Degradable Drug Carriers: Vanishing Mesoporous Silica

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Degradable Drug Carriers: Vanishing Mesoporous Silica Nanoparticles Karin Möller, and Thomas Bein Chem. Mater., Just Accepted Manuscript • Publication Date (Web): 29 Apr 2019 Downloaded from http://pubs.acs.org on April 29, 2019

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Chemistry of Materials

Degradable Drug Carriers: Vanishing Mesoporous Silica Nanoparticles Karin Möller* and Thomas Bein* Department of Chemistry and Center for NanoScience (CeNS), University of Munich (LMU), Butenandtstrasse 5–13, 81377 Munich, Germany Fax: (+49) 89-2180-77622 Emails: [email protected], [email protected] ORCID: T.B 0000-0001-7248-5906 K.M. 0000-0002-2712-6610

ABSTRACT The versatile potential of mesoporous silica nanoparticles (MSNs) as drug delivery agents for cytotoxic or chemically sensitive (macro) molecules has been demonstrated in numerous in-vitro and in-vivo studies. Nevertheless, a translation of MSNs into clinical applications still appears to be difficult for several reasons – one prominent concern being the uncertainty about the fate of these nanoparticles in the body. The degradability of drug carriers is a prerequisite for avoiding potentially hazardous effects upon application in living systems. Furthermore, a timely degradation might even enhance medical efficacy through efficient drug release. Knowledge about the stability of drug carrier systems and about the parameters that might influence their degradation process is therefore very valuable for developing optimal carrier designs. Hence, the hydrolytic stability/degradation of MSNs is expected to be a key feature regarding potential medical applications of mesoporous silica. So far, conclusive studies addressing the hydrolytic or bio-degradability of MSNs are limited and the available data sometimes appear to be contradictory. Here, we performed a comprehensive evaluation comparing the degradability of a number of different MSNs under biomedically relevant conditions by using low particle concentrations. We synthesized MSNs at acidic, neutral or basic pH. MSNs at basic pH were prepared as pure silica MSNs, as hybrid MSNs containing functional aminoand mercapto-groups, as well as containing additional redox-sensitive disulfide entities, all integrated via co-condensation. These samples were synthesized following a common recipe, even when changing the particle size, in order to minimize the influence of particle preparation on the dissolution kinetics. The degradation process was followed in different buffers over short and long exposure times using pristine particles or MSNs decorated with a variety of frequently used surface attachments. The quantitative assessment of the degradation process by ICP (Inductively Coupled Plasma - Optical Emission Spectrometry) was complemented with Transmission Electron Microscopy (TEM) as well as UV-VIS and FTIR spectroscopy. Cross-polarized and directly polarized 29Si Solid State NMR was applied to identify differences in connectivity in the silica network. We find that the dissolution rate at low concentrations is predominantly governed by (i) the silica network connectivity, determined by the synthesis pH and co-condensation, and (ii) by the silica building blocks. Thus, co-condensed MSNs with “interrupted” networks made under basic conditions degrade 1 ACS Paragon Plus Environment

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fastest and nearly completely within a few hours independent of particle size, while additional disulfide linkers in the pore walls retard this process. This is strongly contrasted by the behavior of purely siliceous MSNs, which are very stable when made under acidic conditions but show increasing degradability when made at higher pH. Hence, in this study we demonstrate that the aqueous stability of mesoporous silica nanoparticles can be widely tuned from almost complete to nearly no degradability under medically relevant conditions. These results establish a new set of design rules for the adaptation of multifunctional MSNs to the requirements of desired scenarios in targeted drug delivery.

Introduction Drug delivery (DD) has developed as the major area of application for mesoporous silica nanoparticles (MSNs), reflected in more than 2300 published articles starting around 2003.1-4 Even earlier, it had been demonstrated that bulk mesoporous silica was suitable for absorbing large amounts of drugs such as ibuprofen.5 However, a prerequisite for many in vitro and in vivo applications are small and tunable particle sizes between 50 to 200 nm as present in MSNs, especially to sustain good blood circulation and to (hopefully) achieve favorable biodistribution. These nanoparticles are promising drug carrier vectors since they comprise inexpensive, ready-made, scalable and long-term storable multi-purpose compartments, which can be decorated with numerous molecular functionalities both inside their pore system and at their external periphery. Furthermore, MSNs offer very high surface areas and thus loading capacities that can be easily be tuned with respect to surface properties as well as pore/particle sizes to admit guest molecules of different charges, solubility and molecular dimensions.6 The broad scope of applications in drug delivery is documented in a number of recent review articles describing more generally the use of MSNs in DD,7-11 or focusing specifically on their potential in cancer therapy,12-16 for delivering nucleic acids,17, 18 or for delivering poorly water-soluble drugs.19 An excellent comprehensive recent article spans the discussion from MSN and MON (mesoporous organosilica nanoparticles) synthesis, their biological properties including biodegradation, to delivery strategies and biomedical applications.20 It is nicely complemented by a review concentrating on the biocompatibility and drug delivery with silsesquioxane nanoparticles.21 Despite these numerous reports on successful in vitro and even in vivo drug delivery with MSNs, there are today no clinical trials involving MSNs, in contrast to over 1600 trials at various stages involving just liposomes. Only small-sized (< 10 nm) dense silica nanoparticles used as imaging aid have recently been approved by the FDA as an investigational new probe in one clinical trial.22, 23 Reasons for this reluctance are probably mostly related to the biodistribution of MSNs and their possible aggregation behavior in different body fluids. Further issues regard their stability/degradability in body fluids, which could result in particle accumulation in the human body causing unwanted side-effects. To gain more confidence in the biocompatibility of MSNs and thus to open an avenue for clinical translations it will be necessary to address these critical aspects of MSNs.24-26 Hence, we urgently need more information on the degradability of MSNs under bio-medically relevant conditions.

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Dissolution conditions for silica Silica is present in the human body in hair and nails, it is important for the growth of bone and connective tissue and it is recognized as safe by the FDA. Silica is taken up in the body as siliceous acid, the degradation product of silica. However, besides nonporous silica nanoparticles, there are a large number of different types of mesoporous silica nanoparticles distinguished through synthesis routes, morphologies, degree of structural order, particle size and pore size. MSNs also come as organic-inorganic hybrids, including organic partitions in their wall structure which are known as MONs, as well as MSNs with functionalized surfaces prepared through in-situ or post-synthesis steps. While the saturation concentration of solid amorphous silica or similarly silica gel was measured to be about 115 ppm at neutral conditions and 25°C27, 28 it was found to vary with the solution pH as well as the synthesis conditions of the particles. Thus, the solubility of each type of MSN will depend on structural parameters and is presumably different for all these forms of nanoscale MSNs. Consequently, not only the stability and resorption will change with the synthesis parameters of MSNs, but also their biomedical performance including possible side effects. While toxicity assessments of MSNs are usually obtained by viability assays on specific cell types, mostly demonstrating the good biocompatibility of MSNs, there is only limited information on the degradability of these silica particles. What is known about the degradation of MSNs Excellent reviews have recently been published discussing the solubility of different forms of silicon and dense silica as well as the biodegradability and renal clearance of inorganic nanoparticles, including (ordered) mesoporous silica nanoparticles.29, 30 However, there are only few articles primarily concerned with the solubility of porous silica nanoparticles and hybrids thereof. For an overview we have summarized the existing literature on the degradability of mesoporous silica and mesoporous organosilicas in Table S1 (see Supporting Information, SI). It is evident that the reported materials differ vastly in their timescale of degradability. Therefore we have included the MSN synthesis parameters as well as the respective dissolution conditions in this table. We also included the analytical methods used since a number of the listed reports rely on qualitative assessments of MSN degradation (TEM), while a quantitative measurement, e.g. via ICP, would allow for a better comparison. An important parameter for dissolution is the MSN concentration. When stored at high concentrations of > 10 mg/mL in ethanol, MSNs can have an extremely long shelf-life. We have measured nitrogen sorption isotherms on a number of our MSN samples that were stored for over 3 years under these conditions and have found nearly identical surface area and pore size values, morphologies as measured by TEM and DLS particle size distributions (see SI, Fig. SI-1 and SI-2). However, MSN concentrations such as those used in drug-delivery experiments are highly diluted in aqueous media and dissolution studies should be conducted accordingly. In vitro experiments have shown that MSNs are usually non-toxic to cells in concentrations up to 100 µg/mL. In vivo studies in mice are performed with MSN concentrations ranging from 20 mg to as low as 2 mg MSN/kg body weight.31-34 With mice weighing about 25 g and having a blood volume between 1.5-2.5 mL, concentrations between 0.25 to 0.025 mg MSN/mL are used, which is close to or even well below the solubility limit of silica. It is thus necessary to study the behavior of silica carrier systems at or below the solubility limit of about 0.1 mg/mL, in buffer solutions of different pH and at 37°C to approach conditions close to in vivo studies. Several of the reports listed in Table S1 3 ACS Paragon Plus Environment


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(SI) discuss data in the range of these conditions. In general it was found that the particle size is of minor importance in contrast to MSN concentration or surface area. Even large micron-sized particles dissolved in a similar time frame as nano-sized particles, namely within a day when kept close to the solubility limit.35, 36 However, the reported degradation rate of different MSNs varies tremendously from complete dissolution in several hours to only partial dissolution in over one week when kept at similar low concentrations.37, 38 These contradictory results stimulated a development of different hybrid silica systems, where either stabilizing organic bridges39 or in contrast destabilizing dopants40-42 were implemented into the silica framework. Reducible disulfide groups were further incorporated to stimulate a site-specific degradation within the cancer cell.43 Scope of the present study An optimum carrier for drug delivery purposes should fulfill seemingly contradictory conditions in being stable (and “stealth”) enough for a prolonged blood circulation, however labile enough to prevent long lasting accumulation of carrier debris. A conclusive judgement about these properties based on literature data is hampered by missing comparative studies and non-standardized experimental conditions. We have started this study on our multifunctional MSN particles that we have used before successfully in drug delivery applications. These MSNs feature a core-shell partitioning of chemical properties made by time-delayed co-condensation reactions. To obtain a comprehensive view on MSN particle degradation we also included more common, completely siliceous MSNs of similar size prepared following a similar synthesis route at basic, as well as neutral pH. For particles made at acidic pH we had to change the reaction conditions. Subsequently, all samples were handled in a similar way regarding template removal and dissolution conditions. Using low MSN concentrations of 0.1 mg / mL, samples were evaluated in different buffers and were analyzed in short term (hours) as well as longer term (days) studies. Earlier work in our group had concentrated on the structural break-down of related MSN particles in simulated body fluid (SBF) solutions at higher concentrations of 2 mg/mL.44, 45 It was found that even under those more concentrated conditions the morphology, i.e. the surface area and pore volume are easily compromised within a few days, or even faster when MSN particles were co-condensed with phenyl groups. This degradation process could be retarded by covering the surface with polyethylene glycol (PEG) ligands. These studies were based on structural methods such as XRD, nitrogen sorption and electron microscopy analysis of remaining solid particles. Here, we greatly extend these studies by following the dissolution process under biomedically relevant low concentrations by various means, focusing on the quantitative assessment of the dissolved silica under various conditions using ICP. We find that the dissolution kinetic of MSNs is widely tunable and that it correlates with the structure of the MSNs, reflected by the connectivity of the silica network.

Results and Discussion Synthesis of the MSN particles MSN particles can be prepared using a variety of reaction conditions. The standard approach in our group is performed at a basic pH, adjusted with triethanolamine (TEA), which allows for a good control over hydrolysis and condensation rates and thus the generation of nanoparticles with variable particle sizes.46 Additionally, our group has established core-shell structured MSN particles by creating MSNs with regio-specific chemical properties. This is done by integrating specific 4 ACS Paragon Plus Environment

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organosilanes either into the inner or the outer region of the particle body via a time-delayed cocondensation procedure47 (a representative synthesis is described in the SI). We have used these core-shell MSN particles in a number of drug-delivery applications. For instance, this approach has allowed us to load very large amounts of siRNA exclusively into the inner pores of MSNs while endosomal escape agents were located on the surface. This way we could achieve a luciferase gene suppression of over 80% by using extremely low MSN concentrations of only 25 µg/mL.48 The spatial structuring of our nanoparticles proved also to be successful in the selective delivery of cis-platin to human ex-vivo 3D lung tumor tissue while sparing the healthy tissue.49 A comprehensive description of possible MSN variations following this core-shell synthesis method and an overview of our drug delivery applications can be found in our recent review.50 A schematic overview over this synthesis approach is depicted in Scheme 1. Scheme 1: Schematics of two possible synthesis routes resulting in MSNs with core-shell partitioned chemical properties: (top) amino-functionalized core covered by a mercaptofunctionlized shell or (bottom) amino-functionalized core containing S-S-bridges in the pore walls, covered by a mercapto-functionlized shell. Two examples of preparation routes are sketched here for particles that are used in our degradation studies. A mixture of TEOS, base TEA and silane R1 (here: aminopropyl triethoxysilane (APTES, 9 mol % of total silica)) is added to the reaction solution containing the surfactant, stirred at 60°C. This forms the inner core of the MSN particle. A second, smaller amount of an equimolar mixture of TEOS and R2, here mercaptopropyl triethoxysilane (MTES, 1 mol % of total silica) is added after 30 minutes, thus forming the MSN shell with oppositely charged residues. This reaction can be varied as depicted in the lower synthesis route in Scheme 1 where another partition, R3, here bis(3-triethoxysilylpropyl)disulfide (BTDS), is simultaneously added with R1 in the first step resulting in MSN particles containing additional disulfide groups in the wall structure. This core-shell MSN configuration can be exploited for spatially-selective labeling or for a covalent attachment of other ligands (such as polyethylene glycol, PEG, avidin etc.), as we have used in our dissolution studies (see below). Completely siliceous MSNs are similarly prepared by using simply 100% TEOS instead of the multicomponent mixtures. To simplify the recognition of the particle composition we will use small icons as seen in Scheme 1 in the following text. The approach of a time-delayed co-condensation allows for a broad variation of MSN particle compositions made in a one-pot synthesis. In order to tune the particle size we can simply vary the TEOS : TEA ratio as shown below. Following the synthesis, a repeated template extraction is always performed with an acidified ethanol mixture under reflux for 45 minutes. Dissolution of co-condensed MSN (MSN-C) and the influence of particle size The co-condensed samples, here containing 9 mol % APTES in the core and 1 mol % MTES in the shell were prepared with different particle sizes and abbreviated as S, M, L = small, medium and large size; C = co-condensation. The average particle size was varied from about 50-60 (sample S-C), to 110 (sample M-C) and 240 nm (sample L-C) as determined from TEM images and measured by DLS in water as shown in Figure 1 a-c (see also SI for a list of the surface properties of all samples in Table S2). The surface areas and pore sizes of these three highly functionalized MSN samples are very 5 ACS Paragon Plus Environment


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similar around 700 m2/g and 4.8 nm, respectively (nitrogen sorption isotherms of all samples and an illustration of the synthesis reproducibility are shown in Fig. SI-3). This large pore size (as compared to about 3.5 nm in standard MSNs) was obtained by adding the pore expansion agent tri-isopropyl benzene (TiPB) to the template cetyltrimethyl ammonium chloride (CTAC). Figure 1 e, f shows a representative nitrogen sorption isotherm and pore size distribution of sample M-C.

Figure 1 a-c) Transmission electron micrographs of MSNs containing 9% NH2-groups in the core and 1% SHgroups in the shell with varying particle sizes, obtained by changing the TEOS : TEA ratio from 1:3 in sample S-C (a), to 1:10 in sample M-C (b) to 1:15 in sample L-C (c); d) corresponding particle size distributions (DLS measurements performed in water), e) representative nitrogen sorption isotherm and f) corresponding pore-size distribution of sample M-C. In our dissolution experiments we prepared a stock solution containing 0.1 mg MSN/mL of the respective sample and placed, for each data point, 3 aliquots of 2 mL into 2 mL Eppendorf tubes that were then shaken at 37°C in water or buffer solutions for the desired time period. These aliquots were rigorously centrifuged and 1 mL of the supernatant was carefully removed for subsequent ICP analysis regarding the Si content. After removing the residual supernatants the solid residues were taken up in ethanol and exemplarily studied with TEM. In Figure 2 we give an overview over a number of different dissolution experiments that were followed quantitatively by ICP. In Figure 2a and 2b we show the influence of the particle size using the samples as shown above in Figure 1. Figure 2a shows the long-time dissolution behavior of the bare co-condensed particles. These particles were stirred between 4 h and 2 days in simulated body fluid (SBF). All three samples show a surprisingly high degree of dissolution ranging between 70 and 98% already after 4 h, showing no trend for the particle size (Figure 2a). Also under other dissolution conditions as described later (Figure 2b and 2c), we find that the particle size of our co-condensed MSNs has no dominant impact on the degradation process. Subsequent studies were thus performed only with medium-sized samples M-C.


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Figure 2: Dissolution of co-condensed large (L), medium (M) and small-sized (S) MSNs as measured by ICP a) L, M, S MSN particles in SBF; b) particles with covalently attached PEG to the outer surface; c) comparison of the short-term dissolution behavior of sample M-C when performed in SBF or water; d) short-term measurements of sample M in water with surface decorations as listed in the legend. The effect of the particle size has also been studied in several previous publications (see Table S1, SI), however, only with purely siliceous MSNs. Nevertheless, in accordance with our studies particle size was not found to affect the dissolution kinetics. However, the reported time span to almost complete dissolution of these particles varies greatly in the different reports. It lasted up to 6 days for MSN particles sized between 20 to 80 nm as analyzed by Yamada et al..51 Their results might have been influenced by their experimental conditions, as they studied the dissolution with samples kept in dialysis bags without stirring. Other researchers also found long dissolution times of even over 8 days with similar particles. In contrast, Braun et al. measured degrees of dissolution of 80 to 100 % already after 6 h with purely siliceous MSNs, independent of particle size ranging between 80 to 1500 nm.37 Their samples were stirred in buffer solutions at 37 °C at concentrations of 0.1 mg/mL, similar to our measurements. Another study reported that the aspect ratio had more influence than the particle size, e.g. that spherical particles (80 nm) degraded faster than rods (200 and 400 nm), however incompletely and over a long time period of 60 days.52 7 ACS Paragon Plus Environment


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The influence of surface coverage with PEG (L-M-S) MSN-C-PEGout Since our functionalized MSNs were prepared via a co-condensation approach, they inherently carry functional groups on the surface (in the pore system and/or on the outer particle surface), other than in most other studies where purely siliceous MSNs were used. In drug delivery applications, functional groups are usually exploited for the attachment of labeling or targeting agents or for the covalent coupling of guest molecules in the particle interior. In order to investigate if covalent attachments at these surface groups might change the dissolution process, we decorated the outer, the inner, or the combined functional groups of our MSN-C particles with a number of different ligands. In experiments shown in Figure 2b we coupled polyethylene glycol (PEG) via mercaptoreactive linkers (methoxypolyethylene glycol maleimide, MW 750 g/mol) to the exterior of the same samples (samples L-, M-, S-C-PEGout) as shown in Figure 2a. PEGylation has been reported to achieve a more favorable blood circulation for MSNs, with less accumulation in liver and spleen and a slower degradation than particles without PEG.53 Moreover, these MSNs did not show tissue toxicity after 1 month. The data in Figure 2b confirm that the dissolution rate is slightly reduced in the presence of the PEG linker. A similar trend was observed in our earlier dissolution studies using higher MSN concentrations of 2 mg/mL.44 However, the influence of PEG is not significant since a high dissolution degree between 60 to 75% is reached here also already after 4 hours, again showing no obvious dependence on particle size. We have further investigated the bare sample as well as the PEGylated sample shown above after 2d and 5d, respectively with TEM and by EDX analysis after separating their solid residues from the supernatants by prolonged centrifugation. In both samples, we found mainly salt residues originating from the buffer solution and only traces of Si, thus confirming our ICP results that show a near complete dissolution (see SI, Figures S-4). However, these buffer residues can easily obscure any analysis with TEM. We have therefore tested if the salt concentrations in these buffer solutions change the degradability of our samples. For that purpose we studied the short-term behavior of sample M-C in SBF buffer as used before and compared it to a pure aqueous solution. As seen in Figure 2c) we obtained nearly identical results. Thus, in the following we performed all dissolution experiments in water. Investigation of internal and external surface coverage (MSNs -C-ATTOin, -C-ATTOin/out and -C-ATTOin/PEGout) To investigate if a covalent coupling of hydrophobic residues to the inner amino groups or outer mercapto groups influences the dissolution process, we attached amino- or mercapto-reactive dyes to sample M-C, either only on the inside (M-C-ATTOin,) or inside-out (M-C-ATTOin/out, with ATTO488 NHS or ATTO-488 MAL, respectively) or dye to the inside and PEG to the outside (M-CATTOin/PEGout). Samples were again investigated for their short-term behavior in water, starting at 30 minutes and up to 4 h and were analyzed by ICP. As shown in Figure 2d, we do observe a short delay in dissolution in the first two hours, but then again a rapid dissolution that reached 70 to near 100 % after 3-4 h when ATTO dyes were attached. Only when PEG was covalently bound on the outside of the particles we noticed a shielding effect against hydrolytic degradation, now even more pronounced as in Figure 2b) where no ATTO-dye was attached in the inside of the particles.


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We analyzed the short-term solid residues of the M-C-ATTOin sample by TEM, and results are summarized in Figure 3: Compared to the starting material M-C in Figure 3a, the radial pore structure and the spherical particle shape are dramatically altered after only 3 h stirring at low concentration in water. The residue shows a complete loss of porosity, a rougher particle shape of slightly smaller size and often a denser outer layer around the particles. This restructuring starts already after 1 h (see also SI, Figure S-5). The sample is nearly completely dissolved after 1 d and at this point only a few flakes can be found by TEM as shown in Figure 3c, confirming the fast degradation found by ICP. A similar structural evolution was observed recently by Shi et al., who studied the dissolution of hollow, grafted mesoporous silica particles in PBS, DMEM and in contact with cell cultures. However, their particle restructuring occurred on a longer time scale lasting between 1 and 2 weeks, likely owing to the purely siliceous nature of the particles and their higher silica concentration of 0.4 mg/mL, above the solubility product. Interestingly, the authors found an increasing degradation rate depending on the medium, ranging from PBS to DMEM to intracellular medium, and they proposed a degradation governed by particle hydrolysis as opposed to a biodegradation route involving biological activity.54 We analyzed the supernatants of our degraded samples also with DLS for the presence of remaining particles or fragments thereof. We just noticed spurious particles, detectable only at high instrumental amplification, and found no indication for smaller fragments. This supports a degradation mechanism via rapid molecular hydrolysis into silicic acid rather than fragmentation (see SI, Figure S-5).

Figure 3: Transmission electron micrographs of co-condensed sample M-C with amino groups in the core and mercapto groups in the shell, a) Pristine sample M-C after synthesis, b,c) M-C-ATTOin with ATTO dye attached inside after b) 3 h and c) after 1 d stirring at 37°C in water. 9 ACS Paragon Plus Environment


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Influence of pore-gating agents biotin, biotin-avidin or a lipid bilayer (samples M-C-ATTOin/biotin-out, M-C- ATTOin/biotin-avidin-out, M-C ATTOin/DOTAPout) The covalent dye-labeling of the MSN particle interiors enabled us to additionally follow the dissolution process with UV-VIS spectroscopy. For this, supernatants of the dissolved samples were retrieved at intervals between 1 and 20 hours to follow concentration changes of the detached dye. We used this method to study the influence of frequently used pore-gating agents such as biotinavidin or the lipid bilayer DOTAP. To do so, we coupled biotin covalently to the external MSN mercapto groups via a pyridyl-dithiol linker. Subsequently, we attached avidin by exploiting its affinity to biotin. This large protein has also been used as capping agent in drug delivery applications with MSNs and might be expected to provide shielding against degradation around the silica particles.55 To study the effect of an alternative peripheral layer structure on dissolution kinetics, we also formed a lipid bilayer shell around the MSN-C particles using the lipid DOTAP, which has been used before as protecting/pore-closing gate layer around silica particles (see SI for synthesis). The UV-VIS measurements of supernatants of these samples are summarized in Figure 4. The sample composition is schematically represented by our sample icons and the progress of dissolution is visualized by time-based color coding of the spectra. All samples show a fast increase in intensity of ATTO residues in solution with time, independent of the external functionalization. A maximum of the dye concentration was reached after similar reaction times between 3 and 4 h under all circumstances, no matter if the outer surface was left untreated (Figure 4a), covered with biotin (Figure 4c), with additional avidin (Figure 4d) or enclosed by a DOTAP lipid bilayer (Figure 4b). Hydrolysis and thus dissolution occurs fast and apparently unhindered in the different samples when stirred in water at this low concentration. At most it is slightly delayed when a biotin-avidin complex is present on the periphery. Hence, the different surface decoration does not seem to strongly influence the dissolution process. We hypothesize that all of the above surface decorations are to some degree permeable for water and thus cannot prevent its access into the inner pore system of the MSNs.


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Figure 4: UV-VIS analysis of the dissolution process of different derivatives of sample M-C-ATTOin with a covalently anchored dye at the particle core. Spectra of the respective supernatants are shown. Samples are as synthesized (M-C-ATTOin, a), covered by a DOTAP layer (M-C-ATTOin/DOTAP, b) or with biotin anchored at the surface (M-C-ATTOin/biotin, c) which is additionally complexed by avidin on the shell (M-C-ATTOin/biotin-avidin, d). Dissolution was performed in water at 37°C at a particle concentration of 0.1 mg/mL. Influence of MSN concentration The absorption maximum of the ATTO-dye 488 at 501 nm as observed by UV-VIS increases on a comparable time scale as the Si content in the supernatants of sample M-C-ATTOin when measured by ICP (see SI, Figure S-6a). The similarity between both curves points to a rapid dissolution of the inner structure of the MSNs and explains the drastic restructuring observed in the TEM images (Figure 3). We used this efficient UV-VIS method for measuring the degradation for following the dissolution as a function of MSN concentration: we compared solutions with the usual MSN concentration (sample M-C-ATTOin) of 0.1 mg/mL after 3 h stirring with samples of 0.2 and 0.4 mg/mL concentration. When doubling the sample amount to 0.2 mg/mL instead of seeing a correlated increase in dissolved silica, we only observed an absorption maximum nearly identical to the sample with 0.1 mg/mL in the UV absorbance spectrum . With a fourfold MSN concentration even a decrease in intensity in intensity is seen (SI, Figure S-6c). Thus, apparently the dissolution rate is reduced when the MSN concentration is increased. Accordingly, for the more concentrated samples we found increasing solid residues in the Eppendorf tips after centrifugation. We attribute these findings to surpassing the solubility limit of silica in these more concentrated solutions. This would also explain the observed long shelf-life of these particles when kept at high concentration. Similar trends regarding the concentration dependence of dissolution were also found with our 11 ACS Paragon Plus Environment


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hybrid samples containing disulfide groups in the MSN walls (see below) and were also reported by others, for instance in an early study by the group of Shi.35 They examined 200-300 nm sized unfunctionalized siliceous MSNs in SBF buffer at 37 °C between 0.1 to 0.5 mg/mL concentration and found dramatic differences when the dissolved silica was measured by ICP. The most dilute particles were dissolved to over 90 % within 3 h compared to only 35% at the highest MSN concentration. Their template-extracted MSNs were also much more labile than calcined MSNs, which dissolved by just 30% even after 15 days. Consequently, dissolution studies have to be performed at conditions that closely resemble the final application. Furthermore, degradation results can only be compared when studied at similar concentrations. Can blood serum change the dissolution behavior? So far we have discussed the dissolution behavior of MSNs in water or buffered solutions, conditions that are quite different from those that exist in the human body. To also compare the dissolution behavior under simulated biological conditions, we examined the stability of three selected samples upon exposure to fetal bovine serum (FBS), which contains many proteins that are encountered when samples are administered systemically. Samples included the purely siliceous sample Si-B, and two samples where either (i) the inner pore or (ii) the outer shell is protected by (i) a phenyl residue (sample M-C-Ph; 1 % phenyltriethoxysilane core/1 % SH shell ) or (ii) a peripheral PEG linker (sample M-C-PEG; 9% NH2 core /1% PEG shell, the latter coupled via mecapto groups). An amount of 1 mg sample each was stirred for 1 hour in FBS at 37°C, washed and then finally studied in aqueous solution as done before (0.1 mg/mL, 37°C). Aliquots from the time series were analyzed by ICP and the results are shown in Figure 9. Sample Si-B did show a similar low dissolution rate before and after being exposed to SBF. Similarly, the 1% co-condensed sample M-C-Ph was only dissolved to an extent of about 30 % after 3 days. Surprisingly, sample M-C-PEG (green curve) was much more stable after this treatment. Here, we measured only 38 % dissolved silica after 3 h, which increased to 48 % after 3 days. This is in strong contrast to near complete dissolution after 3 h of the same sample with or without PEG when never exposed to FBS (see also Figure 5 and included points, after 3 d, in Figure 9).

Influence of synthesis pH and co-condensation – comparison with purely siliceous MSNs Nearly all reports on dissolution deal with completely siliceous MSNs, often prepared under different reaction conditions. We wanted to evaluate if the process of molecular co-condensation might influence the stability of these MSNs. To this end, we prepared purely siliceous MSNs following the same synthesis procedure at basic pH as before and replaced the amino-and mercapto-silanes by equimolar amounts of TEOS (sample Si-B: siliceous basic). Furthermore, we prepared plain silica MSNs at a different synthesis pH simply by drastically reducing the concentration of the base TEA in our standard recipe, until a neutral synthesis pH was reached (sample Si-N: siliceous neutral). In order to also have a comparison with a mesoporous silicate sample made under strongly acidic conditions, we adapted a recipe by Gao et al.56 to obtain MSN particles of comparable size (sample Si-A: siliceous acidic, see SI for synthesis, Figure S-7 for TEM and DLS data of all siliceous samples before and during dissolution). These three plain silica samples and additionally a co-condensed sample M-C were then stirred in water as before and analyzed with ICP at different time points between 1 and 7 days. We also 12 ACS Paragon Plus Environment

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included a second co-condensed M-C sample to show deviations between identically synthesized samples. Notably, the plain siliceous sample prepared at basic pH (Si-B, Figure 5, green dots), made without co-condensation showed a pronounced resistance against dissolution. Increased degradation starts very much later and reaches only 59% after 7 days as compared to 80-98% in the co-condensed samples. Furthermore, when analyzing the siliceous MSN particles made at a lower pH, e.g. under neutral and acidic reaction conditions, we found an even smaller tendency to dissolve, decreasing to 17 % (Si-N) and only 8% (Si-A) dissolution after 7 days (Figure 5, red and blue dots).

Figure 5: Dissolution measurements of plain siliceous MSNs synthesized under basic (Si-B), neutral (Si-N) and acidic (Si-A) conditions in comparison to two different batches of co-condensed samples M-C made at basic pH, as measured by ICP (0.1 mg MSN/mL in water). In contrast to the rather stable siliceous samples, the co-condensed MSN samples showed a nearly complete dissolution in water, similar to the behavior in SBF buffer as displayed before in Figure 2. We note that the BET surface area of the acidic sample Si-A is the lowest (306 m2/g) when compared to the samples made at neutral pH (Si-N, 540 m2/g) or basic conditions (Si-B, 1130 m2/g; see SI, Table 2). However, the highly degradable co-condensed sample (M-C, 694 m2/g) has also a smaller surface area than the purely siliceous samples made under basic conditions and still dissolves most rapidly, indicating that this factor cannot explain these large differences in stability. We carefully separated the remaining solid residues of the more stable sample Si-N and analyzed it with TEM as shown in Figure 6. Striking differences are observed in comparison with the cocondensed samples M-C as documented above in Figure 3. Notably, nearly no structural changes occur in the siliceous sample Si-N made at neutral pH, even after prolonged stirring over 7 days at low concentration,. The pore structure in this sample is still clearly maintained, quite in contrast to 13 ACS Paragon Plus Environment


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sample M-C. The sample is still nicely dispersed and not completely disintegrated as observed for the co-condensed particles. We could also retrieve solid residues of the siliceous sample Si-B made at basic pH. This sample shows a higher sensitivity to morphology changes (see SI, Figure S-5). It transforms into doughnut-shaped particles with a thinner particle center after 1 day, similar to the co-condensed sample. However, this silica sample Si-B is still much more stable than the cocondensed samples M-C made under basic conditions and retains its transformed but still porous particle structure even at day 7

Figure 6: TEM micrographs of siliceous sample Si-N synthesized at neutral pH. a) as synthesized, b) after 1 day, c) after 7 day stirring in water at 37°C. These results clearly show that changes in the MSN synthesis conditions can cause dramatic variations in the degradability of MSNs. Influence of the inclusion of reducible disufide groups in co-condensed MSN pore walls (M-C-S) The frequently observed high stability of pure silica MSNs was a reason for several research groups to insert biodegradable groups, preferably disulfide-containing silanes, into the silica network to achieve a better particle fragmentation and thus potentially less bio-accumulation.57 Conceptually, disulfide groups are expected to facilitate particle decay via reductive bond-cleavage initiated by glutathione (GSH). GSH is present in all cells, but at increased concentration in nearly all cancer cells58, and thus could ideally induce a regio-selective cargo release from these hybrid silica nanoparticles upon fragmentation inside the cytosol. The successful implementation of disulfide bridges into silica had been shown earlier for small nonporous Stöber particles,59 and was later established for periodic mesoporous organosilica (PMO) nanoparticles by the group of Durand,60 as well as for hollow mesoporous organosilicas (HMONS) in Shi´s group.61 Very recently, tetra- or di14 ACS Paragon Plus Environment

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sulfide-bridged silanes have been used for partial replacement of TEOS in MSNs and in silica nanoshell constructs.43, 62-66 In the existing literature above, it is suggested that inclusion of these disulfide groups can induce sample degradation through reductive S-S bond cleavage.43, 60 A demonstration of the degradation process is usually performed upon exposure to GSH and by showing changes in particle morphology through disintegration into small fragments, as captured in TEM images. Sample degradation was typically reported to occur within 2 to 14 days, in some instances suggesting a near complete dissolution (see SI, Table S-1, hybrid mesoporous silicas). A recent study reported by the group of Shi documents the degradation products of their disulfide-containing 50 nm MSN particles also by using TEM images, but complemented by quantitative analysis via ICP, and further by comparison to purely siliceous MSNs.67 They used a low particle concentration (0.1 mg/mL) in SBF solution containing the reducing agent GSH. While TEM images suggest substantial particle disintegration of the S-S-MSNs, ICP showed only about 45% dissolved silica after 2 days, somewhat more than the pure silica MSNs (30%). Furthermore, the dissolution degree of the S-S-MSNs was nearly unchanged for up to 7 days, with or without GSH treatment and increased only after 14 days in GSH solution. This indicates that the complete particle degradation into silicic acid is likely not primarily triggered by the presence of disulfide groups in the MSNs. As shown below, our studies related to disulfide (or tetrasulfide) groups in MSNs confirm these conclusions. We have recently implemented disulfide- bis-alkoxy silanes into our core-shell cocondensed MSN-C systems.50 Now we ask how these disulfide groups influence the degradation process when increasing concentrations of di- or tetra-sulfide spacers are introduced into either our usual co-condensed MSNs (M-C-S) or into purely siliceous MSNs (Si-S) made under similar reaction conditions. To this end, we replaced varying amounts of TEOS in our basic recipe with BTDS or BTES (bis-[(triethoxysilyl) propyl]-disulfide or -tetrasulfide, S or S4, respectively), represented in Scheme 1 as R3, following the bottom route. The substitution of 10 or 30 mol% of the silica precursor with BTDS in the synthesis solution results in mesoporous MSNs with high surface areas between 890 and 700 m2/g, even if a concomitant co-condensation was performed. However, less porous MSNs with just 290 m2/g were obtained when 50 mol% BTDS was used in the reaction solution (see SI, Table S3). Surface areas also decreased when smaller particle sizes were prepared or when the longer tetrasulfide spacer was included. These latter low surface area samples were not included in our studies.



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Figure 7: a,b) TEM micrographs of -NH2 and -SH co-condensed sample with 10 or 30 mol% of TEOS replaced by the disulfide-bridged silane BTSE (samples M-C-S10 and M-C-S30). c) ICP results comparing co-condensed samples (M-C, blue) with disulfide-enriched samples M-C-S (red or green) when dissolved in water at pH = 6.0 without GSH present, or d) in a 10 mM aqueous solution of the reducing agent GSH at pH 6.2 in PBS buffer. TEM pictures of these co-condensed samples indicate that the radial pore architecture is retained when 10 % of TEOS is replaced with BTDS (M-C-S10). Higher S-S contents, replacing 30% of TEOS, produce a more irregular structure, possibly creating thicker walls (M-C-S30, Figure 7). The successful S-S inclusion is also reflected by increasing weight losses in the TGA (not shown) as well as by systematic changes in the Raman spectrum (see SI, S-9). Degradation studies with the 10 % and 30 % S-S samples (M-C-S10 and M-C-S30) were performed as described above, and the ICP results are shown in comparison to the co-condensed sample M-C with no disulfides in its structure (Figure 7 c). Notably, we observe that the disulfide linker slows down the dissolution significantly with respect to the parent sample M-C, and that the S-S content makes no difference - both samples behave similarly. Surprisingly, this holds true even when the samples are stirred under reducing conditions with a 10 mM GSH solution in PBS (pH 6.2, Figure 7 d). Bondbreakage and an increased degradation is not observed. The dissolution degree is even slightly less than without GSH and is unaffected by the S-S concentration in the MSN. Even in extended time studies for up to 7 days under varying conditions, including GSH, we find that the mesoporous particles containing disulfides are more stable than the parent co-condensed MSN-C sample. Only when we inserted a tetrasulfide bis-silane did the degradation increase to about 80 % after 7 days in the presence of GSH, and thus becomes similar to the parent sample (see SI, Fig. S-10). We assume 16 ACS Paragon Plus Environment

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that the disulfide groups stabilize the MSN particles due to a higher organic content introduced through the propyl bridges and that the S-S bonds are likely hidden within the silica walls. Quignard et al. reported similar findings with solid Stöber particles, namely that the dissolved silica content decreased with an increasing content of disulfide groups in their particles when stirred in acetate buffer at pH 4.5. They registered nearly no differences in degradation with particles containing 10 up to 40 % S-S in the presence of DTT within the first 10 days.68 A slow degradation of S-S bonds caused by steric hindrance was also proposed by Elzes et al. in their cystamine disulfide-based polymers (polyamido amine) when they successively implemented increasing numbers of methyl groups close to the disulfide linkage.58 Additional evidence for a decreasing degradability in our samples upon S-S inclusion is provided through TEM, DLS and UV-VIS studies (see Supporting Information). We could never observe fragmentation of our particles without or with exposure to GSH (using TEM, see Figures S 11-13 ) and noticed only a very slight indication for S-S bond reduction under reductive conditions in highly substituted (30 or 50 % S-S or S4) samples (using UV-VIS and Raman spectroscopy, see Figures S-14 to S-16). In summary, all our experiments with disulfide-containing samples indicate that the particle degradation process is significantly delayed when compared to the co-condensed sample M-C. An efficient stabilization of the M-C samples is already obtained by replacing just 10% of TEOS by BTDS (M-C-S10). Do structural differences dominate the degradability of MSNs? All the MSN samples discussed here, except the purely siliceous sample Si-A made at acidic pH, have been prepared and extracted under conditions as similar as possible. Why do they behave so differently when it comes to dissolution at low concentration? To better understand these differences, we performed solid state magic-angle spinning NMR measurements. As representatives for the different synthesis conditions we selected the two purely siliceous samples made at acidic and basic pH (Si-A and Si-B), which are contrasted to our parent co-condensed sample M-C (made at basic pH) and compared to the co-condensed sample M-C-S30 made with an additional 15 % of the SS precursor. A combination of 29Si-1H CP (cross-polarized) and 29Si DP (directly-polarized) SS-MAS NMR spectroscopy was employed for these studies. CP-SS-MAS NMR is commonly used to detect the presence of functionalized silica generated either by post-synthesis grafting or through cocondensation. This method excites the abundant protons of the organic residues and transfers their polarization to the silicon atoms being two to three bonds apart, thus probing predominantly the silicon atoms in close proximity to hydrocarbons while de-emphasizing Q4 silicon atoms. For a quantitative evaluation of the silica condensation it is necessary to directly excite the Si spins by DPMAS-NMR, at the expense of very long accumulation times due to the slow relaxation times. The respective NMR spectra of our samples (CP-MAS-NMR: blue spectra, DP-MAS-NMR: red spectra) are shown in Figure 8, where we included the nomenclature for the possible coordination spheres of the silicon atoms.



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Figure 8: Magic Angle Spinning Solid State NMR data; 1H -> 29Si cross-polarized in blue, directly polarized 29Si NMR in red; a, b) purely siliceous sample prepared at acidic pH, sample Si-A, c, d) purely siliceous sample prepared at basic pH, sample Si-B, e, f) co-condensed sample with 9% aminopropyl silane at basic pH, sample M-C and g, h) sample M-C-S30, co-condensation with 9% aminopropyl silane and 15% disulfide precursor BTDS. The chemical shifts and relative concentrations of different silicon species extracted from the integrated peak areas are collected in Table S-4 in the Supporting Information. As expected, for the purely siliceous MSN samples Si-A and Si-B prepared at acidic (a, b) and basic conditions (c, d) we find only resonances at the typical chemical shifts for completely coordinated and partially hydroxylated silicon in both, the CP and in the DP-NMR spectra.69 For these two siliceous samples we note the substantial difference in the degree of condensation of the Si-O-Si network expressed in the ratio Q3/Q4, which is 0.42 in Si-A but much higher in Si-B (0.77), indicating the abundant presence of hydroxyl groups and concomitantly lower network connectivity in the latter sample. The group of Zhao found a value of about Q3/Q4 = 0.4 for their siliceous MSNs made at an oil/water interface at nearly neutral conditions using the same catalyst TEA as in our study. They proposed that a low level of crosslinking favors faster degradation.70 In accordance with this hypothesis, we view the high value of Q3/Q4 = 0.77 in our Si-B sample, indicating lower network connectivity, as the main reason for its increased hydrolytic degradability in comparison with sample Si-A. How do the co-condensed MSN samples fit into this picture? As described above, the co-condensed samples M-C containing additional amino- and mercapto- groups (and their surface-decorated derivatives) show a profoundly higher degradability than the purely siliceous samples. For this MSN-C sample, we see strong new T2 and T3 signals in the CP spectrum in Figure 8e, showing that the cocondensation of the two organosilanes with TEOS was successful. If their integrated intensities in the DP spectrum are considered (see SI, Table S-4) we find 2.7 % (T2) and 3.1 % (T3), respectively. The corresponding sum of 5.8 % is less than the sum of 9 mol% APTES and and 1 mol% MPTES as offered in the synthesis, thus a slightly incomplete co-condensation has occurred. Some of the surface hydroxyls were consumed by the co-condensation as reflected by a slight reduction of the Q3 18 ACS Paragon Plus Environment

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intensity. We consider the organically functionalized T3 and T2 sites in the silica network as large defects interrupting the silica network in the MSN walls, hence contributing to the high sensitivity towards hydrolytic degradation. A recent study using two-dimensional 29Si-29Si dynamic nuclear polarization (DNP) enhanced NMR on co-condensed MSNs found evidence for self-condensation or clustering of APTES with partial embedding in the MSN walls.71 These results corroborate our hypothesis, viewing the co-condensed organic species as interruption of the silica network. Considering the disulfide-bridged silanes of sample M-C-S30, they are clearly incorporated into the MSNs to a large degree as demonstrated by the NMR spectra in Figure 8 g and h. Prominent T3 CP and DP signals occur at similar chemical shifts as for APTES and MPTES, such that they cannot be discriminated from each other. Quantitative analysis shows the presence of 33.8 % T3 signal, which is very close to the offered amount of 15 % BTDS (or 30 % with regard to Si atoms) even if we assume the same partition of 5.8 % APTES/MPTES combined in this signal. As long as the hydrophobic disulfide bridge is not cleaved, it will keep the network connected and intact at this site. We therefore view the inclusion of such a bridged hydrophobic defect to be the cause for the higher hydrolytic stability of the sample M-C-S30 in comparison with the parent sample M-C. Lastly, the implemented amino- and mercapto-groups can induce a higher polarity at the surface due to their potential deprotonation or protonation in comparison to plain siliceous MSNs as illustrated by zeta potential measurements (see SI, S-17), which might further contribute to their pronounced hydrolytic degradability. In order to substantiate our hypothesis that co-condensation introduces defects in the particle wall structure and consequently facilitates hydrolytic break-down we varied the concentration of aminogroups in the particles, thus assuming a decrease in defect structure. Instead of the usual 9% (M-C) we reduced this content to 6% (M-C-6) and followed the dissolution behavior. Figure 9 summarizes the ICP-results of both samples and compares them to those of the purely siliceous sample Si-B. As predicted, we observe a diminished dissolution in sample M-C-6 relative to M-C and a drastically reduced dissolution kinetics in the purely siliceous sample Si-B. This clearly shows the dominant influence of the degree of co-condensation and once more documents the higher stability of purely siliceous MSN particles. This higher stability of Si-B is further supported in Figure S-18 where we intentionally varied the MSN concentration between 80 to 120 µg/mL. In all cases we observed a similarly slow dissolution behavior ranging between 10 % (1 day) to maximal 50 % after 5 days.



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Figure 9: ICP results comparing the influence of co-condensation on the dissolution kinetics. Blue: sample M-C-9 containing 9% amino-groups in the core, green: sample M-C-6 containing 6% aminogroups in the core and pink: the purely siliceous sample Si-B. Taken all these results together, we recall that the morphology and pore sizes are very similar in all of the MSN samples discussed here (except sample Si-A), hence these factors (as well as the particle size) can be excluded as origin of the observed differences in stability. Furthermore, using these particles and covalently attaching various frequently used ligands either on the inside, and/or the periphery of the particles we could show that a) nearly no changes in dissolution rate are observed besides a stabilization when a PEG-layer is present on the outside, b) that the dissolution occurs mainly from the inside and that c) the pore structure is lost first. By implementing disulfide-bridged organic linkers into the pore wall of these co-condensed particles we could show that this retards the dissolution kinetics, likely brought about by the hydrophobicity of these bridging linkers. By comparing the co-condensed samples with purely siliceous MSN particles made by using the same synthesis routine we could show that these siliceous particles are more stable and, supported by Solid State NMR measurements, that this is most likely based on fewer defects in the pore structure. The same argument holds also true for the more stable silica MSNs made under neutral or even acidic conditions. We have thus comprehensively shown that the dominant factor supporting degradation is the presence of defects in the MSN wall structure, which are predominantly present in co-condensed samples. Thus, the network connectivity and the presence of hydrophobic partitions in the particles dictate the fate of the MSNs under dilute aqueous conditions. This proposed mechanism is schematically summarized in Scheme 2.


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Scheme 2: MSN particles synthesized under acidic conditions have the highest degree of connectivity and thicker pore walls. Increasing the synthesis pH increases the concentration of hydroxyl groups and reduces the wall thickness; co-condensation with organosilanes creates additional defects in the pore walls, causing kinetic instability against hydrolytic attack under dilute conditions.

Do we have to consider protein adsorption when studying particle stability? In this manuscript we have discussed the dissolution behavior of MSNs in water or buffered solutions, conditions that are quite different from those that exist in the human body. Considered as an outlook, in order to get a more realistic picture of the dissolution behavior under simulated biological conditions, we performed preliminary experiments using fetal bovine serum (FBS). We tested the stability of three selected samples after being exposed to FBS, which contains many proteins that are encountered when samples are administered systemically. With these samples we observed a drastically reduced dissolution rate (shown in Figure S-19 in the SI). Also the cocondensed, PEG-decorated sample M-C-PEG was only dissolved to 48% after 3 days when exposed to FBS (as opposed to about 80% without FBS) even though PEGylation was previously shown to strongly reduce the binding of proteins to nanoparticles.72 However, the formation of protein coronas specifically on silica nanoparticles is a complex process and is expected to be dependent on a variety of physical parameters of the specific nanoparticles.73, 74 The adsorption of proteins can even be intentionally exploited for achieving a high targeting efficiency. 75 However, our results suggest that protein adsorption can strongly influence the degradation of MSNs and we suggest that additional studies regarding the fate of different MSNs in biological environments are needed to ultimately judge and control their behavior in vivo.

Conclusions Mesoporous silica nanoparticles are inexpensive multipurpose carrier systems that can be produced at low cost in large quantities and that have an extremely long shelf-life of several years when kept under controlled conditions. However, when used in low concentrations, as is typical for medical 21 ACS Paragon Plus Environment


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applications the solubility of these versatile carrier systems can span a wide range such that they are degradable within time periods of only hours to over a week. In our comprehensive study we established a platform enabling a reliable comparison of different MSN particles by following a common synthesis approach. We varied the synthesis pH, the particle size, the particle composition and also the particle surface decorations. Importantly, by using quantitative elemental analysis we could show that neither the particle size nor different covalent surface attachments have a major impact on degradability. In contrast, the synthesis conditions (pH) and the particle composition (modified via co-condensation) determine the fate of MSN particles. The least degradable particles are purely siliceous MSNs made under acidic synthesis conditions (stable for more than a week), while MSNs made under basic conditions and particularly by inclusion of co-condensed functional groups can be dissolved in water within hours. Moreover, we implemented disulfide-bridged silanes into our highly degradable MSN particles and could show that these moieties slow down the degradation process even under reducing conditions, which we attribute to their hydrophobic nature and enhanced network connectivity. Quantitative NMR results provide strong evidence for the decisive role of silica network connectivity regarding the degradation rate under hydrolytic conditions. Hence, we have established a convenient protocol to tune the degradability of MSNs by either changing the MSN synthesis pH and/or by introducing an increasing number of defect sites through co-condensation. These synthetic strategies allow us to adapt the degradability of mesoporous silica nanoparticles to different scenarios in drug delivery.

Supporting Information Supporting information includes: A tabulated review of existing publications concerning the degradation behavior of mesoporous silicas, including metal-doped and periodic-/disordered mesoporous organosilicas (PMOs , MONs, S-S containing MSNs); Experimental section: synthesis procedures and tabulated properties of MSNs, additional analytical results, including nitrogen sorption, ICP, UV-VIS, Raman spectroscopy, FTIR, TEM, DLS and solid state NMR measurements (CP and DP 29Si MAS NMR). Acknowledgements We thank the Deutsche Forschungsgemeinschaft (DFG) for financial support (SFB 1032). Additional support is gratefully acknowledged from the Excellence Cluster Nanosystem Initiative Munich (NIM) and from the Center for NanoScience Munich (CeNS).


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TOC text: Drug carriers should be non-toxic and ideally leave the body without a trace. We establish that mesoporous silica nanoparticles (MSNs), having a shelf-life of several years under suitable conditions, can vanish completely in several hours via dissolution in aqueous phase and that the rate of degradation can be widely tuned through the specific synthesis conditions. An in-situ cocondensation of silica with functional silanes in the MSNs prepared at basic pH enables the highest degradation rate within a few hours. In contrast, purely siliceous MSNs made under acidic conditions are nearly unaffected under comparable conditions even after 7 days. Dissolution rates increase in the following order: silica MSNs created under acidic conditions < silica MSNs created at neutral pH < silica MSNs created under basic conditions

Degradable Drug Carriers: Vanishing Mesoporous Silica

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