Confined mesoporous silica membranes for albumin zero-order release

Accepted Manuscript

Confined mesoporous silica membranes for albumin zero-order release

Nicola Gargiulo, Ilaria De Santo, Filippo Causa, Domenico Caputo, Paolo

Antonio Netti

PII: S1387-1811(12)00216-8

DOI: 10.1016/j.micromeso.2012.04.003

Reference: MICMAT 5470

To appear in: Microporous and Mesoporous Materials

Please cite this article as: N. Gargiulo, I. De Santo, F. Causa, D. Caputo, P.A. Netti, Confined mesoporous silica

membranes for albumin zero-order release, Microporous and Mesoporous Materials (2012), doi: 10.1016/

j.micromeso.2012.04.003

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Confined mesoporous silica membranes for

albumin zero-order release

Nicola Gargiuloa,*, Ilaria De Santoa,b,*, Filippo Causaa,c, Domenico Caputoc, Paolo

Antonio Nettia,b,c

a Centro di Ricerca Interdipartimentale sui Biomateriali, Università Federico II, P.le

Tecchio 80, 80125 Napoli, Italy

b Centre for Advanced Biomaterials for Health Care, Italian Institute of Technology,

Largo Barsanti, 80125 Napoli, Italy

c Dipartimento di Ingegneria dei Materiali e della Produzione, Università Federico II,

P.le Tecchio 80, 80125 Napoli, Italy

* These two authors contributed equally to this work.

Corresponding author: Domenico Caputo (e-mail: domenico.caputo@unina.it; address:

Dipartimento di Ingegneria dei Materiali e della Produzione, Università Federico II, P.le

Tecchio 80, 80125 Napoli, Italy; telephone: +397682396; fax: +397682394)

Abstract

In this work, the transport capabilities of anodic alumina membranes confining

SBA-15-like nanochannels in their pores were investigated. The mean pore size of the

confined mesophase was set around 7.0 nm, thus to achieve single passage of the

selected Bovine Serum Albumin (BSA) protein, having comparable hydrodynamic

diameter, through the nanochannels. Shape, size and orientation of the manufactured

mesophase were characterized by means of small-angle X-ray scattering, transmission

electron microscopy and N2 adsorption / desorption at 77 K. The usage of

mesostructure-containing samples having pore tuned over the size of the released BSA

allowed achieving a long-term zero-order release profile by single-file diffusion up to

several weeks, which delivers a constant amount of drug by unit time independently

from drug concentration and drug accessible area, while control anodic alumina

membranes showed a classic Fickian diffusion release. The attained BSA constant

release rate was about 2 μg/day. The possibility of rate tuning could be further exploited

by varying the length of the siliceous nanochannels inside the alumina pores, i.e.

through different amounts of mesostructure filling the host support.

Keywords: Mesoporous materials; Drug delivery; Confined diffusion; Zero-order

release

1. Introduction

The morphological control of porous systems on the nanoscale is a field of research that

recently turned out to be very active. In fact, the development of structures consisting in

ordered arrays of oriented nanochannels would allow to efficiently perform operations

such as the inclusion synthesis of a vast range of nanowire systems through hard

templating, the separation of large biomolecules, or the long-term and controlled

delivery of drugs. A shining example of such structures that is subject of many recent

research activities is represented by hierarchical systems made of mesoporous siliceous

materials confined in the channels of anodic alumina membranes (AAMs) [1, 2]. In

these systems the silica-surfactant nanocomposite is assembled at the alumina pore

walls to form the surfactant-templated silica-nanochannels which are typical of such

mesostructures. These materials can be synthesized by different methods, the most

investigated of which relies on the so-called evaporation-induced self-assembly (EISA)

[3]. In one of the first studies about mesoporous silica confined in AAMs by EISA,

different porous alumina membranes having distinct channel diameters (i.e., from 18 to

80 nm) were used as substrates and dip-coated with a synthesis solution containing

Pluronic P123 as templating agent. The development of different mesostructures (from

single chains of spherical mesopores to concentric or chiral helical mesopores) was

found to depend from the different diameters of the hosting alumina nanochannels [4].

More recently, Bein and coworkers conducted thorough experiments on the formation

of mesophases inside AAMs by means of two-dimensional small-angle X-ray scattering

(2D SAXS) and transmission electron microscopy (TEM) [5]. These studies were then

deepened by performing grazing incidence SAXS (GISAXS) measurements [6], by

formulating a possible formation mechanism for the confined mesostructure [7] and by

investigating the influence of the addition of inorganic salts on the orientation of the

channels in confined mesoporous silica templated by non-ionic triblock copolymer

surfactants [8].

Later on, Bein and coworkers also considered possible applications of confined silica

mesostructures as drug delivery systems (DDSs). These studies reported the adsorption

and the in vitro release kinetics of mesostructure-containing AAMs previously loaded

with vancomycin [9] or ibuprofen [10], showing that so-called hexagonal columnar

systems (i.e., systems in which the cylindrical pores of the confined phase are straight

and parallel to those of the hosting AAM [1]) attain a slower release over an extended

time period, and pointing that major parameters affecting drug release are pore shape,

accessibility, length, and diameter compared to that of the eluting molecule. Indeed, the

release or adsorption of bioactive agents from mesoporous materials depends in a

complex way from the physicochemical properties of both the biomolecule and the

device. Furthermore, the dynamic behavior of the transported molecules depends in turn

on the surface chemistry and size of the pores.

Although the reported works already tested mesostructure-containing AAMs as drug

carriers, none were devoted to the assessment of their transport properties as drug

delivery membranes. The most appropriate configuration of the confined mesophase for

this kind of application is represented by the hexagonal columnar orientation, which can

also be tailored in pore size on the nanometric scale by accurately choosing the

templating non-ionic tri-block copolymer surfactant (e.g., Brij 56 instead of Pluronic

P123 [8]). Indeed, the exploitation of mesostructure-containing AAMs as drug delivery

membranes could be advantageous for the long-term delivery of biomolecules, proteins

in particular. Among the diverse release mechanisms, the most fruitful for

pharmaceuticals applications consists in the zero order release mechanism, which

delivers a constant amount of drug by unit time independently from drug concentration

and drug accessible area [11]. Zero order release can be achieved through a single file

diffusion mechanism of molecules trafficking in a pore having similar size. Indeed, the

single-file diffusion of the released molecule is made possible when the diameter of the

constraining 2D geometry in the membrane is smaller than twice the size of the released

molecule, and, as a result, the effective release rate becomes constant with time due to

the single molecule passage [12].

Recent studies have already demonstrated single-file diffusion of protein drugs through

nanoprous membranes. In particular, membranes consisting of cylindrical block

copolymers nanochannels were developed and obtained single-file diffusion of proteins

through the fine-tuning of the pore membrane over the size of the bioactive agent to

release [13]. The pore size was indeed precisely modulated to that of the released agents

through Au deposition. Other groups showed the constant release rate of proteins

through nanoporous silicon membranes having a slit shaped pore nanometric in height,

although obtained through multiple steps of precision silicon fabrication techniques on

silicon-on-insulator substrates [14].

Here, we investigated the transport capabilities of AAMs confining SBA-15-like

columnar nanochannels in their pores (SBA-15-AAMs). The mean pore size of the

manufactured SBA-15-AAMs was set around 7.0 nm, and showed the long-term

capability of zero-order release of the fluorescent BSA proteins (molecular size 7.2 nm)

up to several weeks.

2. Experimental

2.1 Synthesis of AAM-confined mesoporous silica

The confinement of mesoporous silica in AAMs was performed following the procedure

reported in [8]. First, 2.08 g of tetraethyl orthosilicate (Aldrich) were mixed with 3.00 g

of a 0.2 M HCl (J. T. Baker) aqueous solution, 1.80 g of H2O and 4.00 g of ethanol

(Fluka). This mixture was then heated at 60 °C for 1 h to achieve acid-catalyzed

hydrolysis-condensation of the silica precursor. The pre-hydrolyzed silica was then

mixed with 0.75 g of the non-ionic triblock copolymer surfactant Pluronic P123

(Aldrich) that was preliminarily dissolved in 11.85 g of ethanol. In order to achieve the

hexagonal columnar orientation of the mesoporous siliceous channels, 0.085 g of LiCl

(Aldrich) were added to the resulting mixture [8]. The AAMs used (Anodisc, Whatman)

have a diameter of 13 mm and an average pore size of 200 nm. Two drops of the

precursor mixture were homogeneously spread on the membrane surface and let to

evaporate at 30 °C at about 50% relative humidity. Mesostructure-containing

membranes were then calcined using a heating rate of 0.5 °C/min with annealing

periods of 10 h at 120 °C, and 5 h at 200, 300 and 500 °C, respectively. Because the

confined phase may be thought to resemble SBA-15 mesoporous silica in both its

structure and textural properties [15], the final product was labeled as SBA-15-AAM.

2.2 Characterization of SBA-15-AAM

SBA-15-AAM samples were characterized with SAXS by means of an Anton Paar

SAXSess instrument operating in point-collimation mode with a Genix Microsource

X-ray generator (CuKα radiation) at 50 kV and 1 mA. The experimental setup is similar

to that reported in [8]: in detail, SBA-15-AAM samples were stuck onto a Variostage

X-Rotation cell using a small amount of vacuum grease; the tilting angle was set to 10°

and the data collection was performed in 5 exposures of 120 s each that were

successively averaged into one image.

Moreover, TEM images of SBA-15-AAM samples were collected by means of a Philips

EM 208 instrument equipped with a MegaView camera. Prior to the observation,

membrane samples were embedded within resin and then ultramicrotomied.

In order to analyze the pore structure of the synthesized hierarchical structure, partially

ground samples of SBA-15-AAM were submitted to nitrogen adsorption/desorption at

77 K by means of a Micromeritics ASAP 2020 apparatus. The density functional theory

(DFT) pore size distribution was determined modeling experimental adsorption data

with a kernel function for oxide materials having cylindrical pore geometry.

2.3 Release experiments

In order to assess protein release through nanochannels, SBA-15-AAMs were glued by

a cyanoacrylate solution onto Transwell support, in place of the original membrane,

and between two reservoirs. Control Anodisc membranes, having 200 nm pore size,

were attached to a Transwell support in the same way. The proposed experimental

system envisages the uniaxial flow of biomolecules through the mesoporous membrane.

The membrane separates an initially filled biomolecules-containing reservoir from a

phosphate buffered saline (PBS) solution environment where biomolecules are depleted.

Before loading, membranes were preconditioned in 30% ethanol solution for thirty

minutes and then put in water for about ten days. After preconditioning, the volume

above the membranes was filled with 0.5 ml of 2.5 M fluorescent

BSA-TetraMethylRhodamine protein solution (Molecular Probes, MW 67 kDa),

whereas the lower volume contained 2 ml of PBS plus 0.03% sodium azide. The release

system was kept at 23 °C on a 50 rpm shaking plate. Protein concentration in the lower

reservoir was measured by means of a Perkin-Elmer Spectrofluorimeter (485-535 nm).

The BSA concentration was determined from a 100 L solution aliquot removed from

the sink reservoir and replaced with an equal amount of fresh PBS plus 0.03% sodium

azide at intervals of approximately 48 h and followed up to two months.

3. Results and discussion

3.1. Characterization of SBA-15-AAM

Fig. 1 shows the SAXS pattern of a SBA-15-AAM sample: as reported in the literature

[8], SAXS measurements on siliceous mesostructures confined in AAMs usually result

in diffraction patterns with two or four visible first-order reflections that can be

correlated to the orientation of the mesoporous channels with respect to the

macroporous ones of the hosting AAM. The reflections in the horizontal plane of the

primary beam are called in-plane (ip) reflections, and are detectable when the hexagonal

columnar orientation occurs, but also when the mesophase assumes a closed,

donut-shaped circular configuration [1]. The reflections out of the horizontal plane are

called oop reflections and occur only in correspondence of the circular orientation.

When ionic surfactants (such as cetyltrimethylammonium bromide) are used as

templates for the assembly of the confined phase, the process spontaneously leads to the

hexagonal columnar arrangement of the mesostructure [5]. On the contrary, when using

non-ionic triblock copolymer surfactants (e.g., Pluronic P123) as templates, the

assembly evolves towards the circular phase. The addition of an inorganic salt (LiCl) to

the P123-based sol allows to mimic the behavior of a sol based on an ionic surfactant:

by this way, the resulting confined mesostructure will be characterized by a high

accessible porosity due to its columnar orientation, and also by a high pore size due to

the usage of a long-chain, non-ionic surfactant as template. These observations are

confirmed by the analysis of Fig.1, in which the scattering pattern of the SBA-15-AAM

sample shows distinct ip reflections at a value of the scattering vector q of about

0.51 nm-1 (corresponding to a d-spacing of 12.3 nm), and only weak oop reflections

with almost the same d-spacing (~12.7 nm). As a consequence, the mesophase is mainly

hexagonal columnar, with only a small fraction of hexagonal circular domains: this

result is well comparable with others reported in the literature [8].

[Figure 1]

Fig. 2 shows a TEM image of a SBA-15-AAM sample, in which one of the

200 nm-wide pores of the AAM is filled with the siliceous mesostructure. In previous

papers dealing with similar topics [8], TEM observations consisted in top views of the

membranes, and the samples were prepared by dimple grinding and successive ion

milling. On the contrary, in this case, the sample was embedded within resin and then

ultramicrotomied: this approach, though basically more destructive than the

aforementioned one, allowed to directly observe a cross section of the membrane. In

Fig. 2, the hexagonal columnar orientation of the mesoporous siliceous channels is

clearly visible, confirming what already pointed out from SAXS experiments.

Moreover, the adherence of the confined phase to the AAM channel surface is plainly

demonstrated: as reported in the literature [8], when the confinement of the

P123-templated mesostructure is performed without the addition of inorganic salts to

the precursor sol, the resulting circularly oriented phase turns out to be significantly

detached from part of the wall of the hosting macropores after the assembly process;

such phenomenon is further stressed after the calcination treatment. On the contrary, the

presence of LiCl in the synthesis environment, apart from promoting the columnar

orientation of the siliceous channels, aids to protect the confined phase from such

detachment during both the gelation and the template removal steps.

[Figure 2]

Fig. 3 shows the N2 adsorption/desorption isotherm at 77 K on partially ground samples

of SBA-15-AAM: the shape of the isotherm is coherent with what reported in the

literature [8]. In particular, it is classifiable as IUPAC type IV, which is typical of

mesoporous materials [16]; moreover, a H1-type hysteresis loop is highlighted. The

BET specific surface area turned out to be about 40 m2/g, in fair accordance with the

literature: such result seems odd if compared with those (one order of magnitude higher)

usually associated to bulk mesoporous silica particles, but becomes plausible when

considering that the mesophase content inside the composite membrane may be as low

as 10 wt% [8].

[Figure 3]

Fig. 4 shows the DFT pore size distribution of partially ground samples of

SBA-15-AAM obtained from modeling of experimental data reported in Fig. 3. Again,

the results are coherent with those reported in the literature [8]: the distribution has a

main peak at about 7.0 nm, that actually corresponds to the pore size of the confined

mesostructured phase. The tail that follows such peak in Fig. 4 may be related to defects

originated during the grinding process, but also by the occasional merging of silica

pores. This phenomenon may be considered as a side effect of the increased interactions

of the mesophase with the alumina channel walls due to the addition of an inorganic salt

to the synthesis system. In fact, while in the absence of LiCl the mechanical stress

caused to the circularly oriented siliceous channels during the calcination process leads

to the detachment from the alumina pore walls, the columnar phase originated in the

presence of the inorganic salt remains attached to the alumina, and the mechanical stress

related to the thermal treatment propagate to the silica structure, causing the rupture of a

small fractions of its pore walls [8].

[Figure 4]

3.2. Release experiments

Bovine Serum Albumin (BSA) protein was selected for the evaluation of the transport

properties of the manufactured membrane. Indeed, BSA is characterized by a molecular

weight of 67000 Da and a hydrodynamic diameter of around 7.2 nm [17], which is close

to the main peak value of the mesopore size distribution of SBA-15-AAM samples. The

quality of BSA release profile through SBA-15-AAM was assessed through the

evaluation of the time-dependent transport of proteins across a membrane mounted in a

Transwell support. Fig. 5 shows the release profile of BSA through SBA-15-AAM,

and through the control alumina membrane. Open circles represent the released BSA

amount from the control AAM having nominal pore size of 200 nm, while full stars the

quantity released from a SBA-15-AAM with a mean pore size of around 7.0 nm. No

significant time lags are recognized in Fig. 5, which were instead present in absence of

membranes preconditioning (data not shown).

[Figure 5]

The constant release profile of BSA is obtained in SBA-15-AAM, whereas a classic

Fickian diffusion release profile is obtained for the AAM. In the case of AAM, the

release profile has been adjusted following the mass balance, which applies under the

assumption of Fickian diffusion for a quasi-stationary process [18] reported below:

ln 1− C(t) /C(∞)[ ] = − DnAp /L( )× 1/V1 +1/V2( )× t (1)

In Eq. (1), C(t) is the protein concentration in the sink reservoir at a time t, C(∞) is the

concentration of the system at infinite time, D is the protein diffusion coefficient in the

pores, n and Ap are the number of pores and their section respectively, L represents the

membrane thickness, V1 and V2 are the source and sink volumes.

The BSA diffusion coefficient in AAM could be roughly esteemed from Eq. (1),

attaining 3.5E-9 cm2/s, which is around two orders of magnitude smaller than in bulk,

6E-7 cm2/s. Since the ratio of molecule diameter over pore size is in this case 0.04, such

a reduction of diffusion coefficient cannot be uniquely ascribed to constrained diffusion,

and could be rather due to an over esteem of the pore number in AAMs, which are

declared having a broad pore density range.

On the other hand, in the case of SBA-15-AAM the BSA release profile exhibits a clear

zero order release, which follows the equation

Qt = Q0 + K0 × t (2)

where Qt is the protein amount dissolved at time t, Q0 is the initial amount of drug in the

solution, and K0 is the zero order release constant independent of the solvent accessible

area.

In particular, since during the first two days of observation, the release curve of

SBA-15-AAM shows a higher slope compared to the longer times, probably ascribable

to low sensitivity of the reading apparatus, the linear fit of the profile in Fig. 5 is

evaluated from day three of release attaining a 0.990 r-square, which suggests a zero

order release rate of 1.83 μg/day, and the total released quantity is around 70% of the

protein loaded in the source. Indeed, BSA proteins have comparable size of membrane

pores, of about 7.0 nm. This condition justifies a zero order release rate by the single

file passage of molecules through the nanochannel. This size effect has to be coupled to

considerations regarding surface interactions, which of course cannot be neglected in

the case of high surface to volume ratios accomplished by nanoporous geometries [19].

Indeed, since BSA isoelectric point is 4.7, albumin shows a net negative charge of about

-17e at pH 7. This would suggest that also electrostatic repulsion between a negative

charged SBA-15 inner surface [20] and BSA, avoiding adsorption [21], could play a

determinant role in the zero order release shown.

Zero order release is highly desired in pharmaceuticals applications, since guarantees

constant release rate independently from reservoirs concentrations. Our formulation

attained a constant rate of about 2 μg/day, which is on the same order of magnitude of

what obtained in nanofluidic membranes [22]. This rate could be tuned to satisfy the

small dosages required for long term therapies such as growth factor delivery, useful in

tissue engineering applications for different tissues repair aims, such as vascularization

and bone regeneration. Growth factors as vascular endothelial VEGF and bone

morphogenetic proteins BMPs, control self-renewal, migration, differentiation as well

as other cell fate processes of progenitor cells. In vascularization as well as osteo-

regenerative processes, the release rates are indeed preferably around 100 ng - 10

µg/day/cm3 of injured tissue [23].

However, the obtained rate could be further optimized and adjusted on the specific drug

dosage required for the considered application by fine-tuning the exposed membrane

surface. Otherwise, the amount of silica precursor solution used in the initial sol

quantities, could be lowered thus attaining higher release rates by decreasing the length

of the siliceous nanochannels inside the alumina pores, i.e. filling less alumina

membrane thickness. Indeed, it would be ideally possible to tune the release slope, thus

release velocity, by confining less mesostructure inside the alumina pore in order to

change the effective thickness of the mesoporous channel system.

Since zero order release induced by single molecule passage can be achieved as soon as

the diameter of the constraining 2D geometry in the membrane is smaller than twice the

size of the released molecule, the manufactured pore size of SBA-15-AAM could be

used to perform zero order release of several other proteins, including hemoglobin and

human insulin (hydrodynamic diameters 7.0 and 5.4 nm, respectively). Indeed, the

dimensional range explored covers most of large proteins of pharmaceutical interest,

which the tested BSA belongs to. In addition, it was shown already the achievement of

confined mesoporous silica membranes of about 5.0 nm pore size in the presence of

ionic surfactants (i.e. cetyltrimethylammonium bromide [8-10]). These membranes

could be tested to obtain single passage of smaller proteins, such as lysozyme and

chymotrypsinogen (hydrodynamic diameters 3.8 and 4.8 nm, respectively).

Moreover, it would be interesting to explore the possibility to expand the pores of

SBA-15-AAM by modifying the starting sol, for applying the manufactured membrane

to even larger proteins such as apoferritin and thyroglobulin (hydrodynamic diameters

16.4 and 20.2 nm, respectively).

Different works already demonstrated zero order release for BSA diffusing in

nanochannel of nanometric height, though this result was never achieved so far for

mesoporous membranes produced by EISA. The major advantage in this configuration

is the relatively ease of manufacturing compared to costly and time-consuming

production schemes developed for the microprocessor industry.

4. Conclusions

Samples of anodic alumina membranes confining SBA-15-like nanochannels in their

pores were successfully synthesized and characterized by means of small-angle X-ray

scattering, transmission electron microscopy and N2 adsorption / desorption at 77 K.

SAXS and TEM measurements confirmed the hexagonal columnar orientation of the

mesoporous siliceous channels hosted by the 200 nm-wide alumina pores. The mean

pore size of SBA-15-AAMs, as confirmed by microporosimetric analysis, was set

around 7.0 nm, which is comparable with the hydrodynamic diameter of the Bovine

Serum Albumin protein selected for release testing.

Long-term zero order release of BSA through SBA-15-AAM samples was demonstrated

up to several weeks, obtaining a release rate of about 2 μg/day. The possibility of tuning

the release rate through different amounts of AAM-filling mesostructure could be

exploited. Further investigations are needed in order to test protein biological activity

intactness after several weeks of protein-mesostructure interaction. These findings claim

a deeper insight into transport phenomena through confined silica mesopores in order to

better design drug delivery systems.

Acknowledgements

The authors acknowledge the help provided by Anton Paar GmbH (Austria).

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Figure captions

Figure 1. SAXS pattern of SBA-15-AAM evidencing in-plane (ip) and out-of-plane

(oop) reflections.

Figure 2. TEM image of SBA-15-AAM.

Figure 3. N2 adsorption (solid symbols) / desorption (open symbols) isotherm at 77 K

on partially ground SBA-15-AAM samples.

Figure 4. DFT differential pore size distribution of partially ground SBA-15-AAM

samples.

Figure 5. Release profiles of BSA through SBA-15-AAM (full stars) with a pore size of

7.0 nm, and through the supporting AAM with a nominal pore size of 200 nm (open

circles).

Graphical abstract

Highlights

> Usage of nanoporous silica-alumina composites as drug delivery membranes. >

Investigation about protein transport capabilities of mesophase-confining membranes. >

Long-term zero-order release profile of bovine serum albumin up to several weeks.