaded nanoparticles, confirming that the obtained tumor growth inhibition was related to Dox delivery. More interestingly, the tumor growth inhibition was significantly nanoparticle size-dependent. Dox@PMSN45 showed a higher rate of tumor shrinkage than that observed with Dox@PMSN150. A remarkable reduction in the relative tumor weight was observed for chick embryos treated with Dox@PMSN45 that reached 40% ± 6%, compared to 25% ± 10% of tumor growth inhibition when treated with Dox@PMSN150. The difference was statistically significant (p < 0.05). These observations might be consistent with the increase of EPR effect when the nanoparticle size decreases, leading to more efficient accumulation of nanoparticles into the tumor and to better drug delivery. This is also in agreement with the more pronounced intratumoral Dox release and diffusion that were obtained with small nanoparticles, leading to better tumor growth inhibition. CONCLUSION In this contribution, we have demonstrated that particle size tuning of MSNs and their surface functionalization with phosphonate groups constitute major factors to ensure an efficient drug delivery strategy and to control the drug release rate of doxorubicin. Sub-50 nm MSNs nanocarriers (phosphonated MSNs, 45 nm) have superior ex vitro, in vitro and in vivo drug-elution and delivery performances compared to larger ones. This should be ascribed to the combination of the strong biocompatibility, colloidal stability, efficient passive targeting, intratumoral diffusion, intracellular retention and fast controlled release of such small phosphonated nanoparticles. These small nanoparticles have strong potential for the therapeutic delivery of drugs and may find significant applications in the treatment of different types of cancers. Finally, this investigation of the effect of particle size on drug delivery and elution performances represents a key step toward the development of tailored-made nanovectors for emerging applications in nanomedicine. EXPERIMENTAL SECTION Materials. Tetraethylorthosilicate (TEOS, 98%), ncetyltrimethylammonium bromide (CTAB, 99%), Pluronic F127 (EO106PO70EO106, BioReagent), 3-(trihydroxysilyl)propyl methylphosphonate monosodium salt solution 50% wt in H2O (THMP), (3-aminopropyl)triethoxysilane (APTES) were from Sigma-Aldrich (Canada). Triethanolamine and doxorubicin (Dox, doxorubicin hydrochloride salt > 99%) were obtained from Alfa-Aesar (Canada) and LC laboratories (USA), respectively. Alexa fluor 488 NHS-ester and Alexa fluor 405 goat anti-mouse IgG were purchased from ThermoFisher Scientific/Life Technology (Canada). Mouse G3BP1 (TT-Y, GTPase activating protein (SH3 domain) binding protein) was from Santa Cruz biotechnology, Inc. (Canada). Small-sized MSNs (45 nm diam). CTAB (663.2 mg), F127 (2.68 g) and triethanolamine (15.64 g) were dissolved in EtOH 100% (57 mL) and water (125 mL), followed by overnight stirring. TEOS (2.56 mL) was added, followed by the same

procedure as described above, except for the addition of 200 mL of ethanol (95%) to the translucent, colloidal suspension of the obtained mesoporous material prior to the centrifugation step. The resulting nanoparticles are designated as "MSN45". Intermediate-sized MSNs (300, 150 and 90 nm diam.). A synthesis procedure was modified from the method reported by Kim et al.47 Briefly, CTAB (1.0 g) and F127 ("x" g; x = 2, 4, 10 for MSNs with medium particle size of 300 nm, 150 nm and 90 nm, respectively) were dissolved in EtOH 100% (85 mL) and 2.9 wt% NH4OH solution (213 mL). Then, TEOS (3.86 mL) was added at room temperature (RT) under high stirring rate (1000 rpm) for 1 minute. The reaction mixture was then aged 24 h under static conditions (air, RT). The resulting product was collected by centrifugation (10000 rpm), washed twice with water and dried overnight in air at 65°C. Finally, the product was calcined (air, 550 °C, 1°C min–1, 5 h). The resulting nanoparticles are designated as "MSNx", where "x" is the mean particle size in nm scale (namely, 300, 150, or 90 nm). Large-sized MSNs (500 nm diam.). CTAB (663.2 mg) and triethanolamine (15.64 g) were dissolved in EtOH 100% (57 mL) and 2.9% NH4OH aqueous solution (141 mL). TEOS (2.56 mL) was added, and the product was treated exactly as aforementioned. The resulting nanoparticles are designated as "MSN500". Functionalization of MSNs with phosphonate. 0.13 mL of THMP was dissolved in water (13 mL) and the initially highly basic pH of the solution (pH = 12.1) was adjusted to slight acidic pH (pH should be between 5 and 6). This acidification is very important to avoid silica hydroxylation and dissolution during the grafting process and to catalyze the condensation between silanol groups on silica surface and silanol groups of the phosphonate silane (THMP). Then, this solution was added to the pure MSNx suspension (100 mg in 13 mL of water), followed by overnight reflux at 100 °C. The functionalized MSNs was isolated by centrifugation (7500G, 10 min), washed three times with water, twice with ethanol, and dried. The resulting products are designated as "PMSNx" ("x" = mean initial particle size, in nm). Functionalization of MSNs with fluorophores. Fluorescent nanoparticles were prepared by grafting the fluorophore Alexa fluor 488. For this, APTES (1 %, molar ratio APTES/TEOS) was added to the reaction after the addition of TEOS for MSN150 and MSN45, respectively. Then, a solution of Alexa fluor 488 NHS ester (1 mg mL-1 in anhydrous DMSO) was added to the suspension of MSNx-APTES (100 mg in 10 mL HEPES buffer pH = 8). The mixture was kept under gentle stirring for 1 h, in the dark. The suspension was centrifuged (7500G, 10 min) and the supernatant, discarded. The solid residue was washed three times with HEPES buffer, twice with water, and dried. The resulting nanoparticles are designated as "PMSNx-F". X-ray diffraction characterization. XRD measurements were performed using a Siemens D5000 (reflection, θ–θ configuration; CuKα: λ = 1.541 Å; 40 kV; 30 mA; 1–8° 2θ, step size: 0.02 2θ; 0.02 s/step). The Jade (v 2.1) software coupled with JCPDS and ICDD (2001 version) databases was used to analyze the XRD data. Nitrogen physisorption analysis. Nitrogen physisorption measurements were performed at −196° C with an Autosorb iQ2 (Quantachrome Instrument, Boynton Beach, USA). Before the sorption measurements, the samples were outgassed



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under vacuum at 200 °C for 12 h (for pure MSNs) or 80°C for 10 h (for functionalized MSNs). The surface area (SBET) was determined using the BET equation in the range 0.05 ≥ P/P0 ≥ 0.20 and the total pore volume was obtained at P/P0 = 0.92. Pore diameter was estimated using non-local density functional theory (NLDFT) methods applying adsorption branch model (considering N2 sorption at −196 °C in silica with cylindrical pore geometry).58 Thermogravimetric analysis (TGA). Measurements were performed using a Netzsch STA 449C thermogravimetric analyzer, under airflow of 20 mL/min, with a heating rate of 10 °C/min, between 35 and 700 °C. The percentage of grafted phosphonate groups was calculated based on the mass loss between 200 °C and 630 °C. The residual solvent (e.g. physisorbed water) was excluded from the calculation. TEM size analysis. The nanoparticles were dispersed in water, the suspension (5 µL), deposited on a carbon-coated copper grid and dried for at least 24 h. Images were taken in with a JEM-1230 TEM at an accelerating voltage of 80kV. Particle size distributions were calculated by ImageJ, based on a sample of at least 500 particles, from different images taken over different quartiles. Dynamic light scattering and zeta potential measurements. The hydrodynamic diameter of the nanoparticles was measured by dynamic light scattering (DLS) using a Malvern DTS Nano zetasizer 173° (equilibration time set to 3 min; 3 measurements taken on each sample; only quality criteria data accepted as valid results). Zeta potential dependence on pH was obtained with the same equipment, by measuring the zeta potential in phosphonate buffers (0.1M). NMR characterization. Solid-state magic-angle spinning (MAS) nuclear magnetic resonance (NMR) spectra were obtained on a Bruker DRX300 MHz NMR spectrometer. The 75.4 MHz 13C CP/MAS, the 121.4 MHz 31P MAS, and the 59.6 MHz 29Si MAS NMR spectra were obtained using a 4 mm rotor spinning at 10 kHz. The chemical shifts are reported in ppm relative to adamantane for 13C, to trifluoroacetic acid (TFA) for 31P and to tetramethylsilane (TMS) for 29Si. MSNs drug loading and in vitro drug release assays. Pure MSNx as well as functionalized nanoparticles (PMSNx) were loaded with doxorubicin (Dox). For this, 25 mg of nanoparticles were suspended in a doxorubicin solution (5 mg mL-1 in water at pH = 7.9). The suspension was shaken for 24 h at RT in the dark. Doxorubicin-loaded nanoparticles (Dox@MSNx and Dox@PMSNx) were obtained by centrifugation and washed very carefully five times, then dried under vacuum. The total Dox loading was evaluated by UV-Visible spectroscopy. In vitro release studies: PBS (1×; pH = 7.4) and phosphate buffer solution (1x; pH = 5) were used to mimic in vivo conditions. The Dox-loaded nanoparticles (5 mg) were soaked in 5 mL of each medium at 37 °C. At time points, particles were centrifuged, then 1mL of the supernatant (release medium) was removed, and replaced with fresh medium (1 mL). The percentage of released doxorubicin was evaluated by recording the absorbance of the supernatant at 480 nm, using a Varian UV-VIS-NIR Cary 500 Scan spectrophotometer. Cell viability study. Cell viability was determined by the resazurin-resorufin assay (mitochondrial function). Briefly, human skin melanoma cells (M21) and human fibrosarcoma cells (HT1080, highly tumorigenic) were incubated at 37 °C in 96-well plates for 24 h, 48 h and 72 h, with different concentrations of nanoparticles (pristine and Dox-loaded PMSNx).

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Then, 7-hydroxy-10-oxidophenoxazin-10-ium-3-one (resazurin) was added to the cell suspensions, which is reduced by viable cells into the fluorescent resorufin (7-hydroxy-3isophenoxazin-3-one) derivative. After 2 h, the reaction of resazurin into resorufin was quantified by fluorescence measurements (excitation, 530 nm; emission, 590 nm) with a SpectraMax i3x (Molecular devices). Results were performed in triplicate, and expressed as a percentage of the total fluorescence measured in the control cells (mean ± standard deviation). Nanoparticle cell uptake study. Cells were seeded on glass coverslips in a 24-well plate and cultured at 37 °C in an incubator. After 24 h, the cells were treated with 25 µg mL-1 of nanoparticles (Dox@PMSN45-F and Dox@PMSN150-F) for 2 h and 24 h, washed with PBS at least 3 times and then fixed with 4% paraformaldehyde in PBS for 10 min at room temperature. Finally, the glass coverslips were washed with PBS. Immunofluorescence staining and microscope observation: Following fixation, the glass coverslips were immersed in a solution of BSA 3% + saponine 0.1% and placed in an incubator at 37 °C for at least 45 min. the cells were then incubated at RT for 1h30 with 40 µL of diluted G3BP1 (a protein that localizes into the cytoplasm in proliferating cells). After being rinsed with PBS + 0.05 % tween 80 three times, anti-mouse IgG Alexa Fluor 408 dye was conjugated to G3BP1 by incubating the cells with 40 µL of the corresponding diluted solution (1:1000) at RT for 1h. Finally, glass coverslips were washed, and then mounted on Superfrost glass microscope slides. Images were collected using an Olympus (BX51) fluorescence microscope. The contrast in Figure 8-a and 8-b was enhanced with ImageJ software to better observe the bleu fluorescence. No further adjustments of the fluorescent images were performed for the rest. Transmission electron microscopy cell uptake study: 2 × 105 cells were incubated for 5 h with nanoparticles (PMSN45 and PMSN150; [NPs] = 200 µg mL-1). Then, the cells were collected by centrifugation and fixed using 2.5% glutaraldehyde for 48 h at 4 °C, followed by dehydration in a graded series of ethanol solutions. Finally, they were embedded in epoxy resin and stained with uranyl acetate. Thin sections were processed with an ultramicrotome (Leica JUNG RM2065) and visualized in TEM (Jeol JEM-1230, 80 kV). Chicken embryo chorioallantoic membrane assay. Fertilized chicken eggs were purchased from Couvoirs Victoriaville (Victoriaville, Québec, Canada), and incubated for 10 days in a Pro-FI incubator fitted with an automatic egg turner before being transferred to a Roll-X static incubator for the rest of the incubation time. The eggs were kept at 37 °C in a 60% humidity atmosphere for the whole incubation period. At 10 days, a hole was drilled (Dremel, Racine, WI) on the side of the embryo, and a negative pressure was applied to create a new air sac. A window was opened on this new air sac, which was covered with transparent adhesive tape to prevent contamination. Freshly prepared cell suspensions (M21 or HT1080 cancer cells) were applied directly onto the freshly exposed chorioallantoic membrane (CAM) tissue through the window: 3.5 × 105 cells/egg for HT1080 cells, and 1.5 × 106 cells/egg for M21 cells. Intratumoral injection: on day 17, the embryos were submitted to low temperature conditions (30 min at 4 ºC) to minimize motion. Then, 2.3 µL of a suspension of nanoparticles (unloaded MSN45, unloaded MSN150, Dox@PMSN45, Dox@PMSN150, Dox@PMSN45-F and Dox@PMSN150-F) in nanopure water ([NPs] = 20 mg mL-1 ; injected dose of doxorubicin = 7 µg/tumor) were directly injected in the tumor


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(30G-needle; Hamilton 1710-100 µL syringe; Ultra Micropump III injector; Micro 4 Controller from World Precision Instruments Inc, Sarasota, FL). The embryos were incubated for 1 h and 18 h. At time points (1 h or 18 h), embryos were euthanized by transfer at 4 °C. Then, tumors were collected and 1) submitted to very low temperature conditions (-20 ºC) and then imaged by the IVIS Lumina II imaging system; or 2) fixed in 4% paraformaldehyde, then embedded in paraffin, cut with a microtome in slices of 4 µm and mounted on SuperfrostTM Plus glass microscope slides for immunohistofluorescence study. Intravenous injection: on day 11, 100 µL of a suspension of nanoparticles in saline were injected intravenously into embryos for each experiment (Dox-unloaded PMSN45, Dox-unloaded PMSN150, Dox@PMSN45, Dox@PMSN150; [NPs] = 3.5 mg mL-1 ; [Dox] = 0.5 mg mL-1 ; injected dose of Doxorubicin = 50 µg/egg a limited dose of doxorubicin that could be injected in this model if not, the cytotoxicity of Dox would induce a significant mortality of chick embryos). 100 µL of saline (154 mM + 1% PenStrep (peniciline + streptomycin)) were also injected as a control. The embryos were incubated for 7 days, at which time they were euthanized by transfer at 4°C. Tumors (n = 11 for each condition) were collected, and the tumor weights were recorded. Data represent the mean ± standard deviation. Immunohistofluorescence. For staining human cancer cells in the chick embryo’s chorioallantoic tumor model, tumor slices were washed once with toluene for 8 min, twice with EtOH 100% for 1 min, once with EtOH95% for 1 min and once with water for 2 min. Then, tumor slices were immersed in a solution of BSA 3% + saponine 0.1% and placed in an incubator at 37 °C for at least 45 min. After that, tumor tissue were incubated at RT for 1h30 with 40 µL of diluted G3BP1 (a ubiquitously protein that localizes to the cytoplasm in proliferating cells) and then washed two times with PBS + 0.05 % tween. 50 µL of anti-mouse IgG Alexa Fluor 408 (1:1000) was added to conjugate this secondary antibody dye to G3BP1 at RT. After 1h, tumor slices were washed 4 times with PBS + 0.05 % tween and then glass coverslips were mounted. Images were collected using an Olympus (BX51) fluorescence microscope. The contrast in Figure 11-c was enhanced with ImageJ software to better observe the red fluorescence. No further adjustments of the fluorescent images were performed for the rest.

ASSOCIATED CONTENT Supporting Information. Supporting information includes: TEM images; fitting of in vitro Dox release data; fluorescence images of HT1080 cells; hydrodynamic diameters and polydispersity indexes of both pure and phosphonated nanoparticles; half maximal inhibitory concentrations (IC50, mean ± SD) of Dox@PMSN45, and Dox@PMSN150; details related to the incubation of NPs with cancer cells; details concerning THMP conventional post-grafting strategies. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author *Freddy Kleitz: [email protected] *Marc-André Fortin: [email protected]

ACKNOWLEDGMENT

The authors acknowledge the financial support from the National Science and Engineering Research Council (Canada) and the Fonds Québécois de la Recherche sur la Nature et les Technologies (FRQNT Team grant 2013-PR-167010). Meryem Bouchoucha is grateful to FRQNT for a PhD fellowship. The authors would like to thank Dr. Todd Galbraith (CHU-LOEX, Quebec) for the his assistance during the ex vivo fluorescence imaging study, Prof. Ryong Ryoo and Dr. Yongbeom Seo (KAIST, Korea) for inset HRTEM images as well as Ms Caroline Germain (student from INSA, Rouen) for assistance in the preparation of particles.

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FIGURES

Figure 1. TEM images of the different MSNs synthesized in this work, with their corresponding size distribution: a) and b) MSN45; c) MSN90; d) conventional MSN150; e) MSN300; f) MSN500. The insets (in a, c and d) are HRTEM images.



a)

c)

b) (211)

(220) (420) (332)

0.14 MSN500 MSN150 MSN90 MSN45

1600 1400 1200 1000 800 600

4

2θ (°)

8

0.08 0.06 0.04 0.02

200

6

0.10

400

0.00

0

2

MSN500 MSN150 MSN90 MSN45

0.12

d(d) (cm3 Å-1 g-1)

MSN500 MSN150 MSN90 MSN45

Adsorbed volume (cm3 g-1)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 12 of 22

0.0

0.2

0.4

0.6

0.8

1.0

Relative pressure (P/P0)

2

4

6

8

Pore width (nm)

Figure 2. a) Powder XRD patterns of MSNx; b) N2 physisorption isotherms of MSNx, the isotherms for MSN90, MSN150 and MSN500 samples are offset vertically by 200, 400 and 600 cm3 g- 1 STP, respectively; c) corresponding NLDFT pore size distributions.


12

Page 13 of 22

b)

a) MSN45 MSN90 MSN150 MSN300 MSN500

40 ii) 20

0

MSN45 MSN90 MSN150 MSN300 MSN500

60

Intensity (a.u.)

Number (a.u.)

60

40 ii) 20

i) 10

0 100

1000

i) 10

100

1000

Hydrodynamic diameter (nm)


c)

d) MSN45 MSN90 MSN150

60

MSN45 MSN90 MSN150

60

50

50

Intensity (a.u.)

Number (a.u.)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


40 30 20

40 30 20

10

10

0

0 10

100

1000


10

100

1000


Figure 3. DLS analyses of pure (MSNx: a and b) and phosphonated (PMSNx; c and d) nanoparticle suspensions in aqueous (i) and saline solutions (ii; vertical offset).


13


b) a) pH 5_Dox@MSN90

40

pH 5_Dox@MSN150 pH 7_Dox@MSN45 pH 7_Dox@MSN90

30

pH 7_Dox@MSN150

20

pH 7_Dox@PMSN45 pH 7_Dox@PMSN90 pH 7_Dox@PMSN150

100

pH = 5

80 i)

100 Cumulative release / %

pH 5_Dox@MSN45

pH 5_Dox@PMSN45 pH 5_Dox@PMSN90 pH 5_Dox@PMSN150

Cumulative release (%)

50

Cumulative release (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 14 of 22

60

80 60 40 20

40

0 0

10

2

4

6

8

10

Time / h

pH = 7

20 0 0 20 40 60 80 100 120 140

Time (h)

0 0

20

40

60

80

100

120

140

160

Time (h)

Figure 4. Doxorubicin release profiles from pure MSNx (a) and PMSNx (b). Inset figure in b (i) shows the Dox release profiles from PMSNx up to 10 h.


14

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 5. a) 29Si MAS NMR spectrum of pure MSNx; b) 31P MAS NMR spectrum of PMSNx; c) 13C CP/MAS NMR spectrum of PMSNx and d) 29Si MAS NMR spectrum of PMSNx. Similar spectra were obtained regardless the size of the nanoparticles. e) Zeta potential profile of the nanoparticles. f) Schematic representations of the surface of MSNx and PMSNx, with their corresponding active sites and respective pKa.


15


Figure 6. TEM images of HT1080 and M21 incubated with PMSN45 (a and b, respectively) and PMSN150 (d and e, respectively); e) PMSN45 nanoparticles taken up in vesicles and f) PMSN150 nanoparticles taken up in vesicles.

24 h

120

120

80 60 40 20

80 60 40

0 0

1.9

3.9

7.8 15.5 31.5 62.5 125 250

0

Concentration (µg mL-1)

c)

120

100

1.9

3.9

7.8 15.5 31.5 62.5 125 250

Concentration (µg mL-1)

d)

100

Cell viability (%)

HT1080 cells

PMSN150

b)

20

0

120

72 h

100

Cell viability (%)

Cell viability (%)

M21 cells

48 h

PMSN45

a)

100

Cell viability (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 16 of 22

80 60 40 20

80 60 40 20 0

0 0

1.9

3.9

7.8 15.5 31.5 62.5 125 250

0

1.9

3.9

7.8 15.5 31.5 62.5 125 250

Concentration (µg mL-1) Concentration / µg mL-1 Figure 7. Cell viability assay of M21 and HT1080 cells incubated with PMSN45 (a and c, respectively) and PMSN150 (b and d, respectively) for 24 h, 48 h and 72 h.


16

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 8. Fluorescence images (obtained at 60×) of M21 cells incubated with Dox@PMSN45-F and Dox@PMSN150-F for 2 h 30 (a and b, respectively) and 24 h (c and d, respectively). Red fluorescence: Dox; green: Alexa-fluor488 grafted on nanoparticles; yellow/orange: merged of red and green signals; blue: anti-mouse IgG Alexa Fluor408 (stains the cytoplasm of human cancer cells). e-h) cross-section images analysis based on line-scanning profile of green and red fluorescence intensity (green and red curves, respectively).


17


HT1080 cells

M21 cells a)

100

100

b) 24 h

24 h

60 40 Dox@PMSN150 Dox@PMSN45 Free Dox

20

100

80

Cell viability (%)

Cell viability (%)

80

0 0.1


20 0

0.01

1

10 Dox concentration (µg mL-1)

0.1

Dox concentration (µg mL-1)

c)

100 Dox@PMSN150 Dox@PMSN45 Free Dox

48 h

60 40 20

80

Cell viability (%)

Cell viability (%)

80

1

d)

48 h


20

0

0

100

0.01

1


0.1


e)

100

Dox@PMSN150 Dox@PMSN45 Free Dox

60 40 20 0

1

f) 72 h

72 h

80

Cell viability (%)

0.1

Cell viability (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 18 of 22

80

Dox@PMSN150 Dox@PMSN45 Free Dox

60 40 20 0

0.1

1


0.01

0.1


1

Figure 9. In vitro cytotoxicity of Dox@PMSN45, Dox@PMSN150 and free Dox against M21 cells and HT1080 cells at different Dox concentrations for 24 h (a and b, respectively), 48 h (c and d, respectively) and 72 h (e and f, respectively).


18

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 10. Ex vivo fluorescence images of M21 CAM tumor model treated with Dox@PMSN45 and Dox@PMSN150 after 1 h (a and b, respectively) and 18 h (c and d, respectively) of post-intratumoral injection. Images were obtained with IVIS Lumina II imaging system.


19


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 20 of 22

Figure 11. Fluorescence images of M21 CAM tumor treated with small Dox-loaded nanoparticles (45 nm) and large Dox-loaded nanoparticles (150 nm) after 1 h and 18 h of post-intratumoral injection. Red: Dox; green: Alexa-fluor488 grafted on nanoparticles; blue (dark or pale or flashy blue): anti-mouse IgG Alexa Fluor408 (selectively stains human cancer cells and the tumor tissue in the CAM tumor model); yellow/pale turquoise: merged of the red, green and blue fluorescence.


20

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Relative tumor weight (%)

Page 21 of 22


100

**

80

**

60

40

Figure 12. HT1080 CAM tumor weight at t = 7 days (n = 1 for each condition), normalized to the weight of tumors at t = 0. Embryos were treated with Dox-containing MSNs (Dox@PMSN45 and Dox@PMSN150), as well as their "pure" (non-Dox) counterparts (PMSN45; PMSN150). *: Results were statistically significant (p < 0.05).

TABLES Table 1. Physicochemical parameters of different sized nanoparticles obtained from nitrogen physisorption measurements.

Pure nanoparticles (MSNx)

Phosphonated nanoparticles (PMSNx)

BET Surface Area [m2 g-1]

Pore Volume [cm3 g-1]

NLDFT Mean Pore size [nm]

MSN45

1131

0.96

3.6

MSN90

1269

0.95

3.4

MSN150

1299

0.91

3.4

MSN300

1241

0.93

3.4

MSN500

1300

0.93

3.4

PMSN45

850

0.67

3.2

PMSN90

930

0.70

3.0

PMSN150

970

0.71

3.0


21


Page 22 of 22

Table 2. Release kinetics parameters.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Release exponent n

Kinetic constant k [wt.% (hn)-1]

pH 5_Dox@ PMSN45

0.50

38.9

pH 5_Dox@ PMSN90

0.51

25.1

pH 5_Dox@ PMSN150

0.58

14.1

pH 7_Dox@ PMSN45

0.7

6.9

pH 7_Dox@ PMSN90

0.71

4.6

pH 7_Dox@ PMSN150

0.75

3.3

Sample

pH 5

pH 7

Release kinetics parameters were obtained for profiles up to 90% of total amount released.

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Size-Controlled Functionalized Mesoporous Silica Nanoparticles for

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