Water Di ﬀ usion Modulates the CEST E ﬀ ect on Tb(III)-Mesoporous Silica Probes

: The anchoring of lanthanide(III) chelates on the surface of mesoporous silica nanoparticles (MSNs) allowed their investigation as magnetic resonance imaging (MRI) and chemical exchange saturation transfer (CEST) contrast agents. Since their e ﬃ ciency is strongly related to the interaction occurring between Ln-chelates and “bulk” water, an estimation of the water di ﬀ usion inside MSNs channels is very relevant. Herein, a method based on the exploitation of the CEST properties of TbDO3A-MSNs was applied to evaluate the e ﬀ ect of water di ﬀ usion inside MSN channels. Two MSNs, namely MCM-41 and SBA-15, with di ﬀ erent pores size distributions were functionalized with TbDO3A-like chelates and polyethylene glycol (PEG) molecules and characterized by HR-TEM microscopy, IR spectroscopy, N 2 physisorption, and thermogravimetric analysis (TGA). The di ﬀ erent distribution of Tb-complexes in the two systems, mainly on the external surface in case of MCM-41 or inside the internal pores for SBA-15, resulted in variable CEST e ﬃ ciency. Since water molecules di ﬀ use slowly inside silica channels, the CEST e ﬀ ect of the LnDO3A-SBA-15 system was found to be one order of magnitude lower than in the case of TbDO3A-MCM-41. The latter system reaches an excellent sensitivity of ca. 55 ± 5 µ M, which is useful for future theranostic or imaging applications. PEG5000-COOH Organo-Modiﬁed


Introduction
The distinctive properties of mesoporous silica nanoparticles (MSNs) in terms of their tunable mesoporosity, high thermal stability, chemical versatility, and good biocompatibility, have come to receive increasing attention by the scientific community [1,2]. In particular, the use of mesoporous silica for the preparation of novel diagnostic and biomedical probes has seen an exponential growth of literature reports in the last decade [2][3][4][5]. Among the possible applications, the magnetic resonance imaging (MRI) and theranostic fields have taken advantage of the presence of empty channels and high specific surface areas for the development of efficient and more sensitive MRI contrast or theranostic agents [6][7][8][9][10].
On the other hand, in 2000, Balaban et al. [11] reported for the first time the feasibility to generate contrast in MR images through the application of CEST probes [12][13][14]. In the last decade, a number of molecules have been investigated as possible CEST agents. In particular, both diamagnetic (e.g., polysaccarides, peptides, etc.) [12,15,16] and paramagnetic (Ln-based PARACEST complexes) [12,13,[17][18][19] have been described as efficient for in vitro and in vivo contrast generation and as responsive MRI probes [20][21][22][23][24]. The peculiarity of such agents is that they generate a frequency-encoded contrast, thus the contrast arises only if the proper radiofrequency pulse is applied.
Then, the pH was raised to 8.5 and the mixture was stirred for 2 h. Centrifugation at 10,000 rpm for 5 min at 298 K allowed the separation of Tb(OH) 3 from the solution. Finally, the solvent was removed under reduced pressure leading to the formation of the desired product as a powder. ESI-MS: m/z calc. for Tb(III)-DO3A (C 20

Synthesis and Functionalization of the Mesoporous Silica Supports
MCM-41 was purchased from Sigma-Aldrich Co. LLC. SBA-15 was prepared following a procedure reported in the literature [35]. In detail, 4.0 g of Pluronic P123 were dissolved in 30 mL of water and 120 mL of HCl 2N with stirring at 308 K. Then, 8.5 g of TEOS were added to the solution and stirred at 308 K for 24 h. The mixture was aged at 373 K in autoclave for 24 h. The solid product was filtered and washed several times with H 2 O. The calcination of the solid was carried out increasing the temperature at 1 K/min under air flow from room temperature to 823 K and heating the material at 823 K for 5 h. NH 2 /MCM-41 and NH 2 /SBA-15: the calcined MCM-41 (1.0 g) and SBA-15 (1.0 g) samples were firstly treated under vacuum at 250 • C for 2 h to activate the surface. Then, the materials were suspended in toluene (100 mL) under N 2 and 3-aminopropyltriethoxysilane (40 wt %) was added. The mixtures were stirred at 323 K for 20 h. The suspension was then filtered, and the products washed with diethyl ether to remove the unreacted silane. Finally, the samples were dried at 333 K for 2 h.

Anchoring of Tb(III)-DO3A Complexes and PEG5000-COOH to the Organo-Modified MSNs
NH 2 /MCM-41 (300 mg) and NH 2 /SBA-15 (300 mg) were suspended in DMF (20 mL) for 30 min. In parallel, the Tb(III) complex (260 mg) containing a free carboxylic acid was dissolved in DMF (30 mL) and activated by adding one molar equivalent of HATU and DIPEA. The solutions of the activated complexes were added to the suspensions of NH 2 /MCM-41 and NH 2 /SBA-15 and stirred at 323 K for 24 h. The suspensions were then filtered, and the solids were washed several times with H 2 O to remove the unreacted complex. Both the solids were functionalized with PEG molecules. In detail, 210 mg of TbDO3A-MCM-41 and TbDO3A-SBA-15 were suspended in 10 mL of water. In parallel, 50 mg of PEG5000-COOH were dissolved in 10 mL of water in the presence of 19.2 mg of EDC and 46.3 mg of Sulfo-NHS. The solution was added to the previous suspensions and stirred at 298 K for 20 h. Finally, the suspensions were filtered and the powders were washed with water (20 mL) in order to remove the unreacted PEG molecules and dried at 323 K for 4 h.

Characterization of Materials
The amount of Tb(III) in the final hybrid material was measured by ICP-MS technique. The elemental analyses were performed with a Thermo Scientific (Waltham, MA, USA) X5 series inductively coupled plasma mass spectrometer. Prior to the analysis, the samples were mineralized by dissolution in 1:1 HNO 3 /HF at 373 K for 24h.
Electrospray ionization mass spectra (ESI MS) were recorded on an SQD 3100 mass detector (Waters), operating in positive or negative ion mode, with 1% v/v formic acid in methanol as the carrier solvent.
HRTEM images were collected on a JEOL 3010 high resolution transmission electron microscope operating at 300 kV. Specimens were prepared by dispersing the samples by sonication in isopropanol and by depositing few drops of the suspensions on carbon-coated grids.
Infrared (IR) spectra were recorded under vacuum conditions in the range 4000-400 cm −1 at 4 cm −1 resolution using a Bruker Equinox 55 spectrometer. N 2 physisorption measurements were carried out at 77 K in the relative pressure range from 1 × 10 −6 to 1 P/P 0 by using a Quantachrome Autosorb 1MP/TCD instrument. Prior to the analysis, the samples were out gassed at 373 K for 3 h (residual pressure lower than 10 −6 torr). Apparent surface areas were determined by using the BET equation, in the relative pressure range from 0.01 to 0.1 P/P 0 . Pore volume and diameter were defined by the Barret-Joyner-Halenda (BJH) approach (applied to the desorption branch) for MCM-41 derivative samples and by NLDFT method for SBA-15 and functionalized systems.
Dynamic light scattering (DLS) measurements were performed by using a Zetasizer NanoZS at 298 K (Malvern Instruments; Malvern, UK) operating in a particle size range from 0.6 nm to 6 µm, equipped with a He-Ne laser with λ = 633 nm. Before the analysis, the samples were dispersed in PBS solution (20 mg/mL).

CEST Experiments
For CEST experiments, TbDO3A-MCM-41 and TbDO3A-SBA-15 MSNs were suspended in water at the concentration of 20 mg/mL and added with xanthan gum (2 mg/mL) to stabilize the suspension. In order to avoid silica aggregation, they were sonicated for 45 min, 59 Hz, power 80%, 313 K in an ultrasonic bath. Samples of Tb(III)-MSNs at variable concentration or pH were prepared for MRI acquisitions.

MRI Acquisitions and Image Analysis
Z-spectra were acquired at 7.1 T on a Bruker Avance 300 spectrometer equipped with a microimaging probe. A frequency-offset range of ± 150 ppm was investigated. A typical RARE spin-echo sequence (RARE factor varying from 4 to 32 depending on the T 2 of the sample) with an echo time of 3 ms and a TR value of 5 s was used. An isotropic 64 × 64 acquisition matrix with a FOV of 30 mm and a slice thickness of 1 mm were used. The whole sequence was preceded by a saturation scheme consisting of a continuous rectangular wave pulse 2 s with a proper radiofrequency B 1 . The Z-spectra were interpolated by smoothing splines to identify the zero-offset on a pixel-by-pixel basis of the bulk water and, then, to assess the correct ST% value over the entire range of frequency offsets investigated [36]. Custom-made software, compiled in the Matlab platform (Mathworks Inc., Natick, MA, USA), was used. CEST effect is calculated by using the following formula: where M S is the intensity of the bulk water NMR signal after the irradiation on resonance (∆ω) of the mobile proton pool and M 0 is the intensity of the bulk water NMR signal after the irradiation at −∆ω. T 2w images were acquired by using a standard T 2w RARE sequence with the following parameters (TR = 5000 ms, TE = 5.5 s, FOV = 1 cmx1 cm, slice thickness = 1 mm, RARE factor = 4-32 depending on the T 2 of the sample). T 2 values were measured by using a MSME sequence (TR = 2000 ms, 10 variable TE ranging from 11 to 500 ms, FOV = 1 cm × 1 cm, slice thickness = 1 mm).
The functionalization reactions were monitored by infrared spectroscopy (Figure 2). In particular, the IR spectra of MCM-41 and SBA-15, collected at room temperature under vacuum, showed a typical band at 3745 cm −1 due to the stretching of isolated silanol groups (Si-OH) and a wide absorption centered at 3500 cm −1 attributed to the Si-OH groups involved in hydrogen bonds [37]. The amount of OH groups exposed on the silica surface was quantified by thermogravimetric analysis, (323-1273 K) under Argon flow, and resulted to be for MCM-41 and SBA-15, 2.16 and 4.50 per nm 2 , respectively (Supplementary Figure S2). A drastic decrease of these IR absorptions was observed after reaction with aminopropyltriethoxysilane as a consequence of the consumption of the silanol groups (Figure 2A,B). The peaks typical of the amino groups, 3370 and 3300 cm −1 assigned to the asymmetric and symmetric stretching modes and 1595 cm −1 due to the bending of NH 2 , were observed in both the organo-modified solids. Thermogravimetric analysis allowed estimating the amount of NH 2  The functionalization reactions were monitored by infrared spectroscopy (Figure 2). In particular, the IR spectra of MCM-41 and SBA-15, collected at room temperature under vacuum, showed a typical band at 3745 cm −1 due to the stretching of isolated silanol groups (Si-OH) and a wide absorption centered at 3500 cm −1 attributed to the Si-OH groups involved in hydrogen bonds [37]. The amount of OH groups exposed on the silica surface was quantified by thermogravimetric analysis, (323-1273 K) under Argon flow, and resulted to be for MCM-41 and SBA-15, 2.16 and 4.50 per nm 2 , respectively (Supplementary Figure S2). A drastic decrease of these IR absorptions was observed after reaction with aminopropyltriethoxysilane as a consequence of the consumption of the silanol groups (Figure 2A,B). The peaks typical of the amino groups, 3370 and 3300 cm −1 assigned to the asymmetric and symmetric stretching modes and 1595 cm −1 due to the bending of NH2, were observed in both the organo-modified solids. Thermogravimetric analysis allowed estimating the amount of NH2 groups on the MSNs surface as, approximately, 2.0 and 4.0 mmol g −1 for SBA-15 and MCM-41, respectively (Supplementary Figure S3).
Finally, IR spectra of the materials after both chelate conjugation and pegylation (hereafter named TbDO3A-MCM-41 and TbDO3A-SBA-15) are characterized by the presence of two absorptions: i) at 1650 cm −1 , attributed to the stretching of amide groups formed by the coupling of both Tb(III)-complexes and PEG to the surface and ii) at 1615 cm −1 assigned to the stretching modes of COOunits of the Tb(III)-DO3A chelates (Figure 2A,B). The spectra also showed the typical bands of the amino functionalities (i.e., absorption at 1595 cm −1 ), indicating that part of the total amount of -NH2 groups did not participate into the conjugation reaction. The amount of Tb in the final materials was measured by inductively coupled plasma mass spectrometry (ICP-MS) and resulted to be 0.08 and 0.23 mmol g −1 for TbDO3A-MCM-41 and TbDO3A-SBA-15, respectively.
The distribution of the Tb(III)-chelates in the two organo-modified silica was evaluated by N2 physisorption analysis at 77 K. MCM-41, NH2-MCM-41 and TbDO3A-MCM-41 showed a type "IV" isotherm (IUPAC classification) with an "H2" hysteresis loop (Supplementary Figure S4) [38]. MCM-41 showed a specific surface area (SSA) (estimated by Brunauer-Emmett-Teller, BET method) of 905 m 2 g −1 and a pore diameter of 2.8 nm (see Figure 3 and Table 1). The anchoring of NH2 groups led to a reduction of both the volume and diameter of pores of about 25% and 15%, respectively.  (Figure 2A,B). The spectra also showed the typical bands of the amino functionalities (i.e., absorption at 1595 cm −1 ), indicating that part of the total amount of -NH 2 groups did not participate into the conjugation reaction.
The amount of Tb in the final materials was measured by inductively coupled plasma mass spectrometry (ICP-MS) and resulted to be 0.08 and 0.23 mmol g −1 for TbDO3A-MCM-41 and TbDO3A-SBA-15, respectively.
The distribution of the Tb(III)-chelates in the two organo-modified silica was evaluated by N 2 physisorption analysis at 77 K. MCM-41, NH 2 -MCM-41 and TbDO3A-MCM-41 showed a type "IV" isotherm (IUPAC classification) with an "H2" hysteresis loop (Supplementary Figure S4) [38]. MCM-41 showed a specific surface area (SSA) (estimated by Brunauer-Emmett-Teller, BET method) of 905 m 2 g −1 and a pore diameter of 2.8 nm (see Figure 3 and Table 1). The anchoring of NH 2 groups led to a reduction of both the volume and diameter of pores of about 25% and 15%, respectively. The amount of Tb in the final materials was measured by inductively coupled plasma mass spectrometry (ICP-MS) and resulted to be 0.08 and 0.23 mmol g −1 for TbDO3A-MCM-41 and TbDO3A-SBA-15, respectively.
The distribution of the Tb(III)-chelates in the two organo-modified silica was evaluated by N2 physisorption analysis at 77 K. MCM-41, NH2-MCM-41 and TbDO3A-MCM-41 showed a type "IV" isotherm (IUPAC classification) with an "H2" hysteresis loop (Supplementary Figure S4) [38]. MCM-41 showed a specific surface area (SSA) (estimated by Brunauer-Emmett-Teller, BET method) of 905 m 2 g −1 and a pore diameter of 2.8 nm (see Figure 3 and Table 1). The anchoring of NH2 groups led to a reduction of both the volume and diameter of pores of about 25% and 15%, respectively.    TbDO3A-MCM-41 showed a significant decrease (almost 50%) of both specific surface area and pores volume ( Figure 3A, Table 1). In agreement with parent systems [25,30,31] it can be inferred that the complexes are mainly anchored on the external surface of MCM-41 ( Figure 1) and part of them are in close proximity to the pores entrance, promoting a partial blocking of the channels. Also, SBA-15 and the functionalized systems showed type "IV" isotherms but with an H1 hysteresis loop, representative of mesoporous cylindrical or rod-like pores (Supplementary Figure S5) [30]. As shown in Table 1 and Figure 3B, the functionalization of SBA-15 with amino groups led to a decrease of both SSA and pores size, and a further restriction of the pores diameter was observed after conjugation of Tb(III)-DO3A chelates and PEG molecules (from 7.6 to 6.5 nm). These data are in line with those already reported for similar systems suggesting that an important fraction of Tb(III) complexes are confined inside the channels.
It has already been shown for Gd(III)-complexes anchored onto MCM-41 and SBA-15 MSNs that the different localization of the chelates greatly influences the relaxometric properties of the materials [30]. Thus, the CEST properties of TbDO3A-MCM-41 and TbDO3A-SBA-15 were investigated in order to obtain further insights into the physical properties of such nanomaterials, and especially to evaluate the influence of the reduced water diffusion into the pores on the CEST effect.
Although the particle dispersion and stability were improved by the presence of the PEG chains, the quite large amount of MSNs used (20 mg/mL) required the addition of 2 mg/mL of xanthan gum to favor the stability over time of the suspension and thus the accurate CEST measurement. DLS analysis measured at 298 K in PBS buffer evidenced the presence of micrometric aggregates for both the functionalized materials with a heterogeneous distribution of the particles size mainly evident for the pegylated TbDO3A-SBA-15 suspension (Supplementary Figure S6).
The CEST properties of TbDO3A-MCM-41 and TbDO3A-SBA-15 were evaluated at 7.1 T, 21 • C, pH 7 by acquiring Z-spectra on a Bruker 300 MHz spectrometer equipped with a micro-imaging probe. The RARE (rapid acquisition with refocused echoes) sequence, typically employed for CEST MR imaging, was preceded by the pre-saturation scheme that consists in a continuous rectangular block pulse (B 1 in the range 12-42 µT, irradiation time 2 s). The ST%-and Z-spectra of both Tb-MSNs and the control silica (without anchored complexes) with B 1 = 24 µT are shown in Figure 4A  In the case of non-functionalized MSNs, no CEST effect is detectable, given the close proximity of the chemical shift values of water and hydroxyl protons in the absence of a paramagnetic center (data not shown). Conversely, when TbDO3A complexes are anchored to the silica surface, a largely shifted CEST signal is present for both materials. The CEST peaks appear broader at higher concentrations of MSNs, for the presence of micrometric aggregates of the nanoparticles, as suggested by DLS, and the T2 shortening. The detection threshold for TbDO3A-MCM-41 and TbDO3A-SBA-15 was assessed by progressive dilution of silica suspensions in saline phosphate buffer (PBS) solution ( Figure 4C).
As shown in Figure 4C, for TbDO3A-MCM-41 the ST% is higher than 5% (considered as threshold for the visualization) up to a silica concentration of 0.7 mg/mL, that corresponds to a Tb(III) concentration of 55 ± 5 μM. These results are in line with those recently reported for the analogue MSN system without PEG chain, thus showing that PEG does not influence CEST effect. Differently, for TbDO3A-SBA-15 the ST% is higher than 5% up to a silica concentration of 2.3 mg/mL that corresponds to a Tb(III) concentration of 530 ± 10 μM. Therefore, the latter system requires ten times higher concentration to be detected by CEST-MRI.
As shown in Figure 4D, the chemical shift of MSNs changes with the concentration of the It is well known that saturation efficiency (ST%) increases by enhancing the applied B 1 presaturation fields [39]. However, the most important limitation is the occurrence of a high tissue energy deposition (i.e., high specific absorption rate, SAR) when strong irradiation powers are employed. This hampers the use of high B 1 value for in vivo applications of CEST methodology [40]. The B 1 value of 24 µT has been largely used in preclinical CEST studies by using PARACEST agents and nanosystems [39,[41][42][43]. For this reason, we choose to use this irradiation power.
In the case of non-functionalized MSNs, no CEST effect is detectable, given the close proximity of the chemical shift values of water and hydroxyl protons in the absence of a paramagnetic center (data not shown). Conversely, when TbDO3A complexes are anchored to the silica surface, a largely shifted CEST signal is present for both materials. The CEST peaks appear broader at higher concentrations of MSNs, for the presence of micrometric aggregates of the nanoparticles, as suggested by DLS, and the T 2 shortening. The detection threshold for TbDO3A-MCM-41 and TbDO3A-SBA-15 was assessed by progressive dilution of silica suspensions in saline phosphate buffer (PBS) solution ( Figure 4C).
As shown in Figure 4C, for TbDO3A-MCM-41 the ST% is higher than 5% (considered as threshold for the visualization) up to a silica concentration of 0.7 mg/mL, that corresponds to a Tb(III) concentration of 55 ± 5 µM. These results are in line with those recently reported for the analogue MSN system without PEG chain, thus showing that PEG does not influence CEST effect. Differently, for TbDO3A-SBA-15 the ST% is higher than 5% up to a silica concentration of 2.3 mg/mL that corresponds to a Tb(III) concentration of 530 ± 10 µM. Therefore, the latter system requires ten times higher concentration to be detected by CEST-MRI.
As shown in Figure 4D, the chemical shift of MSNs changes with the concentration of the nanoparticles. However, the chemical shift of TbDO3A-SBA-15 MSNs increases much more than in the case of TbDO3A-MCM-41 MSNs probably as a consequence of the presence of micrometric aggregates of the SBA-15 nanoparticles.
Finally, the pH dependences of CEST signals for both materials were also assessed showing that the ST% effect slightly changes in the 6.0-8.5 pH range ( Figure 4E). A possible explanation of the slight reduction of the ST% effect at higher pH values can be the partial deprotonation of the silanol groups. Samples at pH < 6 were not investigated in order to avoid substantial alterations of the silica surface and thus to change the interaction between the silanol groups and the LnDO3A complexes.
Furthermore, both systems clearly act as T 2 contrast agents because of the presence of Tb-complex. Finally, the pH dependences of CEST signals for both materials were also assessed showing that the ST% effect slightly changes in the 6.0-8.5 pH range ( Figure 4E). A possible explanation of the slight reduction of the ST% effect at higher pH values can be the partial deprotonation of the silanol groups. Samples at pH < 6 were not investigated in order to avoid substantial alterations of the silica surface and thus to change the interaction between the silanol groups and the LnDO3A complexes.
Furthermore, both systems clearly act as T2 contrast agents because of the presence of Tbcomplex. T2w and ST%-maps of TbDO3A-MCM-41 and TbDO3A-SBA-15 at variable concentration or pH are reported in Figure 5. T2 measurements of specimens are reported in Figure S9

Discussion
Some years ago, we reported that LnDO3A-MCM-41 MSNs can be efficient CEST-MRI contrast agents [25]. In particular, the paramagnetic metal of LnDO3A complexes anchored on the silica surface can interact, in a revertible way, with multiple -OH moieties of silanol groups on the particle surface. This interaction generates a very sensitive CEST system, much more than the classical monomeric PARACEST agents (e.g., LnHPDO3A complexes), but with a chemical shift closer to

Discussion
Some years ago, we reported that LnDO3A-MCM-41 MSNs can be efficient CEST-MRI contrast agents [25]. In particular, the paramagnetic metal of LnDO3A complexes anchored on the silica surface can interact, in a revertible way, with multiple -OH moieties of silanol groups on the particle surface. This interaction generates a very sensitive CEST system, much more than the classical monomeric PARACEST agents (e.g., LnHPDO3A complexes), but with a chemical shift closer to water resonance.
Different lanthanides (i.e., Eu, Tm, Tb, Gd) were investigated and the Tb-complex was demonstrated to be the best one, as a result of the larger chemical shift and excellent sensitivity down to the mM range. Starting from those results, herein we compared two different kind of MSNs, i.e., MCM-41 and SBA-15 functionalized with a Tb(III)-DO3A derivative. The synthesis followed experimental procedures previously reported and the anchoring process was monitored by IR spectroscopy and thermogravimetric analysis. The data suggested that a large fraction of the NH 2 groups exposed on the silica surface were involved in the reaction with the Tb(III)-complex and the functionalized PEG molecules. The N 2 physisorption data clearly indicated that the location of the complexes is different in the two solids. In case of MCM-41, characterized by pores with 2.8 nm diameter, the Tb(III)-chelates are distributed on the external surface or in the pores entrance as testified by the strong reduction of the pore volumes. In contrast, the complexes are located mainly inside the channels of the SBA-15, as it is composed by large pores (diameter of 8.5 nm).
In this kind of CEST-MRI nanoprobes, the CEST effect arises because of the multiple interaction between the silanol groups in proximity to the anchoring site and the LnDO3A metal center. In addition, another hypothesis of the large CEST efficiency might be related to an increased prototropic exchange rate [25]. However, in both cases, the difficulty of the water molecules to reach the paramagnetic centers inside the silica pores strongly hampers the efficiency of such mechanisms. For this reason, the CEST efficiency of TbDO3A-MCM-41 is much higher (one order of magnitude) than the one displayed by TbDO3A-SBA-15. In particular, a ST = 5% is reached when TbDO3A-MCM-41 have silica concentration of 0.7 mg/mL (corresponding to 55 ± 5 µM of Tb). On the contrary, for TbDO3A-SBA-15, ST = 5% is reached at a silica concentration of 2.3 mg/mL (corresponding to 530 ± 10 µM of Tb).
These results indicate that MCM-41 MSNs are more efficient than SBA-15 MSNs because most of the Tb-complexes are in the external surface of silica nanoparticles, hence water can easily interact with them.
In contrast, in the case of SBA-15 MSNs, the Tb-complexes are mainly located inside the pores where the water diffusion is hindered and, therefore, they are in part silent in terms of CEST effect.

Conclusions
This work shows how physico-chemical properties of the nanosystems play a pivotal role in generating the CEST effect. Hence, much attention has to be devoted to the properties of the MSNs, which are not only a carrier of the paramagnetic complexes, but strongly contribute in modulating the ST effect. This is important for understanding the determinants of the efficiency of such systems as MRI contrast agents. Furthermore, besides the use of this information for generating highly sensitive and efficient MRI contrast agents, CEST analysis was demonstrated to be a suitable and easy tool for investigating the water diffusion in silica channels, thus providing insights into physico-chemical properties of this kind of nanosystems.