Silica nanocarriers with user-defined precise diameters by controlled template self-assembly

Journal Pre-proofs Silica nanocarriers with user-defined precise diameters by controlled template self-assembly Tânia Ribeiro, Ana Sofia Rodrigues, Sebastian Calderon, Alexandra Fidalgo, José L.M. Gonçalves, Vânia André, M. Teresa Duarte, Paulo J. Ferreira, José Paulo S. Farinha, Carlos Baleizão PII: DOI: Reference:

S0021-9797(19)31354-2 https://doi.org/10.1016/j.jcis.2019.11.036 YJCIS 25653

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Journal of Colloid and Interface Science

Received Date: Revised Date: Accepted Date:

8 August 2019 1 November 2019 11 November 2019

Please cite this article as: T. Ribeiro, A. Sofia Rodrigues, S. Calderon, A. Fidalgo, J.L.M. Gonçalves, V. André, M. Teresa Duarte, P.J. Ferreira, J.P.S. Farinha, C. Baleizão, Silica nanocarriers with user-defined precise diameters by controlled template self-assembly, Journal of Colloid and Interface Science (2019), doi: https:// doi.org/10.1016/j.jcis.2019.11.036

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Silica nanocarriers with user-defined precise diameters by controlled template self-assembly Tânia Ribeiro,a,b,† Ana Sofia Rodrigues,a,b,† Sebastian Calderon,c Alexandra Fidalgo,a José L. M. Gonçalves,a,b Vânia André,b M. Teresa Duarte,b Paulo J. Ferreira,c,d,e José Paulo S. Farinha *,a,b and Carlos Baleizão *,a,b a

Centro de Química-Física Molecular and Institute of Nanosciences and Nanotechnology, Instituto

Superior Técnico, Universidade de Lisboa, 1049-001 Lisboa, Portugal b

Centro de Química Estrutural, Instituto Superior Técnico, Universidade de Lisboa, 1049-001

Lisboa, Portugal c International Iberian Nanotechnology Laboratory, Av. Mestre José Veiga, 4715-330 Braga, Portugal d

Materials Science and Engineering Program, The University of Texas at Austin, Austin, Texas

78712, United States e

Mechanical Engineering Department and IDMEC, Instituto Superior Técnico, University of Lisbon,

Av. Rovisco Pais, 1049-001 Lisboa, Portugal † These authors contributed equally to this work. *E-mail: [email protected] (JPSF) and [email protected] (CB). Abstract Mesoporous silica nanoparticles (MSNs) feature ideal structural properties and surface chemistry for use as nanocarriers of molecules, polymers and biomolecules in cutting-edge applications. One important challenge remaining in their preparation is the ability to tune their diameter in the range of a few tens of nanometers, with narrow size dispersity, preferably using a simple, sustainable and scalable synthetic process. This work presents a fully controllable low-temperature and purely aqueous sol-gel method to prepare MSNs with user-defined diameters from 15 nm to 80 nm and narrow size dispersity. The method also allows modification of the pore structure and offers the possibility of incorporating a luminescent species in the silica network for optical traceability. Control was achieved by tuning the colloidal stability of the assembly of cylindrical micelles that template the MSN synthesis. Using CTAB cylindrical micelles as template and sodium hydroxide (NaOH) as catalyst, precise diameter control was achieved either by changing the pH (that controls micelle surface charge) or by adding salt at constant pH (to tune the ionic strength and charge screening). This new sustainable MSN synthesis method provides full control over the nanoparticles diameters and can be used as platform for the application of these user-defined nanoparticles in different fields. 1

Keywords: mesoporous silica nanoparticles; MSNs; particle size tuning; pore morphology change; CTAB micelles; micelle stabilization; supramicellar assembly; fluorescent label Introduction Mesoporous silica nanoparticles (MSNs) are exciting materials due to their high internal surface area and pore volume, tunable pore size, colloidal stability, and the possibility of selectively functionalizing the inner (pore) surfaces and the external particle surface [1]. This structural versatility has been key to the application of these nanoparticles in catalysis [2-4], drug delivery [1,5-15], and biomedicine [6,16,17]. Since the discovery of MCM-41 by Mobil researchers [18,19], significant progress has been made in controlling the characteristics of mesoporous silica materials, mainly the particle size and pore structure [20]. In the preparation of these materials, hydrolysed silicate species condensate around a supramicellar assembly of cylindrical ionic surfactant micelles that act as template. This process depends on several experimental parameters, such as the pH and temperature, the type and concentration of the silicate precursors and surfactant used, etc [21-23]. The versatility of MSN preparation also allows their properties to fit tuned for different application requirements. For example, MSN for biomedical applications are often required to have diameters under 100 nm to facilitate cell uptake and help reaching targets such as the cell nucleus, or crossing the blood-brain barrier or the placenta barrier [6,24,25]. Additionally, control of the diameter is also paramount to meet the ever-stricter standards for in vivo use [16]. Several strategies to prepare MSNs with diameters under 100 nm have been described, but none provides a simple and reproducible control over the particle diameter in a range of sizes. The reported strategies rely on: i) the use of a dilute reactant solution and subsequent neutralization of the medium at short reaction time [26]; ii) the change of the silicate/surfactant ratio [27,28]; iii) the use of a binary surfactant system [29,30]; iv) the change of the alkali concentration to modulate the silicate hydrolysis/condensation rate [25,31]; v) the use of weak bases as catalysts (such as sodium acetate or basic amino acids) [2,32]; or vi) the use of triethanolamine as chelating/retarding agent to control silicate hydrolysis/condensation [33]. In these approaches, either the synthetic procedure is not straightforward, or the size dispersion of the nanoparticles is too high. The preparation of MSNs is based on the use of a template, formed by the supramicellar assembly of cylindrical micelles (Scheme 1). The template, in the case of MCM-41 type 2

materials an ionic surfactant (normally a tetraalkyl ammonium cation with a hydrophobic alkyl chain), self-assembles into cylindrical micelles above the CMC. This shape is obtained if the critical packing parameter of the surfactant is between 0.33-0.5 [34]. The pore network of the final material results from the assembly of the individual micelles, which is determined by their colloidal stability. The colloidal stability of the micelles in turn depends on the surface charge of the polar part of the surfactant, and the balance between the attractive and repulsive interactions mediated by the solvent (water). If the colloidal stability of the cylindrical micelles is low, these will aggregate to reduce surface energy, increasing the supramicellar aggregation number. This low colloidal stability can be induced by neutralizing the micelle surface charges. On the other hand, if the colloidal stability is high (more effective intermicellar electrostatic repulsion), micelle aggregation is reduced and the supramicellar aggregation number decreases.

colloidal stability decrease

cylindrical micelles

micelle assembly

silica condensation

Scheme 1. Mechanism of formation of mesoporous silica nanoparticles and influence of supramicellar assembly in MSNs diameter. The final diameter of the MSNs depends on the size of the supramicellar aggregates, because once the silica precursor is added, the hydrolysis/condensation reactions lead to silica formation around the micelles. The final size of the MSNs will also depend on the balance between the kinetics of the silica formation reactions and the effect of surface tension. Although some of the different strategies mentioned above to tune the diameter of MSNs under 100 nm are apparently unrelated, they are all based on controlling the supramicellar assembly through charge screening of the cylindrical micelles. The concentration and strength of the (base) catalyst leads to different silicate hydrolysis rates, and due to the isoelectric point of silica (pH ≈ 2), those species are deprotonated (charged), strongly influencing the aggregation number of the supramicellar assemblies [22,35]. Additionally, the balance between the supramicellar assembly process and the kinetics of silica formation is also 3

affected by the surfactant and silicate concentrations. Therefore, the structural equilibrium of the supramicellar assembly (and consequently, its aggregation number and, ultimately, the particle size) is extremely sensitive to changes in the reaction system. The aim of our work was to develop a fully controllable method for the preparation of MSNs with sizes under 100 nm and low size dispersity. The method is exclusively aqueous and allows the incorporation of a fluorescent molecule (in the present work, a perylenediimide derivative, PDI) in the silica network to produce luminescent MSNs for particle tracking. By changing only the base concentration, we are able to control the supramicellar assembly of the surfactant cylindrical micelles that originate the template, and therefore the diameter of the MSNs. This allows us to precisely control the MSN diameter down to 18 nm, with low size dispersity. We further illustrate the fundamental importance of the colloidal stability of the micelles and their supramicellar assemblies on the mechanism of nanoparticle formation and the control of their size, by achieving complete size control only by changing the ionic strength of the system. Materials and methods Materials Cetyltrimethylammonium bromide (CTAB, Sigma, BioUltra for molecular biology, ≥99%), tetraethyl orthosilicate (TEOS, Aldrich, 98%), sodium hydroxide (NaOH, Eka pellets), sodium chloride (NaCl, Merck, 99.5%), sodium hydrogen phosphate (Na2HPO4, Riedel-de Haën, 99%), absolute ethanol (Scharlau, 99.9%), tetrahydrofuran (THF, Acros organics, 99.5+%), hydrochloric acid (HCl, Analar normapur®, 37%) were used as received. Deionized water from a Millipore system Milli-Q18 MΩ cm was used in all synthesis steps. Perylenediimide derivative (PDI) was synthesized according to the literature [36]. Synthesis of MSNs (adapted from the literature [13]). In a polypropylene flask, CTAB (0.5 g) was dissolved in water (240 mL), at 30 ºC with vigorous stirring. After 10 min, 1.75 mL of a solution of sodium hydroxide (0.2, 0.4, 0.7, 1.0, 1.4, 1.7 or 2.0 M) was added to the mixture, followed by a dropwise addition of 2.5 mL of TEOS. The reaction temperature was raised up to 40 °C, under stirring for 2 hours. After cooling, the dispersion was centrifuged (3000080000 g, 20-40 minutes, depending on the sample) and washed 2 times with a mixture of 1:1 water:ethanol and 1 time with only absolute ethanol. In the last cycle, the supernatant is removed, and the nanoparticles dried at 50 °C in a ventilated oven. Synthesis of f-MSNs. To prepare the fluorescent MSNs, we used the same procedure described above for the synthesis of MSNs. However, in the beginning, the PDI (3.6 mg) was added to 4

a solution of CTAB in THF (5 mL), and after 10 min of stirring at room temperature (and sonication if necessary), the THF was removed, and the synthesis follow the method described above. Synthesis of s1-MSNs. To prepare MSNs with NaCl, we used the same procedure described above for the synthesis of MSNs. After dissolving the CTAB, 0.875 mL of a solution of sodium hydroxide (0.2 M) and 0.875 mL of a solution of NaCl (0.2, 0.5, 0.8, 1.2, 1.8, 2.5, 3.2, 4.8 and 5.9 M) were added to the mixture, and the synthesis follow the method described above. Synthesis of s2-MSNs. To prepare MSNs with Na2HPO4, we used the same procedure described above for the synthesis of MSNs. After dissolving the CTAB, 0.875 mL of a solution of sodium hydroxide (0.2 M) and 0.875 mL of a solution of Na2HPO4 (0.5 M) or 0.875 mL of a solution of sodium hydroxide (1.4 M) and 0.875 mL of a solution of Na2HPO4 (0.5 M) were added to the mixture, and the synthesis follow the method described above. Surfactant extraction. To extract the CTAB, 0.5 g of dried nanoparticles were added to 20 mL of an ethanolic solution of HCl (0.5 M). The dispersion was kept at 40 °C for 4 hours. After cooling, the dispersion was centrifuged (30000-80000 g, 20-40 minutes, depending on the sample) and washed 2 times with a mixture of 1:1 water:ethanol and 1 time with only absolute ethanol. In the last cycle, the supernatant is removed, and the nanoparticles dried at 50 °C in a ventilated oven. Characterization Bright-field transmission electron microscopy (TEM) images and HAADF STEM images were acquired on a double corrected FEI Titan THEMIS operated at 200 kV. The samples were dispersed in solid state without the use of any solvent in order to avoid carbon contamination. Briefly, a small amount of powder is placed in an Eppendorf and shake thoroughly. Thereafter, a lacey carbon grid (TED PELLA, INC.) was introduced into the Eppendorf and gently stirred. The images were recorded using a convergence angle of 21 mrad with a pixel dwell time set at 10µs. A camera length of 115 mm was selected, which collects electrons between 50 and 200 mrad. The HAADF STEM images were processed using Gatan Digital Micrograph V 3.21 and Fiji software packages. The TEM equivalent diameters were obtained in the Fiji program (Feret diameter) from the analysis of at least one hundred particles per batch. The N2 adsorption–desorption isotherms were obtained at 77 K of the degassed samples, using a Micromeritics ASAP 2010. The surface area was estimated from the adsorption branch of the isotherm, in the p/p0 range of 0.05–0.3, using a N2 cross sectional area of 16.2 Å2. The 5

total pore volume was measured at p/p0=0.99. The mesopore volume (Vmp) was estimated from the cumulative desorption pore volume corresponding to the smaller pore region, using the BJH algorithm. The micropore volume estimated from the t-plot, converted to liquid volume at 77 K. The interparticle volume (Vip) was estimated by subtraction of micro and mesopore volumes from the total pore volume. The pore width was obtained from the BJH analysis, using the cumulative desorption pore volumes in the corresponding ranges. Powder X-ray diffraction data were collected in a D8 Advance Bruker AXS theta-2theta diffractometer, with a copper radiation source (Cu K, =1.5406 Å), a secondary monochromator and a scintillation detector, operated at 40 kV and 30 mA, in the 2-theta range 1-10º, with an increment of 0.02º. Dynamic Light Scattering (DLS): Zetasizer Nano ZS (Malvern, model ZEN3600) equipped with a 4 mW He–Ne solid-state laser operating at 633 nm and backscattered light was detected at 173°. To determine the hydrodynamic diameters of the nanoparticles, the autocorrelation function was analyzed by the CONTIN method. Zeta-potentials were calculated from electrophoretic mobility using the Smoluchowski relationship. Disposable folded capillary cells (DTS1070) were used for the measurement of zeta potentials. All measurements were performed in triplicate. The absorption spectra were recorded on a Jasco V-660 spectrophotometer, and the fluorescence measurements were obtained on a Horiba Jobin Yvon Fluorolog 3-22 spectrofluorometer. The pH was measured with a VWR pHenomenal pH1000L pH meter equipped with a VWR pHenomenal MIC 220 glass microelectrode and a VWR pHenomenal PT1000 1M temperature sensor. The initial pH (pHi) was measured before the addition of TEOS and the final pH (pHf) was measured at the end of each reaction. Results and Discussion The development of a reproducible preparation method for MSNs with precise diameters under 100 nm relies on an accurate control of different experimental parameters affecting colloidal stability and reaction kinetics. The diameter of the MSNs is determined by the aggregation number of the supramicellar aggregates of surfactant cylindrical micelles, that depend mainly on the charge balance at the micelles’ surface and the charge screening effect of the media. To understand the mechanism of MSN formation we started by changing the base concentration, maintaining all other experimental parameters constant. Using sodium hydroxide base, cetyltriammonium bromide (CTAB) surfactant, water and tetraethyl orthosilicate (TEOS) silicate precursor, we started from a NaOH concentration of 14.5 mM, 6

which yielded MSNs with 90-100 nm diameter [15], and expanded the procedure to lower NaOH concentrations. The temperature (30-40 ºC) is kept constant during 2 h to complete the reaction. In the end, the particles are cleaned by centrifugation, with ethanol and then water. The CTAB template is extracted from the pores with acidified ethanol, and the nanoparticles are again cleaned by centrifugation with ethanol and water. The influence of base concentration was apparent when comparing the mean diameters obtained by TEM for experiments using different NaOH concentrations (Table 1, Figure 1). The mean diameter decreases as the concentration of NaOH is decreased, from 91 nm (MSN1) for 14.5 mM NaOH, down to 18 nm (MSN-7) at 1.4 mM NaOH, maintaining a very low size dispersity. This is in line with scattered results reported in the literature, and can be explained by the direct variation of the hydrolysis rate of the silicate source with pH (for pH>7) [37], which affects the charge balance at the micelles’ corona and therefore the aggregation of the micelles into bundles that ultimately originate the particles. The effect of pH on the hydrolysis kinetics results in a time-dependent influence on intermicellar repulsions, which can be appreciated by comparing the decrease in the measured pH after TEOS hydrolysis (Table 1) [38]. Table 1. Mean nanoparticle diameters (obtained by TEM) of MSNs and fluorescent-labeled f-MSNs, prepared with different NaOH concentrations, pH measured in the beginning (pHi) and at the end of the reaction (pHf). Sample [NaOH] (mM)

MSN DTEM (nm)

f-MSN DTEM (nm)

pHi

pHf

MSN-1

14.5

91 ± 20

--

12.0

10.4

MSN-2

12.3

74 ± 10

75 ± 11

12.0

10.4

MSN-3

10.1

59 ± 7

56 ± 6

11.9

10.0

MSN-4

7.2

40 ± 4

38 ± 4

11.7

9.5

MSN-5

5.1

31 ± 3

27 ± 3

11.5

8.2

MSN-6

2.9

21 ± 3

25 ± 4

11.3

6.5

MSN-7

1.4

18 ± 2

21 ± 3

10.9

5.4

The synthesis starts with pHi = 11 - 12, and the final pH ranges from pHf = 10.3 (for the highest NaOH concentration) to pHf = 5.5 (for the lowest NaOH concentration). This means that, for all NaOH concentrations tested, more than 98% of the hydroxyl ions present at the beginning of the reaction are consumed during the hydrolysis of TEOS. However, the remaining amount of hydroxyl ions changes several orders of magnitude, from 0.2 mM (in 7

the highest NaOH concentration used) to ca. 310-6 mM (in the lowest). This strongly affects the charge balance at the micelle corona [38], with higher repulsion (stability) at low NaOH concentration resulting in smaller micelle bundles and ultimately, smaller nanoparticles. The hydrodynamic diameter (DH) obtained by dynamic light scattering (DLS, Supporting Information, Table S1), correlate well with the diameter calculated from TEM, showing low sample dispersity. The colloidal stability of the samples were evaluated by determining the zeta-potential. All samples exhibit a zeta-potential around -25±3 mV, a value in line with the obtained in highly stable silica nanoparticles. Bright-field TEM images of the different samples show that the nanoparticles have spherical shape (Figure 1). The particles were also characterized by high angle annular dark field scanning transmission electron microscopy (HAADF-STEM), which provides better massthickness contrast and enables us to distinguish the particle pores (Figure 2) [39]. The HAADF-STEM images of larger particles (MSN-1 and MSN-3, Figure 2A and 2B, respectively) exhibit the expected pore alignment, with the fast Fourier transform (FFT) pattern (inset in Figure 2) showing an hexagonal pore arrangement, typical of MCM-41 materials. On the other hand, HAADF-STEM images of smaller particles (MSN-5 and MSN7, Figure 2C and 2D, respectively) show a misaligned pore structure, with no diffraction patterns in the FFT of the images (insets in Figure 2C and 2D) This indicates a lower organization in the supramicellar aggregates of cylindrical micelles for the smaller nanoparticles, possibly due to the higher strain imposed in the micelle structure leading to some distortion.

8

Figure 1. Bright-field TEM images of the MSNs with different NaOH concentrations: (A) MSN-1, (B) MSN-3, (C) MSN-5 and (D) MSN-7.

Figure 2. HAADF-STEM image of (A) MSN-1, (B) MSN-3, (C) MSN-5 and (D) MSN-7, showing the mesoporous morphology. The inset shows the FFT patterns (scale bar: 1 nm-1). Characterization by nitrogen sorption yielded type IV isotherms for all samples, typical of mesoporous materials (Figure 3A). The textural porosity of the samples, indicated by the hysteresis observed at high relative pressures, and the high interparticle volume, are related to the small particle size. The structural properties (surface area, pore volume and diameter) are 9

compiled in Table 2. The surface area for samples MSN-1 to MSN-5 is around 1000 m2g-1, a typical value for this type of mesoporous materials. The exceedingly small size of the MSN6 and MSN-7 samples leads to a lower organization of the pores (Figure 2 and 3), that result in lower specific surface areas (777 and 628 m2g-1 for MSN-6 and MSN-7, respectively). This results in an increase of the particle density and a decrease in the specific mesopore volume, from 1 cm3g-1 (MSN-1) to 0.41 cm3g-1 (MSN-7). The distortion necessary to achieve a spherical shape (reducing the surface tension) have more impact on smaller particles leading

1800 Pore Volume (cm3/g)

Quantity Adsrobed (cm3/g STP)

to a poor pore organization.

1500 1200 900

10 9 8 7 6 5 4 3 2 1 0

A

0

2

4 6 8 Avarage Diameter (nm)

10

600 300 0 0

0.2

0.4 0.6 0.8 Relative pressure (p/pº)

1

B

MSN-7

Intensity (a.u.)

MSN-6 MSN-5

MSN-4 MSN-3 MSN-2 MSN-1

1.5

3.5

5.5

7.5

9.5

2 (º)

Figure 3. (A) Nitrogen adsorption-desorption isotherms for samples MSN-1 (red), MSN-3 (green) and MSN-5 (blue), and corresponding pore size distribution (inset). (B) PXRD patterns of samples MSN-1 to MSN-7. The mesopore diameters of all samples, determined by the Barret-Joyner-Halenda (BJH) method (inset in Figure 3A, Table 2), are around 2.8-3.0 nm, as expected for the use of a 10

CTAB template (removed without calcination). Micropores were not detected for samples MSN-1 to MSN-5, and have negligible volume (below 0.01 cm3g-1) for samples MSN-6 and MSN-7. Table 2. Pore morphology of MSNs prepared with different NaOH concentrations: surface area (SBET), total volume (Vt), mesopore volume (Vmp), interparticle volume (Vip) and mesopore diameter (Dmp). SBET (m2g-1)

Vt (cm3g-1)

Vmp (cm3g-1)

Vip (cm3g-1)

Dmp (nm)

1

1068

1.12

1.01

0.11

3.0

2

948

0.94

0.74

0.20

2.8

3

991

1.60

0.73

0.87

2.9

4

967

2.18

0.62

1.56

2.8

5

972

2.19

0.61

1.58

2.9

6

777

1.56

0.81

0.75

3.0

7

628

1.27

0.41

0.85

2.9

MSN

The powder X-ray diffraction (PXRD) patterns of all samples (Figure 3B) exhibit a major peak at small angles for the first reflection (d100) with a 2 maximum around 2.1 degrees. For the larger particles (MSN-1), which exhibit a hexagonal pore arrangement (see TEM images in Figure 1 and 2), it corresponds to a pore-pore correlation distance (lattice parameter) of a0 = 4.8 nm, and a pore wall thickness of around 1.8 nm (obtained by subtracting the BJH mesopore width from a0). These values are in line with what is observed in ordered MCM-41 materials [40]. The diffractograms of MSN-1 and MSN-2 show a sharp peak for the (d100) reflection, and Bragg reflections at higher angles (3.5 < 2 < 4.5), resembling the pattern of MSN-41 bulk materials. In contrast, the diffractograms of MSN-3 to MSN-7 consist of a single broad peak for the (d100) reflection, and no further reflections are observed. This peak broadening is due to the increasingly smaller size and lower organization range of smaller particles, MSN-4 to MSN-7. The incorporation of fluorescent molecules in silica nanoparticles yields imaging agents with several advantages over molecular dyes, such as higher brightness, improved photo-resistance of the encapsulated dyes (lower oxygen concentration and diffusivity inside the nanoparticles), increased range of solvents in which they can be used (upon surface modification of the nanoparticles), targeting of the particles in controlled delivery applications, etc [36,41]. In the specific case of MSNs, the addition of a fluorescent molecule 11

with alkoxysilane groups during the synthesis allows their anchoring on the silica structure network [5], with the fluorescent molecule becoming aligned with the pores, impervious to aggregation and self-quenching effects [42]. We prepared a perylenediimide (PDI) fluorescent dye with two terminal alkoxysilane groups in the imide region, and used it as secondary silica source to anchor on the silica network [43]. Perylenediimide derivatives feature large molar absorptivity, near-unity fluorescence quantum yields and high photochemical stability [36]. Since the process to prepare MSNs presented here is exclusively aqueous, to prepare the fluorescent MSNs (f-MSNs) we first dissolve CTAB and PDI in tetrahydrofuran (THF), and subsequently remove this solvent under reduced pressure. The remaining procedure is the same as for the unlabelled samples, including the NaOH concentrations used. The diameters of the f-MSNs are very similar to those obtained for unlabelled MSNs prepared with the same NaOH concentration (Table 1 and Figure 4A). The TEM images of the f-MSN (Supporting Information, Figure S1) show the same pore structure as the unlabelled MSN, indicating that the incorporation of the PDI in the silica network does not influence the MSN formation kinetics. The nitrogen sorption experiments resulted in typical type IV isotherms (Supporting Information, Figure S2), and the surface area, mesopore volume and pore width (Supporting Information, Table S2) are close to those of unlabelled MSNs (Table 2). The PXRD patterns are also similar to the MSN samples. The fluorescent properties of the PDI dye are not affected by the incorporation in the silica network or template extraction, with the f-MSN dispersed in ethanol showing the same excitation and emission spectra of free PDI (Figure 4B), showing that no dye aggregation occurs [44]. We have shown that the diameter of MSN can be accurately tuned by changing the NaOH concentration, which affects the TEOS hydrolysis rate and consequently the number of anionic silicate species on the micelle surface in each moment (at pH>2 the silanol species are deprotonated). However, since the charge balance on the template cylindrical micelles depends on the electrostatic screening of the micelles surface charge by the media, this effect also affects the diameter of the MSNs. To better understand this point, a batch of synthesis was performed at fixed NaOH concentration (0.7 mM - for which most hydroxyl ions are consumed during the TEOS hydrolysis), while changing the ionic strength (I) of the media by adding increasing amounts of sodium chloride (NaCl). All samples (s1-MSNs) were prepared with pHi = 10.8, presenting pHf = 5.4, similarly to MSN-7 (1.4 mM of NaOH, without NaCl).

12

A

100

D (nm)

80 60 40 20 0 0

4

6 8 10 [NaOH] (mM) O EtO EtO Si N EtO 3 O

B

1.0

Normalized fluorescence intensity (a.u)

2

0.8

12

14

O

PDI

OEt N 3Si OEt OEt O

0.6 0.4 0.2 0.0 400

500

600  (nm)

700

Figure 4. (A) Comparison of the mean diameters obtained by TEM for nanoparticles with PDI (fMSN, red circles) and without PDI (MSNs, blue circles), at different NaOH concentrations. (B) Normalized excitation (solid line) and emission (dash line) spectra of free PDI in ethanol (red) and fMSN dispersed in ethanol (blue). The inset shows the structure of the PDI dye The diameters of s1-MSNs (Figure 5 and Figure S3) increase linearly with the concentration of NaCl (Table 3 and Figure S4). The charge balance on the cylindrical micelle surface is clearly affected by the increase in ionic strength, with the larger charge screening of higher NaCl concentrations leading to a decrease in the colloidal stability of the individual micelles, which increases the aggregation number of the supramicellar micellar assemblies (Scheme 1), and ultimately yield larger nanoparticle diameters.

13

Figure 5. Bright-field TEM images of s1-MSNs with different NaCl concentrations: (A) s1MSN-2, (B) s1-MSN-6 and (C) s1-MSN-9. 14

Table 3. Diameters (obtained by TEM, DTEM) and pore morphology (surface area SBET, mesopore volume Vmp, and mesopore diameter Dmp) of s1-MSN and s2-MSN samples.

a

Sample a

[NaCl] (mM)

[Na2HPO4] (mM)

Ic (mM)

DTEM (nm)

SBET (m2g-1)

Vmp (cm3g-1)

Dmp (nm)

s1-MSN-1

0.7

-

7.1

20 ± 1

416

0.04

-

s1-MSN-2

1.8

-

8.2

24 ± 1

389

0.09

2.7

s1-MSN-3

2.9

-

9.3

25 ± 3

341

0.07

2.7

s1-MSN-4

4.3

-

10.7

28 ± 1

306

0.06

2.9

s1-MSN-5

6.4

-

12.9

33 ± 2

263

0.08

3.0

s1-MSN-6

8.9

-

15.4

43 ± 2

200

0.06

3.3

s1-MSN-7

14.3

-

20.9

54 ± 4

149

0.05

2.9

s1-MSN-8

17.2

-

23.8

58 ± 4

162

0.06

3.0

s1-MSN-9

21.1

-

27.7

66 ± 4

156

0.07

3.3

s2-MSN-1

-

1.8

11.8

47 ± 3

681

0.41

2.9

s2-MSN-2 b

-

1.8

16.2

86 ± 5

139

0.09

4.1

[NaOH] = 0.7 mM; b [NaOH] = 5.0 mM; c Ionic strengths estimated from all charges in the

medium except the silicate species. The TEM images show that the particles have spherical shape, even for smaller diameters (Figure 5 and Figure S3). The surface of the s1-MSN (prepared with salt) is smoother and the size dispersity is lower than for the smaller sized MSNs (Figure 1) and the f-MSNs (Supporting Information, Figure S1), which were prepared without salt. A detailed analysis of the HAADF-STEM images (Figure 6) of the different s1-MSN samples reveals that the visible pore structure is not aligned, but rather forms what looks like an entangled wormlike arrangement. This increase in particle size and the evolution to a wormlike pore structure could be anticipated from the literature on the influence of salts on the packing and structure of cationic micelles, including CTAB [45-48], which reports that an increase in salt concentration shields the repulsion between CTAB molecules and can lead to a transition from cylindrical to wormlike micelles.

15

Figure 6. HAADF-STEM image of (A) s1-MSN-9 and (B) s1-MSN-2, showing the mesoporous morphology. The inset shows the FFT patterns (scale bar: 1 nm-1). Nitrogen sorption experiments reveal that the s1-MSNs pore morphology (Figure 7A and Table 3) is very different from that of salt-free MSNs, with type V isotherms with H1 hysteresis (Figure 7A), consistent with interparticle capillary condensation in agglomerates of approximately uniform spheres. The surface area is inversely proportional to NaCl concentration up to 14.3 mM, remaining unchanged for higher values. This reflects the increase in the thickness of pore walls (see PXDR results below). The mesopore volume is lower than 0.1 cm-3g-1 for all the samples, with negligible micropore volume. This value is very low, compared with the salt-free MSNs (Table 2), indicating that the wormlike pores might be less accessible in the sorption experiments. The pore diameter increases from 2.7 nm (s1-MSN-2, low NaCl concentration) to 3.3 nm (s1MSN-9, 21.1 mM NaCl), an increase also observed in the PXRD experiments (Figure 7B). The PXRD patterns consist of a single broad peak at 2 = 1.7º for s1-MSN-2 down to 2 = 1.3º for s1-MSN-9, without further reflections at higher angles (even for the larger particles). This data contrasts with the ordered MCM-41 materials data. The peak broadening is widely found in wormlike pore arrangements and the shift of the first reflection towards smaller angles indicates a change in the packing and arrangement of the pores, with a d-space of 5.2 nm for s1-MSN-2 and up to 6.8 nm for s1-MSN-9. The pore wall thickness also increases (the d-space increase 1.6 nm and the pore width increases only 0.6 nm), leading to a decrease in the specific surface area. The difference in the pore arrangement of the s1-MSN samples, relative to salt-free MSNs, are attributed to the electrostatic screening of the micelles surface charge by the salt. The decrease in intermicellar repulsion affects the packing of the micelles, leading to a different pore morphology in the final nanoparticles.

16

Quantity Adsrobed (cm3/g STP)

600

A

500 400 300 200 100 0 0

0.2

0.4

0.6

0.8

1

Relative pressure (p/pº)

Intensity (a.u.)

B

s1-MSN-9

s1-MSN-5

s1-MSN-2

1

3

5

7

9

2 (º)

Figure 7. Nitrogen adsorption-desorption isotherms (A) and PXRD patterns (B) for samples s1-MSN-2 (red), s1-MSN-5 (green) and s1-MSN-9 (blue). In a second round of salty MSNs (s2-MSNs), a salt with a divalent anion (sodium hydrogenphosphate, Na2HPO4) was used to evaluate the effect of the bulky divalent charged anion on the electrostatic interactions at the micelles’ surface. The s2-MSNs show further changes in the nanoparticle morphology (s2-MSN-1, Table 3), with Na2HPO4 leading to an increase in the nanoparticle’s diameters (almost a factor of 2) when compared with NaCl under similar conditions (s1-MSN-2, Table 3). The pore morphology (Figure 8A Figure 9A and Figure S5) is wormlike (unaligned) as in the s1-MSN samples, with a pore width below 3 nm (determined by BET). The first reflection appears at small angles (2 = 1.7º), with peak broadening, and a d-space of 5.2 nm (Supporting information, Figures S6 and S7).

17

Figure 8. Bright-field TEM images of s2-MSNs: (A) s2-MSN-1 and (B) s2-MSN-2.

Figure 9. HAADF STEM image of (A) s2-MSN-1 and (B) s2-MSN-2, showing the mesoporous morphology. The inset shows the FFT patterns (scale bar: 1 nm-1). Due to poor solubility, the Na2HPO4 concentration could not be increased. Instead, the [NaOH] was increased from 0.7 mM to 5 mM (s2-MSN-2, Table 3) while maintaining the Na2HPO4 concentration. Since the initial pH for the s2-MSN-2 experiment is higher, the hydrolysis and condensation kinetics of the silicate species are faster and the electrostatic screening of the media is higher, resulting in a larger final diameter of the nanoparticles, as expected from the trend described for the MSN samples (Table 1). However, the diameter obtained for s2-MSN-2 (Figure 8B, Figure 9B and Figure S5) is nearly 3 times higher than that of MSN-5 (no salt but the same NaOH concentration). On the other hand, the pore structure is similar to that obtained for the s1-MSN samples as expected, although the pore width increases ca. 40%, to 4.1 nm (Table 3, Figures S6 and S7, Supporting information). Additionally, the first PXRD reflection is observed at 2 = 1.18º, with a d-space of 7.5 nm, similarly to those obtained for the s1-MSNs samples with higher NaCl concentration. The s2-MSN-2 experiment shows that the charge and the size of the ions are critical parameters in the packing of the micelles to form the template, an effect only previously observed in studies of the influence of ionic compounds in the growth and packing parameters of cationic surfactant micelles [45-47]. The increase in the nanoparticles diameter when increasing salt concentration, in particular when using a divalent instead of a monovalent anion, clearly indicates that the key point that 18

governs micelle bundle formation by supramicellar assembly is the screening of the charges at the micelles’ surface. The increase in screening results in less repulsion between the micelles, leading to larger aggregation numbers in the supramolecular assemblies that also show lower ordering. This results in nanoparticles with larger diameter and pores with lower alignment. One way to rationalize these results is to use the ionic strength (I) as a measure of the charge screening. We calculate I considering the concentrations of all charged species except the anionic silicate species formed by TEOS hydrolysis – these also affect I before condensation occurs, but their total concentration should be approximately constant for all experiments (same concentration of TEOS), although the concentration at each instant depends on the kinetics of hydrolysis and condensation. The diameters of particles as a function of I show different trends for samples MSN, s1-MSN and s2-MSN (Figure 10). It is possible to distinguish 3 different regimes in Figure 10. For approximately I  11 mM the diameters of the samples with and without NaCl (s1-MSN and MSN, respectively) are similar for comparable I (Figure 10, region A). Above this value of ionic strength, we observe different diameters for MSN and s1-MSN at the same I value (Figure 10, region B). This is probably due to differences in TEOS hydrolysis kinetics, faster in MSN samples due to the higher NaOH concentration. The hydrolyzed TEOS species are deprotonated due to the high pH of the system, and before condensation, they also play a role in the charge screening on the micelles surface [35]. Finally, for s2-MSN, in which Na2HPO4 was used (Figure 10, region C), the diameter is larger than for MSN and s1-MSN at comparable I. In this case, we expect that the effect of screening by the divalent anions strongly influence the charge balance on the micelle surface and the size of the supramicellar assemblies. The fact that this effect is larger than what would be expected from the increase in ionic strength, suggests a specific local effect of the size and charge of the anions, or their hydration state.

19

C

Diameter (nm)

100

B

80 60 40

A

20 0 0

5

10

15

20

25

30

I (mM)

Figure 10. Comparison of the diameters of MSN (different [NaOH], no salt – red circles), s1MSN (fixed low [NaOH], different [NaCl] – blue circles) and s2-MSN (fixed Na2HPO4, different [NaOH] – green circles) as a function of the ionic strength I (calculated from only [NaOH], [NaCl] and [Na2HPO4]). It is possible to distinguish 3 different regimes: A) same diameter for similar I in MSN and s1-MSN; B) different diameters for the same I in MSN and s1-MSN, probably due to differences in TEOS hydrolysis kinetic; C) higher diameters when Na2HPO4 is used. The specific interactions of counterions with surfactant head groups affect their self-assembly properties through intra- and intermolecular forces. The first determine interfacial curvature, while the second determine aggregation [49]. According to the Hofmeister series, that classifies anions as a function of their hydration [50], the divalent anion HPO42- is well hydrated (kosmotropic), increasing the local order of the water molecules through ion-dipole interactions that promote hydrogen bonding. By comparison, Cl- has poorer hydration, having an unordering (chaotropic) effect [51]. Such effects have been known to influence protein interaction, polymer phase transition [52], or surfactant assembly [53], and are expected to also have a role on the micelle assembly by affecting the solvation layer around the micelles. In fact, the increase in water structure around the kosmotropic HPO42- anion destabilizes the hydrogen bonds between water and the polar groups of the template micelles, increasing the cost of hydration [54]. The resulting dehydration of the micelle corona can increase micelle aggregation and consequently the final particle size. The specific effect of adding a kosmotropic anion like HPO42- can be appreciated by representing the particle diameter as a function of [NaOH], i.e., for approximately comparable reaction kinetics (Figure 11). In fact, for the same NaOH concentration, the effect on the particle size of 1.8 mM Cl- is relatively small compared to that of 1.8 mM HPO42-. 20

Diameter (nm)

100 80 60 40 20 0 0

5

10

15

[NaOH] (mM)

Figure 11. Diameters of MSN (no salt – red circles), s1-MSN (1.8 mM NaCl – blue circle) and s2MSN (1.8 mM Na2HPO4 – green circles) as a function of [NaOH] used in the reaction. For comparable reaction kinetics (same [NaOH]) the effect of 1.8 mM Cl- is relatively small compared to that of 1.8 mM HPO42-. The curve is a guide to the eye. Conclusions We present a new low-temperature aqueous sol-gel process for the preparation of MSNs with userdefined diameters under 100 nm and low size dispersity. This control was achieved by tuning the colloidal stability of the surfactant cylindrical micelles forming the supramicellar assemblies that template the MSN synthesis. Lowering the colloidal stability (by reducing micelle surface charge or increasing charge screening through an increase in ionic strength) leads to larger aggregation numbers of the micelle assemblies, and consequently larger MSNs. By promoting the colloidal stability of the micelles (increasing surface charge in low ionic strength media), we obtain the opposite effect which leads to smaller MSNs. In particular, we found that small differences in base concentration were enough to control the MSNs diameter, due to the presence of different amounts of hydroxyl ions in the medium. Also, by maintaining the pH at lower values and increasing the ionic strength (by adding salt), it was possible to increase the nanoparticles diameter. In this case, the pore morphology changes from a hexagonal aligned packing to an unaligned (wormlike) arrangement with an increase in the pore width. Our synthesis procedure also allows the incorporation of silane-modified fluorescent dyes in the silica network for applications requiring particle tracking. This is done during the synthesis and does not affect the control over size, morphology and size dispersity. Our simple and sustainable MSN synthesis method is easily scalable and provides full control over MSN size down to very small diameters (ca. 15 nm), opening excellent prospects for the application of these materials is various fields, from imaging and delivery in biomedical applications, to controlled release of catalysts or anti-corrosion agents, etc. Our results explain scattered experiments 21

reported in the literature on very small MSNs, and thus provide the basis for achieving similar control in other mesoporous materials. Acknowledgements This work was supported by Fundos Europeus Estruturais e de Investimento (FEEI), Programa Operacional Regional de Lisboa-FEDER (02/SAICT/2017), and national funds from Fundação para a Ciência e a Tecnologia (FCT-Portugal) and COMPETE (FEDER) within projects PTDC/CTMCTM/32444/2017

(02/SAICT/2017/032444),

PTDC/CTM-POL/3698/2014,

RECI/QEQ-

QIN/0189/2012, UID/QUI/00100/2019 (CQE), UID/EMS/50022/2019 (IDMEC-LAETA) and UID/NAN/50024/2019

(CQFM-IN).

T.R.

and

A.S.R.

also

thank

FCT

for

Pos-Doc

(SFRH/BPD/96707/2013) and Ph.D. (SFRH/BD/89615/2012) grants. The authors would like to acknowledge that this project has received funding from the EU Framework Programme for Research and Innovation H2020, scheme COFUND – Co-funding of Regional, National and International Programmes, under Grant Agreement 713640. References [1]

C. Argyo, V. Weiss, C. Bräuchle, T. Bein, Multifunctional Mesoporous Silica Nanoparticles

as a Universal Platform for Drug Delivery, Chem. Mater. 26 (2014) 435-451. [2]

Y. Yokoi, T. Karouji, S Ohta, J. N. Kondo, T. Tatsumi, Synthesis of Mesoporous Silica

Nanospheres Promoted by Basic Amino Acids and their Catalytic Application, Chem. Mater. 22 (2010) 3900-3908. [4]

Y.-J. Yu, J.-L-Xing, J.-L. Pang, S.-H. Jiang, K.-F. Lam, T.-Q. Yang, Q.-S. Xue, K. Zhang,

P. Wu, Facile Synthesis of Size Controllable Dendritic Mesoporous Silica Nanoparticles, ACS Appl. Mater. Interfaces 6 (2014) 22655-22665. [5]

A. S. Rodrigues, T. Ribeiro, F. Fernandes, J. P. S. Farinha, C. Baleizão, Intrinsically

Fluorescent Silica Nanocontainers: A Promising Theranostic Platform, Microsc. Microanal. 19 (2013) 1216-1221. [6]

L. Xiong, X. Du, B. Shi, J. Bi, F. Kleitz, S. Z. Qiao, Tunable stellate mesoporous silica

nanoparticles for intracellular drug delivery, J. Mater. Chem. B 3 (2015) 1712-1721. [7]

Y. Wang, Q. Zhao, N. Han, L. Bai, J. Li, J.; Liu, E. Che, L. Hu, Q. Zhang, T. Jiang, S.

Wang, Mesoporous silica nanoparticles in drug delivery and biomedical applications, Nanomedicine 11 (2015) 313-327.

22

[8]

Q. Zhang, X. Wang, P.-Z. Li, K. T. Nguyen, X.-J. Wang, Z. Luo, H. Zhang, N. S. Tan, Y.

Zhao, Biocompatible, Uniform, and Redispersible Mesoporous Silica Nanoparticles for CancerTargeted Drug Delivery In Vivo, Adv. Funct. Mater. 24 (2014) 2450-2461. [9]

J. E. Lee, N. Lee, T. Kim, J. Kim, T. Hyeon, Multifunctional Mesoporous Silica

Nanocomposite Nanoparticles for Theranostic Applications, Acc. Chem. Res. 44 (2011) 893-902. [10]

H. Meng, M. Xue, T. Xia, Z. Ji, D. Y. Tarn, J. I. Zink, A. E. Nel, Use of Size and a

Copolymer Design Feature To Improve the Biodistribution and the Enhanced Permeability and Retention Effect of Doxorubicin-Loaded Mesoporous Silica Nanoparticles in a Murine Xenograft Tumor Model, ACS Nano 5 (2011) 4131-4144. [11]

D. Tarn, C. E. Ashley, M. Xue, E. C. Carnes, J. I. Zink, C. J. Brinker, Mesoporous Silica

Nanoparticle Nanocarriers: Biofunctionality and Biocompatibility, Acc. Chem. Res. 46 (2013) 792801. [12]

P. Yang, S. Gai, J. Lin, Functionalized mesoporous silica materials for controlled drug

delivery, Chem. Soc. Rev. 41 (2012) 3679-3698. [13]

Y. Zhao, B. G. Trewyn, I. I. Slowing, V. S.-Y. Lin, Mesoporous Silica Nanoparticle-Based

Double Drug Delivery System for Glucose-Responsive Controlled Release of Insulin and Cyclic AMP, J. Am. Chem. Soc. 131 (2009) 8398-8400. [14]

L. Pan, Q. He, J. Liu, Y. Chen, M. Ma, L. Zhang, J. Shi, Nuclear-Targeted Drug Delivery of

TAT Peptide-Conjugated Monodisperse Mesoporous Silica Nanoparticles, J. Am. Chem. Soc. 134 (2012) 5722-5725. [15]

J. L. Vivero-Escoto, I. I. Slowing, B. G. Trewyn, V. S.-Y. Lin, Mesoporous Silica

Nanoparticles for Intracellular Controlled Drug Delivery, Small 6 (2010) 1952-1967. [16]

Y. Chen, H. Chen, J. Shi, In Vivo Bio-Safety Evaluations and Diagnostic/Therapeutic

Applications of Chemically Designed Mesoporous Silica Nanoparticles, Adv. Mater. 25 (2013) 3144-3176. [17]

Z. Li, J. C. Barnes, A. Bosoy, F. Stoddart, J. Y. Zink, Mesoporous silica nanoparticles in

biomedical applications, Chem. Soc. Rev. 41 (2012) 2590-2605. [18]

J. S. Beck, J. C. Vartuli, W. J. Roth, M. E. Leonowicz, C. T. Kresge, K. D. Schmitt, C. T.

W. Chu, D. H. Olson, E. W. Sheppard, S. B. McCullen, J. B. Higgins, J. L. A. Schlenker, A New Family of Mesoporous Molecular Sieves Prepared with Liquid Crystal Templates, J. Am. Chem. Soc. 114 (1992) 10834-10843. [19]

C. T. Kresge, M. E. Leonowicz, W. J. Roth, J. C. Vartuli, J. S. Beck, Ordered mesoporous

molecular sieves synthesized by a liquid-crystal template mechanism, Nature 359 (1992) 710-712.

23

[20]

S.-H. Wu, C.-Y. Mou, H.-P. Lin, Synthesis of mesoporous silica nanoparticles, Chem. Soc.

Rev. 42 (2013) 3862-3875. [21]

H.-P. Lin, C.-Y. Mou, Structural and Morphological Control of Cationic Surfactant-

Templated Mesoporous Silica, Acc. Chem. Res. 35 (2002) 927-935. [22]

L. P. Singh, S. K. Bhattacharyya, R. Kumar, G. Mishra, U. Sharma, G. Singh, S. Ahalawat,

Sol-Gel processing of silica nanoparticles and their applications, Advances in Colloid and Interface Science 214 (2014) 17-37. [23]

Y.-D. Chiang, H.-Y. Lian, S.-Y. Leo, S.-G.; Wang, Y. Yamauchi, K. C.-W. Wu, Controlling

Particle Size and Structural Properties of Mesoporous Silica Nanoparticles Using the Taguchi Method, J. Phys. Chem. C 115 (2011) 13158-13165. [24]

C. Muoth, L. Aengenheister, M. Kucki, P. Wick, T. Buerki-Thurnherr, Nanoparticle

transport across the placental barrier: pushing the field forward, Nanomedicine 11 (2016) 941-957. [25]

F. Lu, S.-H. Wu, Y. Hung, C.-Y. Mou, Size Effect on Cell Uptake in Well-Suspended,

Uniform Mesoporous Silica Nanoparticles, Small 5 (2009) 1408-1413. [26]

C. E. Fowler, D. Khushalani, B. Lebeau, S. Mann, Nanoscale Materials with Mesostructured

Interiors, Adv. Mater. 13 (2001) 649-652. [27]

H. Yamada, C. Urata, H. Ujiie, Y. Yamauchi, K. Kuroda, Preparation of aqueous colloidal

mesostructured and mesoporous silica nanoparticles with controlled particle size in a very wide range from 20 nm to 700 nm, Nanoscale, 5 (2013) 6145-6153. [28]

H. Yamada, C. Urata, S. Higashitamori, Y. Aoyama, Y. Yamauchi, K. Kuroda, Critical

Roles of Cationic Surfactants in the Preparation of Colloidal Mesostructured Silica Nanoparticles: Control of Mesostructure, Particle Size, and Dispersion, ACS Appl. Mater. Interfaces 6 (2014) 3491-3500. [29]

K. Suzuki, K. Ikari, H. Imai, Synthesis of Silica Nanoparticles Having a Well-Ordered

Mesostructure Using a Double Surfactant System, J. Am. Chem. Soc. 126 (2004) 462-463. [30]

K. Ikari, K. Suzuki, H. Imai, Structural Control of Mesoporous Silica Nanoparticles in a

Binary Surfactant System, Langmuir 22 (2006) 802-806. [31]

Y.-S. Lin, C. L. Haynes, Impacts of Mesoporous Silica Nanoparticle Size, Pore Ordering,

and Pore Integrity on Hemolytic Activity, J. Am. Chem. Soc. 132 (2010) 4834-4842. [32]

M. Yu, L. Zhou, J. Zhang, P. Yuan, P. Thorn, W. Gu, C. Yu, A simple approach to prepare

monodisperse mesoporous silica nanospheres with adjustable sizes, Journal of Colloid and Interface Science 376 (2012) 67-75. [33]

K. Möller, J. Kobler, T. Bein, Colloidal Suspensions of Nanometer-Sized Mesoporous

Silica, Adv. Funct. Mater. 17 (2007) 605-612. 24

[34]

A. W. Adamson, A. P. Gast, Physical Chemistry of Surfaces, 6th Edition, Wiley, Hoboken,

NJ, USA (1997). [35]

A. Corma, From Microporous to Mesoporous Molecular Sieve Materials and Their Use in

Catalysis, Chem. Rev. 97 (1997) 2373-2420. [36]

T. Ribeiro, C. Baleizão, J. P. S. Farinha, Synthesis and Characterization of Perylenediimide

Labeled Core-Shell Hybrid Silica-Polymer Nanoparticles, J. Phys. Chem. C 113 (2009) 18082-18090. [37]

C. J. Brinker, Hydrolysis and condensation of silicates: effects on structure, Journal of Non-

Crystalline Solids 100 (1988), 31-50. [38]

H. Chaimovich, J. B. S. Bonilha, M. J. Pollti, F. H. Quina, Ion exchange in micellar

solutions. 2. Binding of hydroxide ion to positive micelles, J. Phys. Chem. 83 (1979) 1851-1854. [39]

S. Velasco, T. Ribeiro, J. P. S. Farinha, C. Baleizão, P. J. Ferreira, On the Structure of

Amorphous Mesoporous Silica Nanoparticles by Aberration-Corrected STEM, Small 14 (2018) 1802180. [40]

M. Kruk, M. Jaroniec, Characterization of Highly Ordered MCM-41 Silicas Using X-ray

Diffraction and Nitrogen Adsorption, Langmuir 15 (1999) 5279-5284. [41]

A. M. Santiago, T. Ribeiro, A. S. Rodrigues, B. Ribeiro, R. F. M. Frade, C. Baleizão, J. P. S.

Farinha, Multifunctional Hybrid Silica Nanoparticles with a Fluorescent Core and Active Targeting Shell for Fluorescence Imaging Biodiagnostic Applications, Eur. J. Inorg. Chem. (2015) 4579–4587. [42]

F. Hoffmann, M. Fröba, Vitalising porous inorganic silica networks with organic functions—

PMOs and related hybrid materials, Chem. Soc. Rev. 40 (2011) 608–620. [43]

T. Ribeiro, S. Raja, A. S. Rodrigues, F. Fernandes, C. Baleizão, J. P. S. Farinha, NIR and

visible perylenediimide-silica nanoparticles for laser scanning bioimaging, Dyes and Pigments 110 (2014) 227-234. [44]

M. Moffitt, J. P. S. Farinha, M. A. Winnik, U. Rohr, K. Mullen, Steady-State and Dynamic

Fluorescence Measurements of a Perylene-Labeled Triblock Copolymer:  Evidence for Ground-State Dye Aggregate Formation, Macromolecules 32 (1999) 4895-4904. [45]

F. H. Quina, P. M. Nassar, J. B. S. Bonilha, B. L. Bales, Growth of Sodium Dodecyl Sulfate

Micelles with Detergent Concentration, J. Phys. Chem. 99 (1995) 17028-17031. [46]

R. Ranganathan, L. T. Okano, C. Yihwa, F. H. Quina, Growth of Cetyltrimethylammonium

Chloride and Acetate Micelles with Counterion Concentration, J. Colloid Interface Sci. 214 (1999) 238-242. [47]

K. Kuperkar, L. Abezgauz, K. Prasad, P. Bahadur, Formation and Growth of Micelles in

Dilute Aqueous CTAB Solutions in the Presence of NaNO3 and NaClO3, J. Surfact. Deterg. 13 (2010) 293-303. 25

[48]

N. C. Das, H. Cao, H. Kaiser, G. T. Warren, J. R. Gladden, P. E. Sokol, Shape and Size of

Highly Concentrated Micelles in CTAB/NaSal Solutions by Small Angle Neutron Scattering (SANS), Langmuir 28 (2012) 11962-11968. [49]

A. Salis, B. W. Ninham, Models and mechanisms of Hofmeister effects in electrolyte

solutions, and colloid and protein systems revisited, Chem. Soc. Rev. 43 (2014)7358-7377. [50]

W. J. Xi, Y. Q. A. Gao, A Simple Theory for the Hofmeister Series, Phys. Chem. Letters, 4

(2013) 4247-4252. [51]

Y. Marcus, Effect of Ions on the Structure of Water: Structure Making and Breaking, Chem.

Rev. 109 (2009) 1346-1370. [52]

G. Marcelo, L. R. P. Areias, M.-T. Viciosa, J. M. G. Martinho, J. P. S. Farinha, PNIPAM-

Based Microgels with a UCST Response, Polymer 116 (2017) 261-267. [53] P. L. Nostro, B.W. Ninham, Hofmeister Phenomena: An Update on Ion Specificity in Biology, Chem. Rev. 112 (2012) 2286-2322. [54]

Y. Zhang, S. Furyk, D. E. Bergbreiter, P. S. Cremer, Specific Ion Effects on the Water

Solubility of Macromolecules: PNIPAM and the Hofmeister Series, J. Am. Chem. Soc. 127 (2005) 14505-14510.

26

Graphical Abstract

27