Article pubs.acs.org/Langmuir

Adsorption of Lysozyme on Hyaluronic Acid Functionalized SBA-15 Mesoporous Silica: A Possible Bioadhesive Depot System Luca Medda, Maria F. Casula, Maura Monduzzi, and Andrea Salis*,‡ Department of Chemical and Geological Sciences, University of Cagliari-CSGI and CNBS, Cittadella Universitaria, S.S. 554 bivio Sestu, 09042- Monserrato (Cagliari), Italy ‡ Department of Applied Mathematics, Australian National University, 0200 ACT, Australia S Supporting Information *

ABSTRACT: Silica-based ordered mesoporous materials are very attractive matrices to prepare smart depot systems for several kinds of therapeutic agents. This work focuses on the well-known SBA-15 mesoporous silica and lysozyme, an antimicrobial protein. In order to improve the bioadhesion properties of SBA-15 particles, the effect of hyaluronic acid (HA) functionalization on lysozyme adsorption was investigated. SBA-15 samples having high (H-SBA) and low (LSBA) levels of functionalization were analyzed during the three steps of the preparations: (1) introduction of the −NH2 groups to obtain the SBA-NH2 samples; (2) functionalization with HA to obtain the SBA-HA matrices; (3) adsorption of lysozyme. All silica matrices were characterized through N2adsorption/desorption isotherms, small-angle X-ray scattering, transmission electron microscopy, thermogravimetric analysis, and Fourier transform infrared spectroscopy. The whole of the experimental data suggests that a high level of functionalization of the silica surface allows for a negligible lysozyme adsorption mainly due to unfavorable electrostatic interactions (H-SBA-NH2) or steric hindrance (H-SBA-HA). A low degree of functionalization of the silica surface brings about a very good performance toward lysozyme adsorption, being 71% (L-SBA-NH2) and 63% (L-SBA-HA) respectively, compared to that observed for original SBA-15. Finally, two different kinetic modelsa “pseudo-second order” and a “intraparticle diffusion”were compared to fit lysozyme adsorption data, the latter being more reliable than the former.

1. INTRODUCTION Smart nanosystems for drug delivery or tissue engineering require sophisticated features according to nanomedicine challanges.1−3 Among many different kind of nanostructures, ordered mesoporous materials (OMMs) represent a very promising chance.4−7 OMMs are synthesized taking advantage of the self-assembly properties of surfactants which act as templates for the polymerization of silica or other oxides precursors.8 Since their discovery, OMMs have attracted the attention of researchers because of their outstanding properties, like high surface area, high pore volume, and narrow pore size distribution.9 In particular, pore size is comparable with the diameter of biomacromolecules like proteins/enzymes.10,11 This has led to an intense research to explore the use of OMMs as supports for enzyme immobilization12−18 or as carriers for the sustained release of therapeutic proteins.19−21 Basically, what distinguishes the two cases is the strength of protein−OMM interactions. These need to be weak for sustained release applications (the protein needs to be slowly released in a physiological environment) and strong for biocatalytic purposes.22−24 Another issue is the location of adsorbed biomacromolecules on the external particle or internal pore surface.25 This is important because the internal surface © XXXX American Chemical Society

better protects the immobilized protein from the external environment (reaction or release medium) which could inactivate it. It has also been shown that pH,26,27 ionic strength,26,28 and salt type,29,30 affect the adsorption parameters (kinetics, equilibrium adsorption isotherms) and also the resulting release.27,28 All these effects modulate the adsorption step31,32 and the consequent application.27,28 However, while for biocatalysis the problems become competence of the chemical engineer (reactor type, etc.), for controlled release applications the chemist has to solve some other issues. Indeed, depending on the type of formulation, parameters like particle size or particle surface need a careful design. The functionalization of mesoporous silica surface is a well established procedure.33,34 However, in order to improve the bioadhesive properties of silica particles, the external surface needs to be functionalized with a chemical group that is recognized by the receptors present in the physiological environment. A natural polymer like hyaluronic acid (HA) is a good candidate to this purpose.35−37 Received: August 13, 2014 Revised: September 26, 2014

A

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washings with Millipore water, and drying under vacuum overnight at room temperature. 2.4. Characterization of the Materials. Textural analysis was carried out on a Thermoquest-Sorptomatic 1990 instrument, by determining the N2 adsorption/desorption isotherms at 77 K. Before analysis, pure silica samples (SBA-15) were heated up to 250 °C at a rate of 1 °C/min under vacuum, while the functionalized samples (SBA-NH2, SBA-HA) were outgassed overnight at 40 °C. The specific surface area, and the pore size distribution were assessed by the Brunauer−Emmett−Teller (BET) and Barrett−Joyner−Halenda (BJH) methods, respectively.40,41 Attenuated total reflectance Fourier transform infrared (ATRFTIR) studies were conducted with a Bruker Tensor 27 spectrophotometer equipped with a diamond-ATR accessory and a DTGS detector. A number of 256 scans at a resolution of 2 cm−1 were averaged from wavenumber 4000 to 400 cm−1. The Opus software was used for data handling. The amount of free amino groups grafted to SBA-15 was determined before the functionalization with HA through titration. In a typical analysis, a mass of 0.1 g of dried (at 100 °C for 4h) sample was suspended in 5 mL of HCl 0.1 M. The suspension was equilibrated overnight at 25 °C, then filtered and washed with a few milliliters of water. The filtrate was weighted and titrated with a 0.1 M NaOH standard solution. Thermogravimetric analysis (TGA) was carried out on a MettlerToledo TGA/STDA 851. Thermal analysis data were collected in the 25−1000 °C range, under oxygen flow (heating rate = 10 °C·min−1; flow rate = 50 mL·min−1). Small-angle X-ray scattering (SAXS) was recorded with a S3MICRO SWAXS camera system (HECUS X-ray Systems, Graz, Austria). CuKα radiation of wavelength 1.542 Å was provided by a GeniX X-ray generator, operating at 50 kV and 1 mA. A 1D-PSD-50 M system (HECUS X-ray Systems, Graz, Austria) containing 1024 channels of width 54.0 μm was used for detection of scattered X-rays in the small-angle region. The working q-range (Å−1) was 0.003 ≤ q ≤ 0.6, where q = 4π sin (θ)λ−1 is the modulus of the scattering wave vector. Thin-walled 2 mm glass capillaries were filled with the sample for the scattering experiments. The scattering patterns were recorded for 1 h. 2.5. Kinetics of Lysozyme Adsorption on SBA-15 and Related Materials. Lysozyme adsorption on SBA-15, SBA-NH2 ,and SBA-HA samples was carried out by suspending 0.2 g of the mesoporous sample in 20 mL of aqueous solution of lysozyme (5 mg/ mL) prepared in a 100 mM phosphate buffer pH 7.0, and soaked for 24 h with shaking at 100 rpm and 25 °C. The concentration of lysozyme was measured by a UV−vis spectrophotometer at λ = 280 nm. The loading of lysozyme (qt) was recorded as a function of time (t), according to the following equation:

HA is a linear polysaccharide composed by disaccharide units containing N-acetyl-D-glucosamide and glucuronic acid and is the simplest member of a group of substances known as glycosaminoglycans. HA is completely absent in fungi, plants and insects but is almost ubiquitous in humans and vertebrates. HA molar mass, which ranges between 105 and 108 Da, plays important physiological roles in living organisms including maintenance of viscoelasticity of liquid connective tissues (i.e., the synovial fluid in the joints or in the vitreous humor), the control of the tissue hydration, water transport, tissue repair, and various receptor-mediated functions in cell detachment, tumor development, and inflammation.38,39 Some recent works have shown that silica nanoparticles functionalized with HA are able to reach cancer cells with a recognition mechanism that permits the release in loco of an antitumor drug.37 In this work we carried out the functionalization of SBA-15 mesoporous silica with HA. We studied the influence of the degree of HA functionalization on the textural (surface area and pore size) and structural features of the mesoporous material and, more importantly, its effect on the adsorption of lysozyme, a model antimicrobial therapeutic protein. The degree of HA functionalization was varied using different amounts of aminopropyltrimethoxysilane (APTES) in the first functionalization step. In order to test the eligibility of SBA-15 as a possible carrier for therapeutic proteins in drug delivery systems, we also studied the effect of the degree of HA functionalization on the adsorption kinetics of lysozyme.

2. EXPERIMENTAL METHODS 2.1. Chemicals. Tetraethoxysilane (TEOS, 98%), pluronic copolymer F127 (EO101PO56EO101), HCl 37%, anhydrous toluene (99.8%), (3-aminopropyl)triethoxysilane (APTES, >98%), triethylamine (>99%), hyaluronic acid (HA) sodium salt from Streptococcus equi (cod. 53747), NaH2PO4 (99%), and Na2HPO4 (99%) were purchased from Sigma-Aldrich (Milan, Italy). Lysozyme (E.C.3.1.1.17) from hen egg white (70 000 U/mg), N-hydroxysuccinimide (NHS, >97%), and N-(3-(dimethylamino)propyl)-N′-ethylcarbodiimide hydrochloride (EDC, > 98%) were purchased from Fluka. Buffer solutions (pH 1, 4, 7, 9, 10) were from Hanna Instruments (Szeged, Hungary). 2.2. Synthesis of SBA-15 Mesoporous Silica. SBA-15 mesoporous silica was synthesized according to the method reported by Zhao et al.9 In a typical synthesis, 4 g of triblock copolymer Pluronic P123 was dissolved in 20 mL of HCl (37%) and 120 mL of distilled water. The resulting mixture was stirred at 35 °C for 16 h. Then 8.5 g of TEOS was added, and the final solution was stirred at this temperature for 24 h. Finally, the mixture was aged into a teflonlined autoclave at 100 °C for 24 h. After filtration and washing, the solid was dried at 40 °C and then calcined at 550° for 5 h. 2.3. HA Functionalization of SBA-15. Two different amounts of APTES (0.1 and 1 mL) were added to a suspension of 1 g SBA-15 in 30 mL of anhydrous toluene under refluxing conditions for 18 h. The resulting amino-functionalized materials, with a low (L-SBA-NH2) and a high (H-SBA-NH2) level of functionalization, were collected by filtration, washed with toluene and acetone to remove the unreacted APTES, and dried overnight under vacuum at room temperature. The grafting of HA to the amino groups of SBA-NH2 was carried out through the procedure reported by Yu et al.37 Specifically, 1 g of SBANH2 powder was dispersed in 100 mL of Millipore water. In another reaction vessel, 20 mL of an aqueous solution containing NHS (0.37 g) and EDC (0.2 g) was mixed to 60 mL of an aqueous solution containing 113 mg of HA. Finally, the two solutions were mixed, and the pH was adjusted to 9.0 by adding triethylamine. The mixture was stirred at 38 °C overnight. The HA-modified SBA-15 samples (L-SBAHA and H-SBA-HA) were collected after centrifugation, three

qt =

[Lyz]0 V − [Lyz]t V msupport

(1)

where [Lyz]0 and [Lyz]t are the lysozyme concentration (mgLyz/ mLsolution) at t = 0 and at time t, respectively, V is the volume of the lysozyme solution (mL), and msupport is the mass of the mesoporous sample (g). The loading at the equilibrium (qe) is calculated by the lysozyme concentration [Lyz]e determined after 24 h of contact. A pseudo-second order model42 was used to analyze the kinetic data: t 1 1 = + t qt qe k 2qe2

(2)

Kinetic data were also analyzed through an “intraparticle diffusion” model:42 qt = xi + kit 1/2

(3)

where ki (mg g−1 min1/2) is the intraparticle diffusion constant, and xi is the intercept of the line. B

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Scheme 1. Scheme of Reaction between the Amino Groups of the SBA-NH2 Particles and the Carboxylic Groups of the HA Biopolymer

Table 1. Characterization of SBA-15, SBA-NH2, and SBA-HA Obtained through Potentiometric Titrations, N2- Adsorption/ Desorption Isotherms, SAXS, and TGA e

sample

−NH2 (mmol/g)

SBA-15 H-SBA-NH2 H-SBA-HA L-SBA-NH2 L-SBA-HA

1.72 0.51 -

a

2

b

surface area (m /g)

d

781 345 302 349 275

BJH

(nm)

6.9 5.9 5.9 6.9 6.9

c

a (nm) 11.9 11.4 11.4 11.6 11.9

d

TGA (mass loss Δm%)

< 200 °C

> 200 °C

TOT

6.0 2.9 5.1 4.9 4.9

1.0 9.4 14.2 5.8 10.8

7.0 12.3 19.3 9.9 15.9

a

Amount of amino groups determined by potentiometric titration. bSpecific surface area calculated by the BET method. cPore diameter calculated by applying BJH method to the data of the desorption branch. dLattice parameter calculated by the equation a = 2d100/√3, where d100 is the spacing of the (100) plane of hexagonal (p6 mm) array of pores. ePercentage mass loss obtained by TGA.

Figure 1. N2 adsorption/desorption isotherms and pore size distribution (inset) of (red line) SBA-15, (blue line) SBA-NH2 and (black line) SBAHA for (A) the high (H-SBA) and (B) the low (L-SBA) functionalized samples.

3. RESULTS 3.1. Structural and Textural Characterization of Original and Functionalized SBA-15. SBA-15 mesoporous silica samples were functionalized with HA according to Scheme 1. In the first step, the SBA-15 surface was modified by the introduction of a propylamino group (SBA-NH2). Then, HA was grafted to SBA-NH2 samples through a reaction between the carboxylic groups of HA and the amino groups of SBA-NH2. To this purpose, the carboxyl groups of HA were first activated with NHS, using EDC as the coupling agent, and then allowed to react with the amino groups of SBA-NH2. In order to investigate the effect of the functionalization degree on the textural and structural properties of SBA-15, we prepared two samples having a high (H-SBA-NH2) and a low (L-SBA-NH2) level of functionalization, by varying the amount

of APTES reactant. The level of functionalization, quantified through acid/base titration, was 0.51 mmolNH2/gSBA and 1.72 mmolNH2/gSBA for L-SBA-NH2 and H-SBA-NH2, respectively, as reported in Table 1. Substantially, the H-SBA-NH2 sample had a degree of functionalization about 3 times higher than that of L-SBA-NH2. Figure 1 shows the nitrogen adsorption isotherms for the SBA-15 sample and the two series of functionalized samples HSBA (Figure 1A) and L-SBA (Figure 1B). All samples show a type IV isotherm with an increase of the gas volume adsorption at a relative pressure around 0.6−0.7. The presence of a hysteresis cycle, defined by IUPAC as H1, is associated with channel-like mesopores. Table 1 reports the textural data obtained using the BET40 and the BJH41 methods. The functionalization with APTES C

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Figure 2. SAXS patterns of SBA-15, SBA-NH2, and SBA-HA for (A) the high (H-SBA) and (B) the low (L-SBA) functionalized samples.

Figure 3. Transmission electron microscopy images of the SBA-15 showing projections of the hexagonal close packing of cylindrical pores of the synthesized mesoporous silica. Scale bar is 100 nm.

Figure 4. FTIR spectra of SBA-15, SBA-NH2, and SBA-HA for (A) the high (H-SBA) and (B) the low (L-SBA) functionalized series.

pore size distribution (see Figure 1 insets). This is a clear evidence that only the external surface of the mesoporous silica particles is involved in the functionalization with HA. The structural characterization of the original and functionalized samples was performed through SAXS technique. The original SBA-15 sample showed a typical pattern of a hexagonal phase with the occurrence of a strong peak, due to the (100) plane, and other two weak peaks, due to the (110) and (200) planes as shown in Figure 2. The same pattern was also obtained after the functionalization steps with APTES and HA for both H-SBA and L-SBA series. This confirmed that the ordered structure was not particularly affected by the functionalization steps. This is furthermore supported by the very small differences of the lattice parameter (a) observed before and after functionalization as reported in Table 1. Indeed all the values are in the range 11.3 nm < a