Journal Pre-proofs

Spray-Drying Water-Based Assembly of Hierarchical and Ordered MesoporousSilica Microparticles with Enhanced Pore Accessibility for Efficient Bio-Ad-sorption

Zhangxiong Wu, Kathryn Waldron, Xiangcheng Zhang, Yunqing Li, Lei Wu,Winston Duo Wu, Xiao Dong Chen, Dongyuan Zhao, Cordelia Selomulya

PII: S0021-9797(19)30995-6DOI: https://doi.org/10.1016/j.jcis.2019.08.084Reference: YJCIS 25340

To appear in: Journal of Colloid and Interface Science

Received Date: 28 May 2019Revised Date: 22 August 2019Accepted Date: 23 August 2019

Please cite this article as: Z. Wu, K. Waldron, X. Zhang, Y. Li, L. Wu, W. Duo Wu, X. Dong Chen, D. Zhao, C.Selomulya, Spray-Drying Water-Based Assembly of Hierarchical and Ordered Mesoporous Silica Microparticleswith Enhanced Pore Accessibility for Efficient Bio-Adsorption, Journal of Colloid and Interface Science (2019),doi: https://doi.org/10.1016/j.jcis.2019.08.084

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Spray-Drying Water-Based Assembly of Hierarchical and Ordered

Mesoporous Silica Microparticles with Enhanced Pore Accessibility for

Efficient Bio-Adsorption

Zhangxiong Wu*‡,†, Kathryn Waldron†, Xiangcheng Zhang‡, Yunqing Li‡, Lei Wu‡, Winston Duo

Wu‡, Xiao Dong Chen‡, Dongyuan Zhao†,ф, and Cordelia Selomulya*†

‡ Particle Technology Engineering Laboratory, School of Chemical and Environmental

Engineering, College of Chemistry, Chemical Engineering and Materials Science, Soochow

University, Suzhou City, Jiangsu 215123, P. R. China.

ф Department of Chemistry and Laboratory of Advanced Materials, Fudan University, Shanghai,

200433, P. R. China

† Department of Chemical Engineering, Monash University, Clayton Campus, Victoria 3800,

Australia.

Corresponding authors:

Zhangxiong Wu, E-mail: zhangwu@suda.edu.cn

Cordelia Selomulya, E-mail: cordelia.selomulya@monash.edu

Abstract: The fast and scalable spray-drying-assisted evaporation-induced self-assembly (EISA)

synthesis of hierarchically porous SBA-15-type silica microparticles from a water-based system is

demonstrated. The SBA-15-type silica microparticles has bowl-like shapes, uniform micro-sizes

(~ 90 µm), large mesopores (~ 9.5 nm), hierarchical meso-/macropores (20 ~ 100 nm) and open

2

surfaces. In the synthesis, soft- and hard-templating approaches are combined in a single rapid

drying process with a non-ionic tri-block copolymer (F127) and a water-insoluble polymer colloid

(Eudragit RS, 120 nm) as the co-templates. The RS polymer colloid plays three important roles.

First, the RS nanoparticles can be partially dissolved by in-situ generated ethanol to form RS

polymer chains. The RS chains swell and modulate the hydrophilic-hydrophobic balance of F127

micelles to allow the formation of an ordered mesostructure with large mesopore sizes. Without

RS, only worm-like mesostructure with much smaller pore sizes can be formed. Second, part of

the RS nanoparticles plays a role in templating the hierarchical pores distributed throughout the

microparticles. Third, part of the RS polymer form surface “skins” and “bumps”, which can be

removed by calcination to enable a more open surface structure to overcome the low accessibility

issue of spray-dried porous microparticles. The obtained materials have high surface areas (315 ~

510 m2 g-1) and large pore volumes (0.64 ~ 1.0 cm3 g-1), which are dependent on RS concentration,

HCl concentration, silica precursor hydrolysis time and drying temperature. The representative

materials are promising for the adsorption of lysozyme. The adsorption occurs at a > three-fold

faster rate, in a five-fold larger capacity (an increase from 20 to 100 mg g-1) and without pore

blockage as compared to the adsorption onto spray-dried microparticles of similar

physicochemical properties obtained without the use of RS.

Keywords: porous silica, hierarchical structure, particle processing, spray drying, bio-adsorption

1. Introduction

The evaporation-induced self-assembly (EISA) is a universal method for the assembly and

processing of mesoporous materials.1-9 For the synthesis of ordered mesoporous silicas (OMSs),

3

the EISA process involves the competition between the condensation of hydrolyzed silica

precursors and the self-assembly of micelles. During the solvent evaporation process, the template

and hydrolyzed silica precursor enriched at the vapor-liquid interface should cooperatively co-

assemble into composite micelles in order to form ordered mesophases.2, 10-14 The formation of

ordered mesophases is much dependent on a range of factors, especially the solvent type, which

has been well studied for thin-film EISA process that involves long evaporation periods to allow

sufficient mesostructure organization.2-4, 6 It is found that the formation of OMSs with water as the

solvent in EISA systems is much challenging, mainly because a high concentration of water

promotes condensation of silica oligomers rather than micelle assembly. On the other hand, the

aerosol-assisted EISA process involves very short evaporation periods in the order of seconds,

which has been applied for the synthesis of OMSs and porous metal oxides of various structures

and particle sizes.1, 9, 10, 12, 15-18 In the aerosol drying process, the effect of solvent can be more

pronounced as it impacts upon the drying rate and thus the assembly process. In both thin-film and

aerosol-assisted EISA processes, the synthesis of OMSs with non-ionic block copolymers as the

templates and water as the solvent is challenging.

Spray drying provides a well-known, rapid, and scalable preparation method for mesoporous

microparticles.1, 9, 15-23 The use of the micro-fluidic jet spray-dryer avoids the drawback of

polydisperse droplets.24 In our previous work, this method has enabled the formation of uniform

“bowl-like” mesoporous MCM-41-type silica microparticles with water as a solvent and CTAB as

a template,25 as well as uniform mesoporous SBA-15-type silica microspheres with ethanol as a

solvent and a non-ionic block copolymer (F127) as a template.26 However, spray-dried SBA-15-

type silica microparticles in a water system has not been obtained, due to the difficulty in making

the hydrolyzed silica precursor cooperatively assemble with the template in a fast evaporating

4

water system. Moreover, aerosol-dried mesoporous microparticles are often produced with a

“skin” over the surfaces of the particles which allows only small molecules to pass through.27-30

Thus regardless of the average mesopore size of the particles, large molecules are difficult to be

adsorbed. Overcoming such an issue in using aerosol-assisted EISA for producing mesoporous

materials is therefore desirable.

A hierarchical pore architecture with small pores establishing the connection between large ones

in three dimensions is desirable for various applications, because this type of structure can produce

mass transport pathways, enhance the diffusion rate of foreign molecules and promote the

interactions of various species within the pore frameworks.31 The combined soft and hard dual-

templating method is generally applicable for the synthesis of various materials with hierarchical

pore structures.32-34 Aerosol-assisted EISA has been adopted to synthesize hierarchically porous

particles,15, 35-38 but with limited focus on the assembly of hierarchical OMSs. Moreover, in

previous reports the hard templates only play a single role of generating large pores with no

obvious influence on the self-assembly behavior of the soft micelles. In a water-based EISA system

with a non-ionic block copolymer as the soft template, a unique chemical environment needs to be

designed in order to promote self-assembly to form ordered mesostructures and to produce

hierarchical structures at the same time. To achieve this purpose, based on our previous research

in synthesizing core-shell microparticles,39-40 the commercial Eudragit RS polymer, poly (ethyl

acrylate-co-methyl methacrylate-co-trimethylammonioethyl methacrylate chloride), could be an

attractive choice. The RS nanoparticles are water-insoluble, stably dispersible in water and

ethanol-soluble. They are compatible with silica sols and surfactant dispersions. Therefore, it is

hypothesized that the co-existence of RS polymer chains and RS nanoparticles can play the roles

5

of modulating the assembly behavior of block copolymers and templating the formation of

hierarchical pore structures.

In this study, a water-based spray-drying route for the synthesis of uniform silica microparticles

with ordered and hierarchical porosity and enhanced pore accessibility to large biomolecules is

presented. Uniform hierarchically porous SBA-15-type silica microparticles are fast assembled

and processed by using the microfluidic-jet spray drying method. The key to the synthesis is the

introduction of a water-insoluble polymer (Eudragit RS) colloidal nanoparticles to the water-based

precursor solution. It plays three vital roles over the EISA process, namely, partial dissolution by

the in-situ generated ethanol from hydrolysis of TEOS to form hydrophobic polymer chains to

swell the F127 micelles and decrease the hydrophilic-hydrophobic balance to promote the

assembly of ordered mesostructure with large mesopores, forming nano-sized hard template to

generating additional large-meso/macropores, and acting as a skin former on particle surface to

circumvent the accessibility issue by opening up the porous structure after calcination.39 The

material structure and physicochemical property are analyzed in detail and their variations with

several typical experimental parameters are discussed. The obtained uniform hierarchically porous

SBA-15-type microparticles are promising for bio-adsorption, as demonstrated by the significantly

improved adsorption capacity and rate toward the adsorption of lysozyme.

2. Experimental section

2.1 Materials

Tetraethyl orthosilicate (TEOS, ≥ 99.0 %), amphiphilic Pluronic block co-polymer F127, and

lysozyme from chicken egg white were purchased from Aldrich Chemicals. The 0.05 ~ 0.5 M

hydrochloric acid solution was prepared by dilution of a 1.0 M analytical reagent grade HCl

6

purchased from Univar with distilled H2O. Eudragit RS ® 30D (denoted as RS), a copolymer of

ethyl acrylate, methyl methacrylate, and a low content of methacrylic acid ester with quaternary

ammonium groups in a ratio of 1: 2: 0.1 (the molecular structure is shown in Figure S1), in a 30

wt% aqueous dispersion (supplied by Evonik Industries) has a uniform colloidal size of 120.2 ±

1.2 nm (measured from a 1.0 w/v% RS dispersion by the dynamic light scattering method on the

Malvern Zetasizer, Nano-ZS). Milli-Q de-ionized water (~ 16 MΩ) and ethanol (Merck) were used

as supplied. A sodium hydrogen carbonate buffer (pH = 10.95) was prepared from sodium

hydroxide pellets (Merck) and sodium bicarbonate pellets (Sigma Aldrich).

2.2 Precursor preparation

For the synthesis of the uniform hierarchically porous SBA-15-type silica microparticles, 13.3

g of TEOS (equivalent to 3.84 g of silica) was pre-hydrolyzed with 6.4 g of 0.2 M HCl in a glass

bottle under magnetic stirring for 2 hours. Meanwhile, 6.4 g of F127 was dissolved in 70 g of

distilled water in a water bath at 35 °C by magnetic stirring for two hours. The above two solutions

were then combined while being stirred, followed by the addition of the RS dispersion. The

solution was then diluted to 10 wt% (total solute concentration, defined as the total solid weight

percentage of SiO2, F127 pure RS in the precursor) using distilled water and stirred magnetically

for a further 30 min to obtain the final precursor. The amount of RS added varied from 0 to 70

wt% of silica added to study its influence on particle structure. For example, with the addition of

70 wt% RS indicated that the amount of pure RS added was 2.69 g. In this case, the final HCl

concertation of the precursor was about 0.01 M. In order to study the influences of another two

experimental parameters on the properties of the resultant materials, two precursors were prepared

by using initial HCl concentrations of 0.05 and 0.5 M with the RS added amount fixed at 70 wt%

7

and the TEOS pre-hydrolysis time fixed at 2.0 h, and another two precursors were prepared by

using a TEOS pre-hydrolysis time of 1.0 and 4.0 h with the RS added amount fixed at 70 wt% and

the initial HCl concertation fixed at 0.2 M, respectively.

2.3 Spray-drying assembly of microparticles

The uniform droplets were formed using the same process as stated previously.24-26 Briefly, the

precursor solution was atomized using the micro-fluidic aerosol nozzle system,24, 41 with an orifice

diameter of 75 µm. The droplets were formed via vibrating piezoceramics, monitored using a

digital SLR (Nikon D90) with a speed light (Nikon SB-400) and micro-lens (AF Micro-Nikkor 60

mm f/2.8D), and optimized via adjustment of frequency of the piezoelectric nozzle under

observation using high-speed photography. The inlet and outlet temperatures of the drying

chamber were fixed at ~160 and 115 °C, respectively. The particle collection efficiency was > 70

wt%, and the collected microparticles were then oven dried at 100 °C overnight to remove excess

water. Calcination was conducted in air environment at 550 °C for 5 hours with a 2 °C min-1 ramp

to remove the F127 and RS components. The final samples were denoted as UHPM-SBA-15-x,

wherein UHPM stands for uniform hierarchically porous microparticles, and x for the added RS

amount in weight percentage related to the silica amount, respectively. When x is 0 wt%, the

obtained sample was denoted as UPM-SBA-15 in which UPM stands for uniform porous

microparticle. Moreover, a sample for lysozyme adsorption control experiments was also prepared

under the same experimental procedure by using a precursor with the same solute compositions

but without RS and with ethanol as the solvent. The sample was denoted as UPM-SBA-15*.

Besides, two more samples spray dried at 170 and 190 °C were prepared to study the influence of

the drying temperature on the properties of the resultant materials.

8

2.4 Measurement and characterization

Samples of (intentionally) crushed and intact particles were placed on carbon tapes attached to

spindles and coated with ~ 1 nm of Pt for morphological examination of microparticles taken using

a FEI Nova Nano SEM 450 Field Emission Scanning Electron Microscope (SEM) or a FEI

Magellan 400 FEG SEM. Energy dispersive X-ray photoelectron spectrometry (EDX) was

conducted on the FEI Magellan 400 FEG SEM operated at 15 kV for a period of 15 min for the

element map distribution and 180 s for the point element quantification. To observe the pore

structure and distribution, samples were prepared by crushing a small quantity of particles in

butanol in an agate pestle and mortar to form a weak suspension of fragments. A droplet was placed

on a carbon-coated copper grid and then air-dried. The samples were examined using a FEI Tecnai

G2 T20 Twin LaB6 transmission electron microscope (TEM) or a FEI Tecnai G2 F20 S-Twin FEG

STEM, operated at 200 kV. Nitrogen sorption isotherms were obtained using a Micromeritics

ASAP 2020 analyzer. The samples were dried in an oven overnight, followed by a pre-treatment

of 30 min at 100 °C and a degas step for 240 min at 180 °C under vacuum, with the exception of

the samples after the adsorption of lysozyme which were dried in a vacuum oven at 40 °C

overnight, prior to degas conditions of 40 °C for 24 hours. Surface areas were calculated using the

Brunauer-Emmett-Teller (BET) method and pore volumes were determined by the adsorbed

amount at p/p0 = 0.99, while the pore size distribution was determined by the adsorption branch of

the isotherm with the Barret-Joyner-Halenda (BJH) method. Mercury (Hg) intrusion and extrusion

analyses were conducted over a wide range of pressures starting with vacuum up to 60,000 psi

with a Micromeritics Autopore III. Samples were oven dried overnight at 180 °C prior to analysis.

Small angle X-ray scattering (SAXS) patterns were recorded using a Nanostar U small-angle X-

9

ray scattering system using the Cu Kα radiation (40 kV, 35 mA). Small-angle XRD patterns were

measured on a Bruker D2 Phase Diffractometer (30 kV, 10 mA).

2.5 Adsorption of lysozyme

The control sample obtained without the use of RS and with ethanol as the solvent (UPM-SBA-

15*) and the representative sample UHPM-SBA-15-70wt% were selected for adsorption test

because the two samples have very similar internal ordered mesoporous structure, pore size,

surface area and pore volume. The adsorption was conducted in a buffer of pH = 10.95, close to

the isoelectric point of lysozyme to reduce protein-protein repulsion. Sub-samples were dried in a

vacuum oven at 40 °C overnight. 200 mg of particles were then added to glass bottles containing

1.2 g of lysozyme dissolved in 100 mL of 100 mM buffer. The bottles were placed in an incubator

shaker at 25 °C and 200 rpm. At specific time intervals, 5 mL suspension was removed and

centrifuged at 6000 rpm for 10 min. The concentration of lysozyme was then determined via UV-

Vis Spectroscopy where 2.5 mL aliquot was measured in a quartz cuvette at λ = 281 nm. After

each test, the aliquot was re-dispersed and added back to the bulk solution. The intraparticle

diffusion model, expressed as qt = kit0.5 wherein qt, t, and ki are respectively the time-dependent

adsorption capacity, adsorption time and rate constant, was adopted to evaluate the adsorption

rates.

3. Results and discussion

3.1 Pore structure of the representative sample

With the aid of the water-insoluble RS polymer, hierarchically porous SBA-15-type silica

microparticles have been successfully obtained via the fast spray-drying EISA from a water-based

10

precursor system. Representatively, the UHPM-SBA-15-70wt% sample obtained with the addition

of 70 wt% RS at a drying temperature of 160 ºC exhibits an ordered hexagonal mesostructure with

the presence of the 100 peak and the combination of the 110, and 200 peaks (Figure 1A, curve a),

typical of SBA-15 materials.42 The d100 spacing is calculated to be ~ 11.6 nm (Table 1). The N2

adsorption/desorption results of the sample exhibit a typical type IV plot, a narrow mesopore size

distribution centered at ~ 9.2 nm, a high surface area of 510 m2 g-1 and a large pore volume of 0.95

cm3 g-1 (Figure 1B, curve a, and Table 1). The mesopore wall thickness is calculated to be ~ 4.2

nm (Table 1). From the high-resolution SEM (HRSEM), TEM and STEM images, the sample

possesses an ordered mesoporous framework interspersed with large-meso/macropores of 20 ~

100 nm (Figure 2a-c, and 3a, d). The ordered mesopores and the large-meso/macropores are

interconnected, and the distance between adjacent large-meso/macropores is only 50 ~ 120 nm

(Figure 2c, 3d). Besides, the large-meso/macropores are also interconnected to some extent (Figure

2c-e, 3a and 3d), which could be helpful for mass diffusion. These large-meso/macropores are

originated from the RS polymer nanoparticles, but are smaller and more broadly distributed,

indicating that the RS polymer nanoparticles can be partially dissolved and shrink to some extent

during the synthesis process. The mercury porosimetry of the sample further reveals the presence

of uniform mesopores and additional broadly distributed large-meso/macropores of 20 ~ 100 nm

(Figure 4a).

3.2 Influencing factors on pore structure

The RS amount added in the precursor solution is the most important factor influencing the pore

structure of the resultant samples. This is because the water-insoluble RS polymer plays a critical

role in assembly of the ordered mesostructure and the formation of large-meso/macropores. For

11

the sample UPM-SBA-15 obtained without the use of RS, although its SAXS pattern is similar to

those of the samples obtained with the use of RS (Figure 1A), it exhibits a larger full width at half

maximum (FWHM) (Table 1), indicating that its mesostructure is not as ordered as the

counterparts obtained with the use of RS. More clearly, the HRSEM (Figure 2h), TEM and STEM

(Figure 3c, f) images of the sample UPM-SBA-15 clearly reveal the presence of a disordered

“worm-like” mesostructure, indicating a lack of complete self-organization of the surfactant-silica

micelles. This is mainly the result of the aqueous system in the droplet promoting the condensation

of the silica oligomers rather than the self-assembly of the micelles, which has been noted

previously.10, 26 Notably, from the SAXS patterns, there is an apparent increasing trend in unit cell

with the increase of RS concentration (Figure 1A and Table 1), indicating that the hydrophobic

cores of the micelles can be increasingly swollen with the increase of RS concentration. In

accordance, the mesopore size dramatically increases from 6.8 to 9.2 nm, along with significantly

increased surface areas (from 299 to 510 m2 g-1) and pore volumes (from 0.38 to 0.95 cm3 g-1),

with the added RS increasing from 0 to 70 wt% (Figure 1B, Table 1). Resultantly, upon the addition

of RS, all materials possess small pore wall thicknesses of 3.5 ~ 4.8 nm, while the UPM-SBA-15

sample has thicker walls of 5.6 nm (Table 1). Therefore, with the use of the water-insoluble RS

polymer, ordered mesoporous silica, analogue of SBA-15 structure, can be produced in an

evaporating water system, and the mesopore size can be enlarged to ~ 9.5 nm without any

hydrothermal treatment.

On the other hand, with increasing content of RS polymer in the precursor solution, the hysteresis

loops in the N2 sorption isotherms are broadened and the isotherms cannot reach the typical plateau

at high relative pressures (Figure 1B, curves a-d), indicating there is an increased presence of large-

meso/macropores. The HRSEM (Figure 2c-h), TEM (Figure 3a-c), STEM images (Figure 3d-f)

12

and mercury porosimetry (Figure 4) confirm the presence of large-meso/macropores of 20 ~ 100

nm in the UHPM-SBA-15 samples. With a higher amount of RS added, more large-

meso/macropores can be observed in the resulted samples. Meanwhile, the large-meso/macropores

in the samples obtained with the addition of 10 ~ 30 wt% RS are separated one another, while

those in the samples obtained with 50 ~ 70 wt% RS added become more interconnected, which is

beneficial for improving mass diffusion. The initial size of the RS polymer colloids is ~ 120 nm,

while there are broadly distributed pores of 20 ~ 100 nm for all the materials obtained with the use

of RS. Roughly, the mesopores in the size range of 20 ~ 50 nm are majorly originated from largely

dissolved RS polymer nanoparticles by ethanol in-situ produced from the hydrolysis of the TEOS.

The macropores of 50 ~ 100 nm are majorly originated from the less dissolved RS nanoparticles

with thermal shrinkage. For every TEOS precursor solution prepared, theoretically, a maximum

of ~ 11.8 g of ethanol can be produced if the TEOS is completely hydrolyzed. The theoretical

solubility of RS in ethanol is 1:5. In this regard, in the precursor solutions with the addition of 10

~ 50 wt% RS (0.38 ~ 1.92 g of pure RS), the RS nanoparticles can theoretically be fully dissolved

in 11.8 g of ethanol. However, complete hydrolysis of TEOS cannot be achieved in the current

synthesis, which makes much less RS dissolved. The solubility of RS in the produced ethanol from

TEOS hydrolysis may also decrease with the presence of the major solvent component water and

other solutes. On the other hand, part of the dissolved RS chains can enter into the F127 micelles,

which makes more RS dissolved. Therefore, only a small fraction of large-meso/macropores can

be observed in the sample obtained with only 10 wt% RS added. The increasing addition of RS

polymer can produce more and broadly distributed large-meso/macropores to generate hierarchical

pore structures in a controllable way.

13

With the other conditions kept the same, the influence of the HCl concertation adopted for the

TEOS precursor preparation has been studied. The same ordered hexagonal mesostructure can be

obtained with the use of a HCl concentration of 0.05, 0.2 and 0.5 M (Figure S2A, S3). The cell

parameter (~ 11.4 nm) of the sample obtained with a 0.05 M HCl is obviously smaller than those

(~ 13.5 nm) of the sample obtained at the higher HCl concentrations. N2 adsorption/desorption

results show that the surface area of the sample obtained with a 0.05 M HCl is slightly higher

compared with the other two samples, while their mesopore sizes (~ 9.7 nm) are almost the same

(Figure S2B, C). Therefore, the silica pore wall thickness of the sample obtained with a 0.05 M

HCl is obviously smaller compared with those of the other two samples, mostly because the lower

HCl concentration leads to a relatively lower degree of TEOS hydrolysis and condensation. On

the other hand, the sample obtained with a 0.05 M HCl shows a much higher pore volume (0.97

cm3 g-1) compared with the two samples obtained with 0.2 and 0.5 M HCl (Figure S2B).

Meanwhile, In the pore size distribution curves (Figure S2C), the volume of the large-

meso/macropores of 20 ~ 100 nm is obviously larger for the sample obtained with a lower HCl

concentration of 0.05 M. The HRSEM images also show that the large-meso/macropores are more

populated for the sample obtained at a lower HCl concentration of 0.05 M (Figure S3). The above

result is because the hydrolysis degree of TEOS is lower with less ethanol produced at a lower

HCl concentration, leading to less dissolved RS nanoparticles and the formation of more large-

meso/macropores in the calcined sample. Therefore, it can be confirmed that the partial dissolution

of RS by the in-situ generated ethanol plays the dual roles of modulating the mesostructure

assembly and templating the hierarchical large-meso/macropores.

With the other conditions kept the same, the influence of the TEOS hydrolysis time has also

been studied. The same ordered hexagonal mesostructure can be observed in the three samples

14

obtained with 1.0, 2.0 and 4.0 h TEOS hydrolysis time (Figure S4A). The 100 diffraction peak of

the sample obtained with a 4.0 h hydrolysis time becomes broadened, indicating that the

mesostructure ordering decreases. This is because the co-assembly between large-sized silica sols

obtained from a long hydrolysis time and the F127 micelles becomes difficult. On the other hand,

while the three samples possess almost the same mesopore sizes (~ 9.7 nm), the sample obtained

with only 1.0 h hydrolysis possesses a slightly higher surface area and a relatively larger pore

volume (Figure S4B, C). Similarly, this is because the hydrolysis degree of TEOS is lower with

less ethanol produced in a shorter hydrolysis period. As a result, less RS nanoparticles can be

dissolved in a short hydrolysis time and formation of more large-meso/macropores in the calcined

sample can be observed (Figure S5).

Finally, with the other conditions kept the same, the influence of the drying temperature (140 ~

190 ºC) has been considered. It is found that the low temperature of 140 ºC cannot make the water

droplets sufficiently dried because of the short residence time (< 2 s), leading to aggregated and

deformed microparticles (data not shown). The ordered hexagonal mesostructure can be

maintained at a drying temperature of 170 and 190 ºC (Figure S6A). The mesostructure of the

sample obtained at 190 ºC becomes less ordered, mainly because the F127 micelle assemblies

become less stable in such a high temperature. On the other hand, the sample obtained at 190 ºC

shows an obviously higher surface area and a relatively larger pore volume compared with those

of the sample obtained at 170 ºC, while their mesopore sizes are the same (Figure S6B, C),

indicating the formation of more large-meso/macropores as verified by the HRSEM images

(Figure S7). This is probably because of the higher degree of water removal and the faster

evaporation of ethanol with less RS dissolved at 190 ºC, leading to the formation of more large-

meso/macropores in the calcined sample.

15

3.3 Particle morphology and the influence of RS

All the microparticles produced via the spray-drying EISA process in this study have a particle

sizes of ~ 90 µm, and are “bowl-like” in shape, typical of skin forming materials (Figure 5 and

S8).43 Such a particle morphology is the result of the formation at the droplet surface of a

viscoelastic shell consisting of densely packed colloidal particles initially balanced by electrostatic

forces resisting the capillary forces until the electrostatic forces are overcome and the shell

buckles.44 This morphology is similar to mesoporous silica particles from our previous studies.25,

26 The addition of RS polymer nanoparticles appears to affect the external appearance of the

particle, giving the surface a “bumpy” appearance before calcination (Figure 5a, b and S8b, d and

f), while the microparticles obtained without the use of RS show smooth particle surfaces (Figure

5d-f). EDX maps and quantitative data of these “bumps” show a much higher concentration of

carbon and lower concentration of silicon than the other areas (Figure 6a-d, S9 and Table S1),

while no such element concentration variation can be found in the non-calcined sample obtained

without the use of RS (Figure 6e-g, Table S1). This result indicates that the “bumpy” regions are

organic (RS polymer/F127) enriched. Before calcination, the microparticles are also more likely

to appear stuck together (Figure S8b, d and f). This is because the polymer-rich “bumps” and skin

at the surfaces increase the stickiness and thus the adhesive strength of the microparticles.

However, after calcination, the surfaces of the microparticles are very smooth, and they are no

longer stuck together (Figure 5c, S8a, c and e), indicating that the polymer-rich “bumps” and the

skin of the microparticles can be easily removed via calcination.

Our previous work has produced microparticles from binary TEOS and RS polymer

nanoparticles in water based sols.39, 40 It is found that the RS nanoparticles can diffuse to the

16

surface, while the silica can diffuse to the center during the drying process, forming core-shell

morphology due to the difference in size of the solutes. In the current study, the point EDX data

of cross-sections and surfaces of the non-calcined microparticles show only a minor increase in

polymer (carbon) on the external surface (Table S1). The RS polymer and F127, indicated by the

carbon element, appears to be well dispersed throughout the microparticles, with exception of the

afore-mentioned “bumps” on the surface (Table S1). There is also a minor trend for increased

presence of Si in the cross-section when compared to the external surface, especially in the samples

obtained with the addition of 30 ~ 70 wt% RS (Table S1), further indicating that the surfaces of

the spray-dried particles are polymer-enriched but the cross-sections are uniform. Besides,

HRSEM images of the calcined samples show no structure differences over the cross-sections of

the microparticles and little difference can be found between the area close to the surface and the

internal structure of microparticles (Figure 2 and S10), unlike the distinctive core-shell

morphology observed previously,39 indicating that the presence of the amphiphilic F127 can bridge

the RS polymer nanoparticles and hydrolyzed TEOS to be uniformly dispersed over the assemble

process.

It should be mentioned that the HCl concentration adopted for the TESO hydrolysis, the TEOS

hydrolysis time, and the drying temperature has no obvious influences on particle morphology of

the resultant UHPM-SBA-15 silica materials (Figure S3, S5 and S7). The drying temperature at

the high temperature of 190 ºC leading to microparticles with multiple dimples on the surfaces

(Figure S7).

3.4 Formation mechanism

17

The formation mechanism of the UHPM-SBA-15 materials is analyzed in detail (Scheme 1).

Initially, the mixing of the solutions of F127, RS and pre-hydrolyzed TEOS leads to the formation

of a homogeneous suspension (Scheme 1a). As poly(propylene oxide) (PPO) is not particularly

soluble in water, F127 in water consists of PPO hydrophobic cores surrounded by the hydrophilic

poly(ethylene oxide) (PEO) chains. Then, over the stirring stage over 30 min, the solutes start to

assemble (Scheme 1b). First, the electrostatic interactions between silica oligomers and PEO

blocks drive the aggregation of F127, and micelles will be formed when the PPO-PPO attractions

dominate the PEO-PEO repulsions.45 Second, the RS nanoparticles are partially dissolved by the

in-situ formed ethanol from hydrolysis of TEOS. Experimentally, about 75 and 20 wt% of the RS

nanoparticles can be dissolved in the precursor solutions with a fixed TEOS amount of 13.3 g and

a RS amount of 10 wt% (0.38 g) and 70 wt% (2.69 g), respectively. The partial dissolution of RS

produces non-uniform RS nanoparticles and water-insoluble RS chains. It has been reported that

solubilized hydrophobic molecules can promote micellization via the expulsion of water from the

micellar cores, which decreases the hydrophilic-hydrophobic balance to make PPO-PPO

attractions dominant over PEO repulsions.45 In our synthesis, the solubilized RS chains can be

partially attracted to the hydrophobic cores of F127, decreasing the hydrophilic-hydrophobic

balance and the critical micelle concentration (CMC) and thus promoting the micellization and

self-assembly processes. Next, during the initial drying stage, the temperature increase can also

promote micellization driven by the enthalpy increase.46, 47 More importantly, with fast solvent

evaporation, micellization, micelle organization and its assembly with silica oligomers can be fast

approached at the air-liquid interface (Scheme 1c). Due to the easier evaporation of ethanol as

compared with water, part of the dissolved RS chains in ethanol forms a RS-rich surface skin along

with fast diffusion and evaporation of ethanol at the interface (Scheme 1c). The remaining RS

18

polymer nanoparticles are interspersed throughout the droplet. Finally, with the continuous solvent

(water) evaporation, the composite micelles are continuously organized with increasing

mesostructure ordering, leading to the formation of an ordered mesostructured silica network

surrounding the RS-swollen assembled F127 micelles and RS nanoparticles of various sizes inside

the dried microparticles (Scheme 1d). On the other hand, part of the large RS nanoparticles is

located on the surface because of their low diffusivity, forming the “bumps” on the surfaces of the

dried microparticles (Scheme 1d). Comparatively, without the assistance of RS, the evaporating

water system is un-favorable for ordered mesostructure assembly. This is because the in-situ

formed ethanol is not enough to optimize the hydrophilic-hydrophobic balance. Over the fast

drying process, the condensation of the silica oligomers is more promoted in the water-based sols

as compared with micellization and the self-assembly, leading to the formation of a disordered

worm-like mesostructure. Besides, without the use of RS, pore swelling and RS nanoparticle

template are absent, leading to the formation of only a small-sized mesoporous silica framework.

In our synthesis, the unique and novel aspects of the assembly process are three-fold. First, the

in-situ formed RS chains can tune the hydrophilic-hydrophobic balance and swell the micelles to

enable the success in assembling ordered mesostructure from F127 and pre-hydrolyzed TEOS in

water and forming larger mesopores without hydrothermal treatment. Second, the remaining RS

nanoparticles endow the final silica microparticles with a hierarchical structure as well as more

open surface porosity. Last but not least, the whole spray-drying EISA process finishes in ~ 2

seconds, enabling a faster approach for producing mesoporous materials compared with

conventional EISA processes conducted in ambient conditions, as well as conventional

hydrothermal synthesis of mesoporous silica materials.

19

3.5 Performance in adsorption of lysozyme

The uniform hierarchically porous SBA-15-type silica microparticles are desirable for bio-

adsorption and bio-separation.48-54 Typically, adsorption of lysozyme on the two samples UHPM-

SBA-15-70wt% and UPM-SBA-15* is compared. The former sample can adsorb significantly

more lysozyme than the latter, with almost a 5-fold increase from 20 to 100 mg g-1 (Figure 7A).

Compared with some prior research regarding the adsorption of lysozyme,48-54 the adsorption

capacity on the sample UHPM-SBA-15-70wt% is comparable. Based on the adsorption capacity

and the initial dosage, the lysozyme loading efficiency in the sample UHPM-SBA-15-70wt% is ~

1.67 %, much higher than that (~ 0.33 %) in the reference sample UPM-SBA-15*. However, the

loading efficiencies are generally low, thus the microparticles are probably not suitable for loading

costly molecules. Nevertheless, the above results clearly confirm that the hierarchical pore

structure can significantly enhance the adsorption performance. After a week of adsorption, the

SAXS pattern of the sample UHPM-SBA-15-70wt% becomes slightly less resolved but still

typical of ordered mesopores (Figure S11A). After the adsorption process, the cell parameter of

the sample keeps the same. Meanwhile, the surface area decreases significantly from 510 to 293

m2 g-1, but the pore volume and pore size increase slightly, from 0.95 to 0.97 cm3 g-1 and from 9.2

to 9.8 nm, respectively. This result indicates that the silica pore walls are generally stable but may

undergo slight dissolution and re-precipitation in the basic buffer solution (pH =10.95).55-56

Considering the low external surface area of the sample and the considerable adsorption capacity,

the lysozyme molecules are mostly adsorbed on the mesopore surfaces inside the microparticles.

On the other hand, after the adsorption process, the sample UPM-SBA-15* also well maintains the

ordered mesostrucure with only slight mesostructure ordering decreases as revealed by the

broadened 100 diffraction peak (Figure S11B, S12). Meanwhile, after the adsorption process, no

20

N2 porosity can be detected for the sample UPM-SBA-15*, indicating the surfaces of the

microparticles might be blocked probably due to lysosome adsorption and slight redistribution of

silica. Given that the molecular size of lysozyme is much smaller than the mesopore sizes of both

the two samples, the significant higher adsorption capacity on the sample UHPM-SBA-15-70wt%

compared with the sample UPM-SBA-15* can be mainly ascribed to the much improved pore

accessibility inside the microparticles. In spray dried microparticles, a dense skin of silica formed

on the surface of microparticles can be often observed, rendering the inner pores inaccessible for

large molecules. For example, Carroll et al. stated that porous structures can be inaccessible as a

result of a solid layer of materials at the surface.26 In our work, such an issue can be overcome by

introducing RS in the precursor to surface skins and bulk nano-templates which can then be

removed in the calcination step to generate an open and hierarchical porous structure.

Figure 7B shows the plots of the intraparticle diffusion model which produces multi-linearity

with each line representing a step in the adsorption process. It is evident that there are two regions

in the plots indicating two adsorption steps. The initial region correlates to the adsorption on the

external surface (film diffusion), while the second region relates to intraparticle diffusion. From a

comparison of the two samples, it is evident that there is less intraparticle diffusion for the sample

UPM-SBA-15* (Table S2). The rate of adsorption is significantly faster for the sample UHPM-

SBA-15-70wt%, with > 3-fold increases in the rate constant over both the two regions. This result

indicates that the hierarchical pore structure can significantly improve the pore accessibility, thus

promoting the mass transfer of large lysozyme molecules into the pore channels inside the

microparticles.

4. Conclusions

21

In summary, we have successfully synthesized uniform hierarchically porous SBA-15-type

silica microparticles in a water based solution by using a rapid and scalable spray-drying EISA

process. These microparticles have uniform micron sizes of ~ 90 µm, and “bowl-like” morphology.

They possess high surface areas of 315 ~ 510 m2 g-1, large pore volumes of 0.64 ~ 0.10 cm3 g-1,

ordered large mesopores of 8.3- 10.0 nm, hierarchical large-meso/macropores of 20 ~ 100 nm, and

open surfaces toward good accessibility into the inner pores. Gradual increase of RS in the

precursor solution has resulted in more swelling of the ordered mesopores, rapid increase of large-

meso/macropores, as well as enhanced surface areas and pore volumes. The ordered mesostructure

and particle morphology can be maintained over a wide range of experimental conditions. The use

of a lower HCl concentration, a shorter TEOS hydrolysis time, and a higher drying temperature all

lead to less dissolved RS nanoparticles and thus the formation of relatively higher surface areas,

large pore volumes due to the creation of more large-meso/macropores. The assembly mechanisms

of these microparticles have been analyzed in detailed. The RS nanoparticles can be partially

dissolved by in-situ generated ethanol from hydrolysis of TEOS to form solubilized hydrophobic

RS chains. These chains can swell the F127 micelles and decrease their the hydrophilic-

hydrophobic balance to promote the micelle assembly into ordered mesostructures with increased

cell parameters. The un-dissolved RS nanoparticles form large-meso/macropores interconnecting

with the ordered mesoporous framework. In addition, part of the RS chains and nanoparticles form

surface “skins” and “bumps”, the removal of which can make the particle surface more open.

Without the use of RS in the precursors, the obtained control samples display a disordered “worm-

like” mesostructure due to the condensation of silica being favored over the self-assembly of the

micelles in water. In the adsorption of lysozyme, the representative hierarchically porous SBA-15-

type silica microparticles has exhibited a > three-fold faster rate of adsorption and a five-fold

22

increase in adsorption capacity (from 20 to 100 mg g-1), and no pore blockage effect after

adsorption as compared with the control sample obtained without the use of RS. Finally, compared

with the typical previous research,1, 2, 4, 25, 26, 30, 42 this study improves the ordered mesostructure

assembly EISA process in water systems, enhances the mesopore sizes without any hydrothermal

conditions and more conveniently creates hierarchical pore structures with improved pore

accessibility of spray-dried microparticles. The method would be applicable for the synthesis of

other hierarchical and ordered mesoporous materials for adsorption, separation and catalytic

applications.

Appendix A. Supporting Information

Acknowledgements: Financial supports from the National Natural Science Foundation of China

(No. 21875153 for Z. Wu) and the Australian Research Council (DP140104062, FT140101256 for

C. Selomulya) are much appreciated. The authors would like to acknowledge Dr. Wenjie Liu for

experimental assistance. The authors acknowledge the use of facilities within the Monash Centre

for Electron Microscopy. K.W. thanks the Australian Postgraduate Award Scheme for the

provision of a scholarship.

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Figures and tables

Figure 1. SAXS patterns (A) and N2 sorption isotherms (B) and the corresponding pore size

distribution curves (Inset B) of the UHPM-SBA-15 samples obtained with the addition of 70 (a),

50 (b), 30 (c), 10 wt% (d) RS, and the UPM-SBA-15 sample obtained without the use of RS (e),

respectively.

31

32

Figure 2. HRSEM images of the UHPM-SBA-15 samples obtained with the addition of 70 (a-c),

50 (d, e), 30 (f), 10 wt% (g) RS, and the UPM-SBA-15 sample obtained without the use of RS (h),

respectively.

33

Figure 3. TEM (a-c) and STEM (d-f) images of the UHPM-SBA-15 samples obtained with the

addition of 70 (a, d) and 30 wt% (b, e), and the UPM-SBA-15 sample obtained without the use of

RS (c, f), respectively.

34

Figure 4. Mercury porosimetry isotherms and the inset pore size distribution curves of the UHPM-

SBA-15 samples obtained with the addition of 70 (a), 50 (b), 30 (c), 10 wt% (d) RS, respectively.

.

.

35

Figure 5. SEM images of the UHPM-SBA-15 sample obtained with the addition of 70 wt% RS

(a-c) and the UPM-SBA-15 sample obtained without the use of RS (d-f) before (a, b, d, e) and

after (c, f) calcination, respectively.

36

Figure 6. SEM images (a, e) and the corresponding carbon (b, f), silicon (c, g) and oxygen (d, h)

element maps of the UHPM-SBA-15 sample obtained with the addition of 70 wt% RS (a-d) and

the UPM-SBA-15 sample obtained without the use of RS (e-h) before calcination, respectively.

37

Figure 7. Time-dependent adsorption capacity curves (A) and the corresponding linear fitting

curves (B) by using the intraparticle diffusion model for the adsorption of lysozyme on the UHPM-

SBA-15 sample (a) obtained with the addition of 70 wt% RS and on the control sample UPM-

SBA-15* (b) obtained without the use of RS and with ethanol as the solvent, respectively.

38

Scheme 1. Schematic representation of the assembly process of the hierarchical porus SBA-15-

type microparticels: the initial mixing stage (a), further hydrolysis of TEOS and partial dissolution

of RS nanoparticles to form RS polymer chains (b), formation and swelling of micelles and the

structure starting to assemble in the initial drying stage (c), the ordered array of swollen micelles

and disordered packing of RS nanoparticles with some RS polymer on surface of particle to

produce “bumps” after the drying process (d).

39

Table 1. Summary of physicochemical properties of the various samples obtained at different

experimental conditions.

sampleSBET

a

(m2 g-1)

Vtb

(cm3 g-1)Dc (nm)

d100d

(nm)ae (nm)

wf (nm)

FWHMg (nm-1)

UPM-SBA-15 299 0.38 6.8 10.8 12.4 5.6 0.213

UPM-SBA-15* 537 0.76 7.9 10.8 12.4 4.5 0.128

UHPM-SBA-15-10 wt% 487 0.68 9.3 11.0 12.8 3.5 0.185

UHPM-SBA-15-30 wt% 435 0.64 8.3 11.3 13.1 4.8 0.199

UHPM-SBA-15-50 wt% 491 0.86 9.5 11.9 13.8 4.3 0.199

UHPM-SBA-15-70 wt% 510 0.95 9.2 11.6 13.4 4.2 0.185

a the BET surface area, b the total pore volume, c the pore size, d the d100 spacing, e the cell parameter of the hexagonal mesostructure calculated by the formula a = 2/ d100, f the pore wall thickness √3calculated by using the formula w = a - D, and g the full width at half maximum of the 100 peaks in the SAXS patterns, respectively. * the sample was obtained with ethanol as the solvent while the other samples were obtained with water as the solvent.

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Spray-drying-assisted EISA assembly of SBA-15-type silica microparticles with water as the solvent is achieved by using a multiple-role polymer colloid. The microparticles possess uniform micro-sizes, ordered and large mesopores, hierarchical porosities and high surface areas. They are promising for lysozyme adsorption with a fast adsorption rate and a high capacity because of the improve pore accessibility.