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O microemulsion: A method for enzyme nanoencapsulation in silica gel nanoparticles
Colloids and Surfaces A: Physicochem. Eng. Aspects 288 (2006) 52–61
Sol–gel synthesis at neutral pH in W/O microemulsion: A method for enzyme nanoencapsulation in silica gel nanoparticles F. Cellesi, N. Tirelli ∗ School of Pharmacy and Pharmaceutical Sciences & Molecular Materials Centre, University of Manchester, Manchester, UK Received 21 November 2005; received in revised form 1 May 2006; accepted 3 May 2006 Available online 16 May 2006
Abstract The classical sol–gel synthesis of silica gels has been adapted to a W/O (micro)emulsion process under conditions that minimize denaturing effects on encapsulated enzymes. We have in particular focused on optimizing the purification procedures with the aim to produce water nanoparticles dispersions from W/O emulsions without the use of precipitation/sedimentation steps. A proof of principle of encapsulation has been conducted using horseradish peroxidase (HRP). Crown Copyright © 2006 Published by Elsevier B.V. All rights reserved. Keywords: Nanoparticles; Silica; Sol–gel; Enzymes
1. Introduction We here present a new method for the encapsulation of active biomolecules (enzymes) within silica nanoparticles. The encapsulation of catalysts in sub-micron structures (nanoencapsulation) permits to combine the advantages of heterogeneous catalysis (protection; recoverability; control over the access to the catalytic centre and hence selectivity enhancement) with pseudo-homogeneous conditions: the catalytic centre is easily accessible to substrates (due to a high surface-tovolume ratio) and it has a high diffusion coefficient (due to the dimensions and the little tendency to sedimentation of the nanocarriers). We are in particular interested in the nanoencapsulation of biocatalysts, where the catalytic centre is generally a sensitive (macro)molecule, such an enzyme. This could possibly hinder all interactions with other (macro)molecules (or with itself), which denature and inactivate the catalyst, without affecting its activity. The reduction in detrimental interactions results also in an increased pH and temperature stability [1]. For exam-
∗ Correspondence to: School of Pharmacy and Pharmaceutical Sciences, University of Manchester, Oxford Road, Manchester M13 9PL, UK. Tel.: +44 161 275 24 80; fax: +44 161 275 23 96. E-mail address: [email protected] (N. Tirelli).
ple, an appropriate nanoencapsulation could protect a potential immunogenic (e.g. bacterial) enzyme from degradation and keep it in an active form, when it circulates in the body fluids of a host organism. Additionally, the carrier structure can add specific biological interactions, e.g. for favoring a site-specific localization. It is noteworthy that in most literature reports biomolecules are conjugated on the surface of nanoparticles [2–4]. These methods cannot be strictly defined “nanoencapsulation”, since the matrix supports but does not encapsulate the active centre, and consequently there is a limited protective effect. On the contrary, we have tackled the problem of completely embedding the active molecule in a protective matrix. For this scope, silica (or silica gel) offers a number of advantages over organic materials [5]. It is non toxic and shows acceptable in vivo biocompatibity; in a physiologically acceptable range of temperature or pH values, it does not appreciably swell nor changes its porosity; finally, the preparation conditions of silica gel are based on mild sol–gel syntheses (generally the hydrolysis of tetraalkoxy silanes), that may allow to retain the activity of the entrapped enzymes [6]. In addition, functionalities can be easily introduced by decorating the surface of the silica nanoparticles exploiting free silanols, e.g. through chemical derivatization (silanization reactions), electrostatic interactions (adsorption of polycations) and also hydrogen bonding. For example, polymers of the Pluronic series (triblock copolymer of the type PEG–PPG–PEG, where PEG and PPG are poly(ethylene glycol) and poly(propylene
0927-7757/$ – see front matter. Crown Copyright © 2006 Published by Elsevier B.V. All rights reserved. doi:10.1016/j.colsurfa.2006.05.008
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Scheme 1. Standard method for the preparation of silica nanoparticle in W/O microemulsion (emulsion): (A) the hydrophobic tetraalkoxysilane (TOS) is added to an enzyme-containing emulsion. (B) When exposed to water, the TOS starts hydrolyzing and during this process the molecule gradually approaches the water domain and enters it when it is in the fully hydrophilic form of silicic acid. (C) A network of silica gel forms via partial condensation of silanol groups of silicic acid, to yield a material with average formula SiO2−x (OH)2x . The material undergoes a transition from viscous droplet to elastic nanoparticles. (D) The nanoparticles are purified by removal of emulsifier, while the silica gel network entraps the enzymes in the nanoparticles bulk.
glycol), respectively) have been found to adsorb on silica in form of bilayer-like structures (or surface micelles). For Pluronic F127 (average molecular weight 12,600 and 70 wt.%) these surface layers are about 5 nm thick [7] and reversibly cover silica nanoparticles with a polymeric barrier that provides steric repulsion during brownian collisions, even in absence of electrostatic repulsive forces [8]. Protein-encapsulating colloidal silica has been produced in most cases by performing the silicon alkoxide hydrolysis in the dispersed domains of water-in-oil (W/O) microemulsions. The alkoxides diffuse into the enzyme-containing water droplets from the hydrophobic phase, are there hydrolysed to silicic acid, which eventually condenses to give a silica gel network [9] (Scheme 1). A major inconvenience of this method is the extreme pH (either strongly acidic, <4, or basic, >9, in the last case usually obtained with concentrated ammonia), which is required for a reasonably rapid hydrolysis reaction [6,9,10]. If it is true that these conditions are likely to denature most enzymes, they are not necessary for the successive condensation of silicic acid to yield a silica gel network, whose rate is maximal at physiological pH. Another similar hurdle: most published preparation methods of silica nanoparticles employ either strongly denaturing organic solvents, such as acetone [11], or basic solutions [6] for their isolation and purification. Last, surprisingly little is reported about the recovery yield of the nanoparticles preparation procedures; this is a non-negligible issue, since purification is often based on the formation of a gel-like or precipitated phase, where nanoparticles are not unlikely to irreversibly agglomerate. Considering the possible high cost of the biomolecules used, this should be avoided as much as possible. We here propose a modified and milder alternative method for enzyme encapsulation within silica gel nanoparticles that (A) uses a sol–gel synthesis at nearly neutral pH (6–8), (B) minimizes the use of denaturing organic solvents or of extreme pH conditions during purification, and ensures quantitative yields both in the removal of organics and in the dispersability in water.
In summary: point (A) was obtained by decoupling the hydrolysis of the silicon alkoxide from its condensation, cleaving the aliphatic chains in a low pH water solution, which is then buffered at pH 6–8 prior to the enzyme addition and is then used for the formation of the W/O emulsion. By varying the organic solvent/surfactant ratio, we showed that the particle size can be modulated from 10 to 50 mm. point (B) aimed at avoiding the fast silanol condensation at nearly neutral pH, which, upon surfactant removal, in our experience always promotes a substantial and irreversible particle aggregation. We have developed a work-up method consisting of two stabilization steps. First, by changing the polarity of the dispersant phase, the emulsifier is partially desorbed and likely forms surface aggregates. Second, the emulsifier aggregates are replaced by those of a biocompatible polymer (Pluronic F127). In this final form the nanoparticles are indefinitely stable against agglomeration, due to the steric stabilization provided by the polymer. The process has been validated for enzyme encapsulation using horseradish peroxidase (HRP) as a model. 2. Experimental 2.1. Materials Tetraethyl orthosilicate (TEOS, purity 98%) was supplied by Aldrich (Buchs, Switzerland). Berol 26 and Berol 267 were kindly donated by Akzo Nobel (Stenungsund, Sweden). n-Hexane, ethanol, methanol, acetonitrile (reagent grade) and hydrogen peroxide 30% solution were supplied by BDH (Laboratory Supplies, UK). Horseradish peroxidase 500 units/mg was purchased from Fluka (Buchs, Switzerland). 1-Methyl-2pyrrolidone (NMP, purity 99%+), Pluronic F127 and Pyrogallol were supplied by Sigma (Buchs, Switzerland). Water was pre-distilled and further purified by a Milli-Q system (Millipore, UK). Phosphate buffered saline (PBS), 10 mM, was prepared from 0.2 g KCl, 0.2 g KH2 PO4 , 2.14 g Na2 HPO4 ·7H2O and 8 g NaCl
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dissolved in 1 L of pure water. The pH was adjusted by dropping dilute solutions of HCl and NaOH. 2.2. Preparation of silica-gel nanoparticles 1.0 mL of TEOS (0.93 g) was mixed with 0.50 mL of HCl 3.5 mM in pure water and stirred for 2 h at room temperature, until complete solubilization. The ethanol produced by hydrolysis was removed by rotary evaporation at 25 mbar, room temperature (water evaporation is supposedly negligible under these conditions). The solution was first buffered at pH 6 adding 0.25 mL PBS 10 mM pH 10 at low temperature (0 ◦ C) and then diluted with an appropriate volume of protein-containing 10 mM PBS at pH 7.4 (in the case of horseradish peroxidase: 10 mg/mL) to reach the desired silica concentration. A volume variable between 250 and 300 L (in dependence on the final target silica concentration) of this solution were quickly injected in an organic solution of 0.50 g of Berol 26, 0.50 g of Berol 267 and 10 mL of n-hexane, previously prepared. After a vigorous stirring the suspension developed into a stable and clear reverse microemulsion. The suspension was kept at room temperature for 90 min before workup, in order to ensure the formation of silica gel nanoparticles through an effective condensation of silanols. 2.3. SiO2 -NPs work-up Twenty milliliters of the nanoparticle suspension were transferred in a rotary evaporator, where roughly half the volume of the hexane was evaporated at room temperature. The concentrated solution was added of 16 mL of a water solution containing 13 wt.% ethanol and 20 wt.% N-methyl pyrrolidone (NMP), and the resulting mixture was tranferred to the rotary evaporator, where the residual amount of hexane was completely removed (room temperature, 30 mbar). The clear suspension (∼20 mL) was diluted with 100 mL of a 0.5 wt.% Pluronic F127, 18 wt.% ethanol and 7 wt.% NMP water solution, previously prepared and kept at 25 ◦ C under stirring. The liquid dispersion was then purified using an Amicon stirred ultrafiltration cell (Millipore, UK) equipped with a regenerate cellulose membrane (100 kDa molecular weight cut-off). Initially, the ultrafiltration cell was continuously refilled first with a EtOH-NMP-F127 water solution (in the proportions above) to keep constant volume and composition of the solution, then (upon completely soluble in water of the filtrate = complete removal of Berol) with a 0.5 wt.% F127 water solution, to remove both NMP and ethanol. Finally, the dispersion was concentrated to a final volume of 20 mL. 2.4. Characterization 2.4.1. Silica gel point determination A Bohlin Gemini parallel plate rheometer, oscillating at 1 Hz frequency under controlled strain (at strain = 10−2 ), was used to monitor the gelation kinetics of silicic acid solutions, identifying the gel times as the crossing points of elastic (G ) and viscous (G ) shear modulus curves.
2.4.2. Dynamic light scattering (DLS) The Z-average size and size distribution of silica gel nanoparticles and nanodroplets in the W/O microemulsion were measured using a Zetasizer Nano ZS Instrument (Malvern Instrument Ltd., UK), with a backscatter detection (173◦ detection optics) and a 633 nm laser beam. Raw data were processed by Malvern DTS software. The viscosity parameter was corrected measuring the sample viscosity on an automated microviscosimeter (Anton Paar, UK). 2.4.3. Transmission electron microscopy (TEM) The nanoparticle dispersions were pipetted on carbon/formvar coated 100 mesh grids, which were therefore washed with distilled water three times, stained in 1% uranyl acetate solution and air dried. Specimens were observed in a Technai 12 electron microscope at 100 kV. 2.4.4. Pyrogallol assay The activity of HRP, in solution or nanoencapsulated in the silica-gel nanoparticles, was evaluated by the pyrogallol assay [12]: the time-dependent oxidation of pyrogallol to purpurogallin catalysed by HRP can be followed monitoring the absorbance of purpurogallin at 420 nm, at 20 ◦ C in PBS 10mM pH 6.0 (through a UV/Vis spectrophotometer Perkin-Elmer Lambda 25 and WinLab software). The peroxidase activity is expressed in term of pyrogallol units, where one unit forms 1.0 mg of purpurogallin at pH 6.0 and at 20 ◦ C. 3. Results and discussion 3.1. W/O emulsion water phase: hydrolysis without gelation The preparation of silica nanoparticles is based on the conversion of the water phase of a W/O emulsion into a silica gel through a sol–gel process; due to the presence of potentially sensitive biomolecules, there are strong limitations for pH and composition of the starting water solution. Most typically synthesized silica sol–gel processes are based on the use of silicon alkoxides that undergo three elementary reactions: (a) hydrolysis: Si(OR)4 +nH2 O → Si(OR)4−n (OH)n + nROH (b) alcohol condensation: Si-OR + HO-Si → Si–O–Si + ROH (c) water condensation: Si-OH + HO-Si → Si–O–Si + H2 O It is important to note that only the condensation reactions are responsible of the gel formation. The pH dependence of this set of reactions has been extensively studied [8,13–17]. The hydrolysis rate reaches its minimum at neutral pH and almost linearly rises when the pH is lowered or increased. The condensation rate, on the contrary, is highest at neutral pH and sharply decreases far from neutrality [8,13]. Often an alcohol is employed for rendering the system homogeneous, since water and aliphatic alkoxysilanes are immiscible; however, the addition of other solvents can be also avoided if the water/alkoxide mixture is vigorously stirred, since hydrolysis itself progressively converts the hydrophobic
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alkoxides into water soluble materials: hydrosilicates and alcohols [18,19]. In summary, the two factors possibly affecting the activity of biomolecules are (a) the pH necessary for alkoxide hydrolysis and (b) the presence or production of alcohols at high concentration. We have overcome the first problem by performing separately the stages of hydrolysis and condensation: at acid pH the alkoxide can be hydrolyzed in the absence of the biomolecule and without causing a rapid condensation of silicic acid. This precursor solution can be buffered later and mixed with that of the desired biomolecule, which is never in presence of an extreme pH. The presence of organic solvents could be minimized too: first, with careful choice of pH and reaction time an homogeneous solution of hydrolyzed tetraethoxysilane (TEOS) can be obtained (in 3.5 mM HCl in 2 h at room temperature, ratio H2 O/TEOS 0.5:1 (v/v), pH 2.5, 20 ◦ C) without the use of additional organic solvents. Second, the slow condensation rate at acid pH allows for enough time to evaporate the large amount of ethanol produced in TEOS hydrolysis at controlled reduced pressure, before buffering the solution at physiological pH (6.8–7.2) and mixing it with a similarly buffered enzyme-containing water solution. The last operations are conducted as rapidly as possible and just above the freezing point of the solution to avoid appreciable silicic acid condensation (maximal at that pH). By controlling the volume ratio between the two solutions it is possible to control the concentration of both enzyme and silica in the final gel; in our experiments we have mostly produced materials with two silica concentrations: 8 and 20 wt.% (expressing the solid content in terms of theoretical weight of SiO2 /overall weight of the water phase). Silica concentrations above 20 wt.% have not been used in this work, mostly because of the speed of the condensation reactions (gel point below 2 min at room temperature), which render critical the timing of all the operations.
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surfactants of the family of alkyl phenol ethoxylates with the general chemical structure H-(CH2 )n -Ph-(OC2 H4 )m -OH (such as Triton X-100 or surfactants of the Berol series), sometimes in mixture with hexanol [23–25]. Non-ionic surfactants were found to provide less polydisperse silica particles, whereas AOT microemulsions are apparently less stable, and produce nanoparticles with a broad size distribution and possible formation of colloidal gels [25]. We have therefore selected non-ionic surfactants and, more specifically, a mixture of two alkyl phenol ethoxylates with different hydrophilic lipophilic balance (HLB) for a fine modulation of the average HLB and therefore also of the size of water droplets (the higher the HLB, the larger is the droplet): Berol 26 (n = 9, m = 4, HLB = 8.9) and Berol 267 (n = 9, m = 8, HLB = 12.3). For most experiments we have used a mixture Berol 26–Berol 267 1:1 wt. to obtain the maximum average HLB (∼10.6) compatible with their solubility in n-hexane. The concentration of surfactants in n-hexane was set at 10% (w/v), a typical value for these preparations [24], since it is optimal for the microemulsion stability and it is low enough for an effective removal of the surfactant during work-up. 3.2.3. Water content As in any W/O microemulsion, keeping constant the emulsifier concentration the droplet dimensions increase steadily with the increase of the water phase volume (to minimize the energetically unfavourable increase of the interface), until phase separation takes place, generally above a limiting value of roughly 50 nm (Fig. 1). Therefore, the volume ratio water phase/organic phase allows a control of the droplet size and, since dimensions are substantially preserved in the condensation stage, that of nanoparticles too (see next session). However, it must be noted that at high concentration the presence of hydrosilicates can influence the droplet diameter too: while droplets containing 8 wt.% silica is substantially identical to those of pure water, at 20 wt.% silica they are considerably smaller, and the microemul-
3.2. W/O microemulsions: control over droplet size The size and size distribution of silica gels nanoparticles are supposedly templated on those of the precursors, the W/O emulsion droplets, thus unstable emulsions will lead to very broad distributions and uncontrolled average size. We have therefore optimized the preparation of stable W/O emulsions with adjustable droplet size in the sub-100 nm range (microemulsions) as a function of the following factors: composition of the hydrophobic phase, structure and concentration of the surfactant and volume ratio between water and hydrophobic phase. 3.2.1. Hydrophobic phase We have chosen n-hexane as organic solvent, since it is one of the common solvent used in reverse microemulsions [20–22], it is widely available and easy to remove (by evaporation). 3.2.2. Emulsifier Silica nanoparticles have been reportedly prepared either using the anionic surfactant Aerosol-OT (AOT), or non-ionic
Fig. 1. Dependence of the hydrodynamic diameter of pure water or silicic acid droplets on the water phase volume. The W/O microemulsion contained 10 mL of n-hexane, 0.50 g Berol 26 and 0.50 g of Berol 267. The last point of each curve represents the threshold of the microemulsion stability before phase separation.
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Fig. 2. Gel point determination for silicic acid solutions at different concentration. A parallel plate rheometer was used to measure the gel points of silica gels, identified as the points where elastic (G ) and viscous (G ) shear moduli assume the same value.
sion is able to disperse more water before phase separation takes place. This may be due to the increased density and viscosity of the water phase (therefore decreased tendency to coalesce), but may also derive from the presence of some partially hydrolyzed TEOS in the water phase, which has surface active properties. 3.3. Sol–gel process in W/O microemulsion: silica gel nanoparticles Hydrolysed TEOS solutions gel very rapidly at room temperature: 8 and 20 wt.% silica reached a gel point (G = G ) in ∼8 and ∼2 min, respectively (Fig. 2). Anyway, in order to be sure to obtain a well cross-linked silica network, in our emulsion experiments we have allowed the condensation to run for 90 min. The initial water droplets and the final silica gel nanoparticles appeared to have identical size and monomodal distributions, both for 8 and 20 wt.% silica nanoparticles. This finding would suggest a simple picture of a topologically confined conversion of a homogeneous solution into a gel. The silicic acid solution, however, is likely to contain silica clusters (despite the low temperature, the pH conditions do favor silanol condensation) and thus be inhomogeneous. The clusters may distribute unevenly in the emulsified droplets, eventually producing harder gel nanoparticles but also some viscous droplets. A rapid verification can be obtained by decreasing the water/oil ratio of the microemulsion after completion of the condensation reaction: the nanoparticle size would not be affected, while the droplets would reduce their dimensions. Indeed the addition of more hexane–Berol solution to the nanoparticle dispersion rendered bimodal their initially monomodal size distribution; a minor component at smaller size appears, whose sensitivity to dilution suggests being formed by liquid droplets (Fig. 3). The presence of un-gelled droplets implies a loss of material to encapsulate, and should be minimized. It appears that their volume fraction diminishes by increasing silica concentration: for nanoparticles with 8 wt.% of silica, the droplets account for a substantive part of the total water phase volume (40–50%), while
for 20 wt.% silica nanoparticles (the highest concentration compatible with our method) this represented a very minor fraction, around 7%, which we estimate acceptable for most applications. 3.4. Purification of silica gel nanoparticles The main target of this phase is the purification of the nanoparticles from solvents and emulsifiers used in their synthesis and deliver them in water dispersion in the least denaturing and most stable conditions. In commonly adopted procedures, nanoparticles are produced under basic conditions (pH >9) and their suspensions are precipitated by the addition of a fairly polar organic sol-
Fig. 3. Left: decrease of the Z-average size of the 20% SiO2 nanoparticles synthesized in W/O microemulsion by progressive dilution with fresh hexane–Berol solution. Dilution factors on the abscissa are reported as ratio volume diluent added/ volume microemulsion. Right: size distribution of the same nanoparticle suspension before and after increasing dilutions. Initially monomodal, the distribution splits into two distinct peaks, respectively at 10 nm and at the initial size of 50 nm. Note that the nanoparticles size axis is reported in logarithmic scale.
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vent (e.g. acetone, ethanol) where both the hydrophobic phase (hexane) and the emulsifier are soluble [26]. After washing the precipitate with fresh solvent to remove all the surfactant, the nanoparticles can be re-dispersed in a basic water solution. The high pH is instrumental to maintain the ζ-potential of the silica nanoparticles sufficiently negative to prevent strong aggregation, and thus to sufficiently slow down the interparticle condensation of superficial Si–OH groups to avoid formation of irreversible gels or precipitates. These procedures, however, are unsuitable in the presence of sensitive biomolecules, since they require at the very least neutral pH, where the ζ-potential value of silica is too low for avoiding irreversible agglomeration in the precipitate, and, as already mentioned we seek to minimize the use of organic solvents in high concentrations. We have therefore developed a process, which (a) is based on a smooth change of the nanoparticle environment, from a hydrophobic- to a waterdispersed state, (b) keeps the nanoparticles always in a stable colloidal suspension throughout the procedure. The crucial point of this procedure is that Berols are difficult to remove from the silica surface in a water environment: they bear oligo(ethylene glycol) structures, which strongly interact with silanols through hydrogen bonding (as generally for amphiphilic derivatives of PEG, such as Pluronics). Even more important, they have a high HLB value, which means that they tend to disperse water instead of being dispersed in water. The addition of fairly limited quantities of other organic components improves the solubility of Berols in the emulsion continuous phase and forces them to form dispersable aggregates (regular emulsions) (Fig. 4). The size of these aggregates can be controlled by varying the amount and the composition of the organic solvent (that is varying the interfacial energy of a Berol layer). Moreover, in some conditions the same behaviour
Fig. 4. DLS analysis of a 10 wt.% Berol water mixtures (Berol 26 and Berol 267 in 1:1 ratio) containing different amounts of ethanol and NMP. The use of ethanol alone is possible only above 20 wt.%, while below phase separation takes place; the size of the aggregates decreases by increasing the amount of ethanol. 18 wt.% EtOH and 7 wt.% NMP (total amount <26 wt.%) is a good compromise since it produces Berol aggregates smaller than silica nanoparticles (25 nm vs. 50–90 nm) while minimizing the quantity of organic solvents.
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can be observed in the presence of hexane, producing hexanecontaining emulsions. For example, 33 wt.% or more concentrated ethanol solutions succeed in dispersing Berol/hexane mixtures (10 and 25 wt.%, respectively) in water, while 20 wt.% ethanol is enough to disperse Berol alone (10 wt.%). Hexane can be removed from the dispersion by evaporation, generally reducing the size of the aggregates. Our target was to use the least denaturing solvents, at the lowest possible concentrations (and for the shortest time), and producing Berol-containing aggregates with the most different dimensions from those of the nanoparticles, for an easy purification via dialysis or ultrafiltration. We have found an optimal performance at 18 wt.% ethanol and 7 wt.% N-methyl pirrolidone, NMP; this solvent mixture offers the best compromise between emulsificability of Berol/hexane mixtures (other solvents, such as acetonitrile and methanol, were ineffective), the size of the aggregates after hexane evaporation and hydrophilicity of the organic components: in water mixtures the denaturing effect of an organic solvent on an enzyme (the cosolvent concentration yielding a 50% reduction of catalytic activity) is often proportional to its octanol/water partition coefficient. Experimentally, we have evaporated part of the hexane from the nanoparticles/hexane dispersions prior to introduce the NMP/ethanol water solution; screening different solvent formulations, we have recorded the formation of a clear and stable emulsion at a 1:2 hexane/water solution volume ratio. In the aggregates (we may imagine them as emulsified droplets) the core is likely composed by ethanol and Berol hydrophobic chains, while, since NMP and Berol are immiscible, we exclude the presence of NMP. The evaporation of hexane from this emulsion did not change its state, nor caused significant silica agglomeration. It is noteworthy that in the absence of NMP the emulsion could not be formed, while the presence of ethanol is not essential; on the other hand, without ethanol silica precipitates after hexane evaporation. At this stage, dynamic light scattering revealed objects having a size substantially identical to that of the nanoparticles in the W/O emulsion (Fig. 5) without any sign of agglomeration. This, however, does not exclude that Berol aggregates may dynamically and reversibly sit on the nanoparticles surface, similarly to what recorded in literature for Pluronics [7], interacting through Berol-silanol H-bonds (Scheme 2, B). Two findings could indeed suggest a similar phenomenon to involve Berol aggregates and silica nanoparticles in our system: (a) If hexane is completely evaporated after emulsification, precipitation occurs. This may be due to the formation of a Berol pseudo-bilayer that can spread better and adhere more firmly to the surface than hexane-rich emulsion droplets (Scheme 2, C ). (b) The dispersion precipitates also by replacing the dispersion organic components with water during ultrafiltration (the hydrophobic aggregates are supposedly smaller than the nanoparticles and can be separated from them through membrane with an appropriate size cut-off, see Fig. 4). In this case we believe this to be caused by agglomeration and
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direct interparticle H-bonding when the emulsion droplet concentration falls but the solvent is still rich in NMP and ethanol (Scheme 2, C ). These interpretations are clearly just hypothetical, but can be substantially confirmed if precipitation could be avoided by replacing the emulsion droplets with other components that are known to have surface interactions with colloidal silica, such as Pluronic F127. During ultrafiltration experiments we have constantly replaced the filtered emulsion with a 0.5%wt Pluronic water solution; the organic components (Berol aggregates, ethanol and NMP) were removed from the system, obtaining silica nanoparticles in a fairly diluted Pluronic solution at neutral pH, where the reduction in electrostatic repulsion is most likely compensated by the steric stabilization provided by the adsorption of Pluronic micelles (Scheme 2, part C). Within the experimental conditions used, the average particle size increased from 50 to 60 nm before ultrafiltration to about 110–120 nm (Fig. 5), a result qualitatively confirmed by transmission electron microscopy (Fig. 6). It must be pointed out that this increase is likely due not only to some aggregation in the process, but also to the presence of Pluronic on the nanoparticles surface. 3.5. Enzyme activity after nanoencapsulation Enzyme-containing silica gel nanoparticles have been prepared following the method described above; the enzyme (horseradish peroxidase) was introduced in the silicic acid solution immediately after buffering. After work-up, HRP activity in the Pluronic-stabilized nanoparticles was assessed with the pyrogallol assay, where hydrogen peroxide oxidizes the colorless pyrogallol to the yellow purpurogallin [12].
Fig. 5. Size distribution of silica gel nanoparticles from the preparation in W/O microemulsion to the final colloidal dispersion in water after workup. Data were obtained from DLS analysis. (- - -) Silica gel nanoparticles, 20 wt.% in SiO2 , synthesized in a W/O microemulsion with 10 mL hexane, 1 g Berol, 250 L of water phase. (. . .) Concentrated silica nanoparticle suspension (1 g Berol, ∼5 mL hexane and 300 L nanoparticles) re-dispersed in 10mL of NMP/ethanol/water solution during work-up. (—) Silica gel nanoparticles dispersed in water–Pluronic solution after work-up procedure. The peak at 20 nm represents Pluronic F127 micelles.
The comparison between the activity of free HRP (4.20 U/mL, measured at HRP concentration of 0.03 mg/mL) with that of the encapsulated one (0.023 U/mL in 8 wt.% silica nanoparticles and 0.320 U/mL in 20 wt.% silica nanoparticles) with the same overall enzyme concentration, shows a sharp decrease for both formulations, particularly dramatic for the 8% silica nanoparticles (Fig. 7).
Scheme 2. Sketch of the work-up process developed. The Berol-coated silicagel nanoparticles are originally dispersed in a hydrophobic phase (A); by evaporating a major part of it and adding a 7% NMP, 18% ethanol water solution an emulsion is generated, whose droplets contain hexane in the core and Berol and organic solvents at the interface (B). As described in the text, complete evaporation of hexane (C ) or complete removal of the organic components through ultrafiltration (C ) cause aggregation of the nanoparticles and precipitation. On the contrary, the replacement of the organic emulsion with a Pluronic micellar dispersion (C) keeps the nanoparticles in a dispersed state.
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Fig. 6. TEM images of silica gel nanoparticles after work-up. Most nanoparticles show a diameter in the range 60–100 nm, but some clusters are visible. The white spots visible in the right image are likely Pluronic micelles.
Three phenomena can possibly determine this decrease of activity: (a) partial loss during the purification, since the relatively small enzyme (MW at about 4 × 104 g/mol, hydrody˚ [27] can permeate through the pores of namic radius ∼30 A) the ultrafiltration membranes with molecular weight cut-off of 10 × 104 g/mol, (b) partial denaturation of the enzyme that may be caused by the sol–gel process in W/O microemulsion, or by the organic solvents used in the purification, (c) intrinsic reduction of activity, because of the immobilization in a matrix.
By measuring HRP activity throughout the process, it is possible to see that the activity of the 8% silica nanoparticles is initially very similar to that of the 20% wt. nanoparticles and drops only during the last stage of purification (Fig. 8); 20 wt.% silica nanoparticles show a negligible decrease. This finding suggests that the purification conditions, common to the two nanoparticles, produce negligible denaturation to the enzyme; on the contrary, the enzyme seems to leak out of the less concentrated nanoparticles. This can happen because either HRP is present in non-gelled water droplets, or it permeates through the
Fig. 7. Left, top: reaction scheme of the oxidation of pyrogallol to purpurogallin catalysed by HRP. Left, bottom: pyrogallol and HRP-containing silica gel nanoparticles in water before and after the addition of hydrogen peroxide, that allows HRP to produce the yellow-colored purpurogallin. Right: spectrophotometric analysis of HRP activity: comparison between free and encapsulated HRP in 8 and 20 wt.% silica nanoparticles (same theoretical). The enzyme concentration is kept constant at 0.03 mg/mL with nanoparticles (if present) at 0.08% (v/v) nanoparticles, 0.5 wt.% pyrogallol and 0.03 wt.% H2 O2 .
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solvents. High solid nanoparticles have shown the best results both in terms of encapsulation efficiency and enzyme activity, using HRP as a model enzyme. A number of questions are now open and are currently under investigation: - How does the enzyme activity depend on the presence of Pluronic on the surface of the nanoparticles and on the presence of aggregates? We are indeed studying the possibility to replace Pluronic steric stabilization with the electrostatic one arising from polyelectrolyte adsorption, with the hope also to reduce particle aggregation during the work-up. - How applicable is this method to the encapsulation of other and possible less stable enzymes? We are expanding these studies to the incorporation of other bacterial and mammalian enzymes. Fig. 8. Spectrophotometric analysis of HRP activity encapsulated in 8 wt.% silica nanoparticles at different stages of the purification process. It is apparent how the enzymatic activity is substantially unaffected during the first stages.
gel structure of the nanoparticles: both phenomena are strongly reduced by increasing the solid content of the nanoparticles and this explains the remarkable difference between 8 and 20 wt.% silica nanoparticles. The reduction in HRP activity observed for the high solid content 20 wt.% silica nanoparticles (roughly one order of magnitude lower than the free enzyme) could be ascribed to denaturation in the sol–gel process (in the purification this has been previously excluded), or to the more difficult access of the substrates to the catalytic centre/lower diffusion coefficient of the enzyme. In particular, the second option would be a result of lower diffusion coefficient of substrates and products in the matrix, slower motions of the enzyme and possibly also difficult permeation of the slightly anionic pyrogallol in an anionic matrix. The first option is less likely, since, opposite to what recorded, one should observe less activity (=more denaturation) in the high solid samples, where the changes induced by the sol–gel process are more dramatic. Moreover, literature about enzyme encapsulation in silica gel matrices does not report significant denaturation caused by the sol–gel process [18,28]. In summary, we are inclined to exclude significant denaturation effects of both sol–gel and work-up procedures and to explain the difference of HRP activity between high and low solid content nanoparticles in terms of enzyme leakage or poor encapsulation efficiency of the immobilization process. 4. Conclusions We have here presented the successful proof of principle of a modified sol–gel process in W/O microemulsion, which minimizes harsh conditions that can possibly cause denaturation of encapsulated enzymes. It has been shown that it is possible to produce and purify silica gel nanoparticles with controlled diameter in conditions of physiological pH and minimizing the presence of organic
Acknowledgements The authors are indebted to Manchester Innovation Limited (now UMIP, Manchester, UK) for the fundamental financial support and to Medipol SA (Lausanne, Switzerland). The authors also gratefully acknowledge the EM Unit, Faculty of Life Sciences at University of Manchester, for the use of the transmission electron microscope, and Dr. Aleksandr Mironov for the preparation of TEM samples and for helpful discussions. References [1] Y. Ray, C. Tu-Chen, D.H. Dupont, Q. Duje, Patent Number: US6716450, 2004. [2] I. Roy, T.Y. Ohulchanskyy, D.J. Bharali, H.E. Pudavar, R.A. Mistretta, N. Kaur, P.N. Prasad, Optical tracking of organically modified silica nanoparticles as DNA carriers: a nonviral, nanomedicine approach for gene delivery, Proc. Natl. Acad. Sci. U.S.A. 102 (2005) 279–284. [3] W.H. Tan, K.M. Wang, X.X. He, X.J. Zhao, T. Drake, L. Wang, R.P. Bagwe, Bionanotechnology based on silica nanoparticles, Med. Res. Rev. 24 (2004) 621–638. [4] A. Rehor, J.A. Hubbell, N. Tirelli, Oxidation-sensitive polymeric nanoparticles, Langmuir 21 (2005) 411–417. [5] C. Daubresse, C. Grandfils, R. Jerome, P. Teyssie, Enzyme immobilization in nanoparticles produced by inverse microemulsion polymerization, J. Colloid Interface Sci. 168 (1994) 222–229. [6] T.K. Jain, I. Roy, T.K. De, A. Maitra, Nanometer silica particles encapsulating active compounds: a novel ceramic drug carrier, J. Am. Chem. Soc. 120 (1998) 11092–11095. [7] M. Malmsten, P. Linse, T. Cosgrove, Adsorption of Peo Ppo Peo block copolymers at silica, Macromolecules 25 (1992) 2474–2481. [8] C.J. Brinker, Sol–Gel Science. The Physics and Chemistry of Sol–Gel Processing, Academic Press Inc., London, 1990. [9] C. Barbe, J. Bartlett, L.G. Kong, K. Finnie, H.Q. Lin, M. Larkin, S. Calleja, A. Bush, G. Calleja, Silica particles: a novel drug-delivery system, Adv. Mater. 16 (2004) 1959–1966. [10] G. Fiandaca, E. Vitrano, A. Cupane, Ferricytochrome c encapsulated in silica nanoparticles: structural stability and functional properties, Biopolymers 74 (2004) 55–59. [11] W. Yang, H. Zheng, W.T. Yuan, J.G. Xu, Silica-hemin composite nanparticles as new biocatalyst to highly sensitive determination of glucose in human serum, Anal. Sci. 20 (2004) 1265–1270. [12] Standard peroxidase activity assay available on Sigma–Aldrich web site: http://www.sigmaaldrich.com/img/assets/17540/HRP Assay pyro.pdf.
F. Cellesi, N. Tirelli / Colloids and Surfaces A: Physicochem. Eng. Aspects 288 (2006) 52–61 [13] C.J. Brinker, Hydrolysis and condensation of silicates: effects on structure, J. Non-Cryst. Solids 100 (1988) 31–50. [14] R.B. Bhatia, C.J. Brinker, A.K. Gupta, A.K. Singh, Aqueous sol–gel process for protein encapsulation, Chem. Mater. 12 (2000) 2434–2441. [15] J. Sanchez, A. McCormick, Kinetic and thermodynamic study of the hydrolysis of silicon alkoxides in acidic alcohol-solutions, J. Phys. Chem. 96 (1992) 8973–8979. [16] T.M. Alam, R.A. Assink, S. Prabakar, D.A. Loy, Identification and characterization of the hydrolysis products in TMOS and MTMS monomers using Si-29 NMR and polarization transfer techniques, Magn. Reson. Chem. 34 (1996) 603–609. [17] J. Sefcik, A.V. McCormick, Kinetic and thermodynamic issues in the early stages of sol–gel processes using silicon alkoxides, Catal. Today 35 (1997) 205–223. [18] J.P. Chen, W.S. Lin, Sol–gel powders and supported sol–gel polymers for immobilization of lipase in ester synthesis, Enzyme Microb. Technol. 32 (2003) 801–811. [19] C.R. Lloyd, E.M. Eyring, Protecting heme enzyme peroxidase activity from H2 O2 inactivation by sol–gel encapsulation, Langmuir 16 (2000) 9092–9094. [20] M.L. Schlossman, Liquid–liquid interfaces: studied by X-ray and neutron scattering, Curr. Opin. Colloid Interface Sci. 7 (2002) 235–243. [21] K. Kurumada, A. Shioi, M. Harada, Dynamical behavior and structure of concentrated water-in-oil microemulsions in the sodium
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bis(2-ethylhexyl)sulfosuccinate systems, J. Phys. Chem. 100 (1996) 1020–1026. C.F. Komives, D.E. Osborne, A.J. Russell, Characterization of a nonionic surfactant reversed micellar system for enzyme catalysis, J. Phys. Chem. 98 (1994) 369–376. F.J. Arriagada, K. Osseoasare, Phase and dispersion stability effects in the synthesis of silica nanoparticles in a nonionic reverse microemulsion, Colloids Surf. 69 (1992) 105–115. C.L. Chang, H.S. Fogler, Controlled formation of silica particles from tetraethyl orthosilicate in nonionic water-in-oil microemulsions, Langmuir 13 (1997) 3295–3307. L.M. Gan, K. Zhang, C.H. Chew, Preparation of silica nanoparticles from sodium orthosilicate in inverse microemulsions, Colloids Surf. A 110 (1996) 199–206. Z.Q. Ye, M.Q. Tan, G.L. Wang, J.L. Yuan, Novel fluorescent europium chelate-doped silica nanoparticles: preparation, characterization and time-resolved fluorometric application, J. Mater. Chem. 14 (2004) 851–856. G. Raviola, Blood-aqueous barrier can be circumvented by lowering intraocular pressure, Proc. Natl. Acad. Sci. U.S.A. 73 (1976) 638– 642. R. Ulbrichhofmann, B. Selisko, Soluble and immobilized enzymes in water–miscible organic-solvents–glucoamylase and invertase, Enzyme Microb. Technol. 15 (1993) 33–41.
Sol–gel synthesis at neutral pH in W/O microemulsion: A method for enzyme nanoencapsulation in silica gel nanoparticles F. Cellesi, N. Tirelli ∗ School of Pharmacy and Pharmaceutical Sciences & Molecular Materials Centre, University of Manchester, Manchester, UK Received 21 November 2005; received in revised form 1 May 2006; accepted 3 May 2006 Available online 16 May 2006
Abstract The classical sol–gel synthesis of silica gels has been adapted to a W/O (micro)emulsion process under conditions that minimize denaturing effects on encapsulated enzymes. We have in particular focused on optimizing the purification procedures with the aim to produce water nanoparticles dispersions from W/O emulsions without the use of precipitation/sedimentation steps. A proof of principle of encapsulation has been conducted using horseradish peroxidase (HRP). Crown Copyright © 2006 Published by Elsevier B.V. All rights reserved. Keywords: Nanoparticles; Silica; Sol–gel; Enzymes
1. Introduction We here present a new method for the encapsulation of active biomolecules (enzymes) within silica nanoparticles. The encapsulation of catalysts in sub-micron structures (nanoencapsulation) permits to combine the advantages of heterogeneous catalysis (protection; recoverability; control over the access to the catalytic centre and hence selectivity enhancement) with pseudo-homogeneous conditions: the catalytic centre is easily accessible to substrates (due to a high surface-tovolume ratio) and it has a high diffusion coefficient (due to the dimensions and the little tendency to sedimentation of the nanocarriers). We are in particular interested in the nanoencapsulation of biocatalysts, where the catalytic centre is generally a sensitive (macro)molecule, such an enzyme. This could possibly hinder all interactions with other (macro)molecules (or with itself), which denature and inactivate the catalyst, without affecting its activity. The reduction in detrimental interactions results also in an increased pH and temperature stability [1]. For exam-
∗ Correspondence to: School of Pharmacy and Pharmaceutical Sciences, University of Manchester, Oxford Road, Manchester M13 9PL, UK. Tel.: +44 161 275 24 80; fax: +44 161 275 23 96. E-mail address: [email protected] (N. Tirelli).
ple, an appropriate nanoencapsulation could protect a potential immunogenic (e.g. bacterial) enzyme from degradation and keep it in an active form, when it circulates in the body fluids of a host organism. Additionally, the carrier structure can add specific biological interactions, e.g. for favoring a site-specific localization. It is noteworthy that in most literature reports biomolecules are conjugated on the surface of nanoparticles [2–4]. These methods cannot be strictly defined “nanoencapsulation”, since the matrix supports but does not encapsulate the active centre, and consequently there is a limited protective effect. On the contrary, we have tackled the problem of completely embedding the active molecule in a protective matrix. For this scope, silica (or silica gel) offers a number of advantages over organic materials [5]. It is non toxic and shows acceptable in vivo biocompatibity; in a physiologically acceptable range of temperature or pH values, it does not appreciably swell nor changes its porosity; finally, the preparation conditions of silica gel are based on mild sol–gel syntheses (generally the hydrolysis of tetraalkoxy silanes), that may allow to retain the activity of the entrapped enzymes [6]. In addition, functionalities can be easily introduced by decorating the surface of the silica nanoparticles exploiting free silanols, e.g. through chemical derivatization (silanization reactions), electrostatic interactions (adsorption of polycations) and also hydrogen bonding. For example, polymers of the Pluronic series (triblock copolymer of the type PEG–PPG–PEG, where PEG and PPG are poly(ethylene glycol) and poly(propylene
0927-7757/$ – see front matter. Crown Copyright © 2006 Published by Elsevier B.V. All rights reserved. doi:10.1016/j.colsurfa.2006.05.008
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Scheme 1. Standard method for the preparation of silica nanoparticle in W/O microemulsion (emulsion): (A) the hydrophobic tetraalkoxysilane (TOS) is added to an enzyme-containing emulsion. (B) When exposed to water, the TOS starts hydrolyzing and during this process the molecule gradually approaches the water domain and enters it when it is in the fully hydrophilic form of silicic acid. (C) A network of silica gel forms via partial condensation of silanol groups of silicic acid, to yield a material with average formula SiO2−x (OH)2x . The material undergoes a transition from viscous droplet to elastic nanoparticles. (D) The nanoparticles are purified by removal of emulsifier, while the silica gel network entraps the enzymes in the nanoparticles bulk.
glycol), respectively) have been found to adsorb on silica in form of bilayer-like structures (or surface micelles). For Pluronic F127 (average molecular weight 12,600 and 70 wt.%) these surface layers are about 5 nm thick [7] and reversibly cover silica nanoparticles with a polymeric barrier that provides steric repulsion during brownian collisions, even in absence of electrostatic repulsive forces [8]. Protein-encapsulating colloidal silica has been produced in most cases by performing the silicon alkoxide hydrolysis in the dispersed domains of water-in-oil (W/O) microemulsions. The alkoxides diffuse into the enzyme-containing water droplets from the hydrophobic phase, are there hydrolysed to silicic acid, which eventually condenses to give a silica gel network [9] (Scheme 1). A major inconvenience of this method is the extreme pH (either strongly acidic, <4, or basic, >9, in the last case usually obtained with concentrated ammonia), which is required for a reasonably rapid hydrolysis reaction [6,9,10]. If it is true that these conditions are likely to denature most enzymes, they are not necessary for the successive condensation of silicic acid to yield a silica gel network, whose rate is maximal at physiological pH. Another similar hurdle: most published preparation methods of silica nanoparticles employ either strongly denaturing organic solvents, such as acetone [11], or basic solutions [6] for their isolation and purification. Last, surprisingly little is reported about the recovery yield of the nanoparticles preparation procedures; this is a non-negligible issue, since purification is often based on the formation of a gel-like or precipitated phase, where nanoparticles are not unlikely to irreversibly agglomerate. Considering the possible high cost of the biomolecules used, this should be avoided as much as possible. We here propose a modified and milder alternative method for enzyme encapsulation within silica gel nanoparticles that (A) uses a sol–gel synthesis at nearly neutral pH (6–8), (B) minimizes the use of denaturing organic solvents or of extreme pH conditions during purification, and ensures quantitative yields both in the removal of organics and in the dispersability in water.
In summary: point (A) was obtained by decoupling the hydrolysis of the silicon alkoxide from its condensation, cleaving the aliphatic chains in a low pH water solution, which is then buffered at pH 6–8 prior to the enzyme addition and is then used for the formation of the W/O emulsion. By varying the organic solvent/surfactant ratio, we showed that the particle size can be modulated from 10 to 50 mm. point (B) aimed at avoiding the fast silanol condensation at nearly neutral pH, which, upon surfactant removal, in our experience always promotes a substantial and irreversible particle aggregation. We have developed a work-up method consisting of two stabilization steps. First, by changing the polarity of the dispersant phase, the emulsifier is partially desorbed and likely forms surface aggregates. Second, the emulsifier aggregates are replaced by those of a biocompatible polymer (Pluronic F127). In this final form the nanoparticles are indefinitely stable against agglomeration, due to the steric stabilization provided by the polymer. The process has been validated for enzyme encapsulation using horseradish peroxidase (HRP) as a model. 2. Experimental 2.1. Materials Tetraethyl orthosilicate (TEOS, purity 98%) was supplied by Aldrich (Buchs, Switzerland). Berol 26 and Berol 267 were kindly donated by Akzo Nobel (Stenungsund, Sweden). n-Hexane, ethanol, methanol, acetonitrile (reagent grade) and hydrogen peroxide 30% solution were supplied by BDH (Laboratory Supplies, UK). Horseradish peroxidase 500 units/mg was purchased from Fluka (Buchs, Switzerland). 1-Methyl-2pyrrolidone (NMP, purity 99%+), Pluronic F127 and Pyrogallol were supplied by Sigma (Buchs, Switzerland). Water was pre-distilled and further purified by a Milli-Q system (Millipore, UK). Phosphate buffered saline (PBS), 10 mM, was prepared from 0.2 g KCl, 0.2 g KH2 PO4 , 2.14 g Na2 HPO4 ·7H2O and 8 g NaCl
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dissolved in 1 L of pure water. The pH was adjusted by dropping dilute solutions of HCl and NaOH. 2.2. Preparation of silica-gel nanoparticles 1.0 mL of TEOS (0.93 g) was mixed with 0.50 mL of HCl 3.5 mM in pure water and stirred for 2 h at room temperature, until complete solubilization. The ethanol produced by hydrolysis was removed by rotary evaporation at 25 mbar, room temperature (water evaporation is supposedly negligible under these conditions). The solution was first buffered at pH 6 adding 0.25 mL PBS 10 mM pH 10 at low temperature (0 ◦ C) and then diluted with an appropriate volume of protein-containing 10 mM PBS at pH 7.4 (in the case of horseradish peroxidase: 10 mg/mL) to reach the desired silica concentration. A volume variable between 250 and 300 L (in dependence on the final target silica concentration) of this solution were quickly injected in an organic solution of 0.50 g of Berol 26, 0.50 g of Berol 267 and 10 mL of n-hexane, previously prepared. After a vigorous stirring the suspension developed into a stable and clear reverse microemulsion. The suspension was kept at room temperature for 90 min before workup, in order to ensure the formation of silica gel nanoparticles through an effective condensation of silanols. 2.3. SiO2 -NPs work-up Twenty milliliters of the nanoparticle suspension were transferred in a rotary evaporator, where roughly half the volume of the hexane was evaporated at room temperature. The concentrated solution was added of 16 mL of a water solution containing 13 wt.% ethanol and 20 wt.% N-methyl pyrrolidone (NMP), and the resulting mixture was tranferred to the rotary evaporator, where the residual amount of hexane was completely removed (room temperature, 30 mbar). The clear suspension (∼20 mL) was diluted with 100 mL of a 0.5 wt.% Pluronic F127, 18 wt.% ethanol and 7 wt.% NMP water solution, previously prepared and kept at 25 ◦ C under stirring. The liquid dispersion was then purified using an Amicon stirred ultrafiltration cell (Millipore, UK) equipped with a regenerate cellulose membrane (100 kDa molecular weight cut-off). Initially, the ultrafiltration cell was continuously refilled first with a EtOH-NMP-F127 water solution (in the proportions above) to keep constant volume and composition of the solution, then (upon completely soluble in water of the filtrate = complete removal of Berol) with a 0.5 wt.% F127 water solution, to remove both NMP and ethanol. Finally, the dispersion was concentrated to a final volume of 20 mL. 2.4. Characterization 2.4.1. Silica gel point determination A Bohlin Gemini parallel plate rheometer, oscillating at 1 Hz frequency under controlled strain (at strain = 10−2 ), was used to monitor the gelation kinetics of silicic acid solutions, identifying the gel times as the crossing points of elastic (G ) and viscous (G ) shear modulus curves.
2.4.2. Dynamic light scattering (DLS) The Z-average size and size distribution of silica gel nanoparticles and nanodroplets in the W/O microemulsion were measured using a Zetasizer Nano ZS Instrument (Malvern Instrument Ltd., UK), with a backscatter detection (173◦ detection optics) and a 633 nm laser beam. Raw data were processed by Malvern DTS software. The viscosity parameter was corrected measuring the sample viscosity on an automated microviscosimeter (Anton Paar, UK). 2.4.3. Transmission electron microscopy (TEM) The nanoparticle dispersions were pipetted on carbon/formvar coated 100 mesh grids, which were therefore washed with distilled water three times, stained in 1% uranyl acetate solution and air dried. Specimens were observed in a Technai 12 electron microscope at 100 kV. 2.4.4. Pyrogallol assay The activity of HRP, in solution or nanoencapsulated in the silica-gel nanoparticles, was evaluated by the pyrogallol assay [12]: the time-dependent oxidation of pyrogallol to purpurogallin catalysed by HRP can be followed monitoring the absorbance of purpurogallin at 420 nm, at 20 ◦ C in PBS 10mM pH 6.0 (through a UV/Vis spectrophotometer Perkin-Elmer Lambda 25 and WinLab software). The peroxidase activity is expressed in term of pyrogallol units, where one unit forms 1.0 mg of purpurogallin at pH 6.0 and at 20 ◦ C. 3. Results and discussion 3.1. W/O emulsion water phase: hydrolysis without gelation The preparation of silica nanoparticles is based on the conversion of the water phase of a W/O emulsion into a silica gel through a sol–gel process; due to the presence of potentially sensitive biomolecules, there are strong limitations for pH and composition of the starting water solution. Most typically synthesized silica sol–gel processes are based on the use of silicon alkoxides that undergo three elementary reactions: (a) hydrolysis: Si(OR)4 +nH2 O → Si(OR)4−n (OH)n + nROH (b) alcohol condensation: Si-OR + HO-Si → Si–O–Si + ROH (c) water condensation: Si-OH + HO-Si → Si–O–Si + H2 O It is important to note that only the condensation reactions are responsible of the gel formation. The pH dependence of this set of reactions has been extensively studied [8,13–17]. The hydrolysis rate reaches its minimum at neutral pH and almost linearly rises when the pH is lowered or increased. The condensation rate, on the contrary, is highest at neutral pH and sharply decreases far from neutrality [8,13]. Often an alcohol is employed for rendering the system homogeneous, since water and aliphatic alkoxysilanes are immiscible; however, the addition of other solvents can be also avoided if the water/alkoxide mixture is vigorously stirred, since hydrolysis itself progressively converts the hydrophobic
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alkoxides into water soluble materials: hydrosilicates and alcohols [18,19]. In summary, the two factors possibly affecting the activity of biomolecules are (a) the pH necessary for alkoxide hydrolysis and (b) the presence or production of alcohols at high concentration. We have overcome the first problem by performing separately the stages of hydrolysis and condensation: at acid pH the alkoxide can be hydrolyzed in the absence of the biomolecule and without causing a rapid condensation of silicic acid. This precursor solution can be buffered later and mixed with that of the desired biomolecule, which is never in presence of an extreme pH. The presence of organic solvents could be minimized too: first, with careful choice of pH and reaction time an homogeneous solution of hydrolyzed tetraethoxysilane (TEOS) can be obtained (in 3.5 mM HCl in 2 h at room temperature, ratio H2 O/TEOS 0.5:1 (v/v), pH 2.5, 20 ◦ C) without the use of additional organic solvents. Second, the slow condensation rate at acid pH allows for enough time to evaporate the large amount of ethanol produced in TEOS hydrolysis at controlled reduced pressure, before buffering the solution at physiological pH (6.8–7.2) and mixing it with a similarly buffered enzyme-containing water solution. The last operations are conducted as rapidly as possible and just above the freezing point of the solution to avoid appreciable silicic acid condensation (maximal at that pH). By controlling the volume ratio between the two solutions it is possible to control the concentration of both enzyme and silica in the final gel; in our experiments we have mostly produced materials with two silica concentrations: 8 and 20 wt.% (expressing the solid content in terms of theoretical weight of SiO2 /overall weight of the water phase). Silica concentrations above 20 wt.% have not been used in this work, mostly because of the speed of the condensation reactions (gel point below 2 min at room temperature), which render critical the timing of all the operations.
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surfactants of the family of alkyl phenol ethoxylates with the general chemical structure H-(CH2 )n -Ph-(OC2 H4 )m -OH (such as Triton X-100 or surfactants of the Berol series), sometimes in mixture with hexanol [23–25]. Non-ionic surfactants were found to provide less polydisperse silica particles, whereas AOT microemulsions are apparently less stable, and produce nanoparticles with a broad size distribution and possible formation of colloidal gels [25]. We have therefore selected non-ionic surfactants and, more specifically, a mixture of two alkyl phenol ethoxylates with different hydrophilic lipophilic balance (HLB) for a fine modulation of the average HLB and therefore also of the size of water droplets (the higher the HLB, the larger is the droplet): Berol 26 (n = 9, m = 4, HLB = 8.9) and Berol 267 (n = 9, m = 8, HLB = 12.3). For most experiments we have used a mixture Berol 26–Berol 267 1:1 wt. to obtain the maximum average HLB (∼10.6) compatible with their solubility in n-hexane. The concentration of surfactants in n-hexane was set at 10% (w/v), a typical value for these preparations [24], since it is optimal for the microemulsion stability and it is low enough for an effective removal of the surfactant during work-up. 3.2.3. Water content As in any W/O microemulsion, keeping constant the emulsifier concentration the droplet dimensions increase steadily with the increase of the water phase volume (to minimize the energetically unfavourable increase of the interface), until phase separation takes place, generally above a limiting value of roughly 50 nm (Fig. 1). Therefore, the volume ratio water phase/organic phase allows a control of the droplet size and, since dimensions are substantially preserved in the condensation stage, that of nanoparticles too (see next session). However, it must be noted that at high concentration the presence of hydrosilicates can influence the droplet diameter too: while droplets containing 8 wt.% silica is substantially identical to those of pure water, at 20 wt.% silica they are considerably smaller, and the microemul-
3.2. W/O microemulsions: control over droplet size The size and size distribution of silica gels nanoparticles are supposedly templated on those of the precursors, the W/O emulsion droplets, thus unstable emulsions will lead to very broad distributions and uncontrolled average size. We have therefore optimized the preparation of stable W/O emulsions with adjustable droplet size in the sub-100 nm range (microemulsions) as a function of the following factors: composition of the hydrophobic phase, structure and concentration of the surfactant and volume ratio between water and hydrophobic phase. 3.2.1. Hydrophobic phase We have chosen n-hexane as organic solvent, since it is one of the common solvent used in reverse microemulsions [20–22], it is widely available and easy to remove (by evaporation). 3.2.2. Emulsifier Silica nanoparticles have been reportedly prepared either using the anionic surfactant Aerosol-OT (AOT), or non-ionic
Fig. 1. Dependence of the hydrodynamic diameter of pure water or silicic acid droplets on the water phase volume. The W/O microemulsion contained 10 mL of n-hexane, 0.50 g Berol 26 and 0.50 g of Berol 267. The last point of each curve represents the threshold of the microemulsion stability before phase separation.
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Fig. 2. Gel point determination for silicic acid solutions at different concentration. A parallel plate rheometer was used to measure the gel points of silica gels, identified as the points where elastic (G ) and viscous (G ) shear moduli assume the same value.
sion is able to disperse more water before phase separation takes place. This may be due to the increased density and viscosity of the water phase (therefore decreased tendency to coalesce), but may also derive from the presence of some partially hydrolyzed TEOS in the water phase, which has surface active properties. 3.3. Sol–gel process in W/O microemulsion: silica gel nanoparticles Hydrolysed TEOS solutions gel very rapidly at room temperature: 8 and 20 wt.% silica reached a gel point (G = G ) in ∼8 and ∼2 min, respectively (Fig. 2). Anyway, in order to be sure to obtain a well cross-linked silica network, in our emulsion experiments we have allowed the condensation to run for 90 min. The initial water droplets and the final silica gel nanoparticles appeared to have identical size and monomodal distributions, both for 8 and 20 wt.% silica nanoparticles. This finding would suggest a simple picture of a topologically confined conversion of a homogeneous solution into a gel. The silicic acid solution, however, is likely to contain silica clusters (despite the low temperature, the pH conditions do favor silanol condensation) and thus be inhomogeneous. The clusters may distribute unevenly in the emulsified droplets, eventually producing harder gel nanoparticles but also some viscous droplets. A rapid verification can be obtained by decreasing the water/oil ratio of the microemulsion after completion of the condensation reaction: the nanoparticle size would not be affected, while the droplets would reduce their dimensions. Indeed the addition of more hexane–Berol solution to the nanoparticle dispersion rendered bimodal their initially monomodal size distribution; a minor component at smaller size appears, whose sensitivity to dilution suggests being formed by liquid droplets (Fig. 3). The presence of un-gelled droplets implies a loss of material to encapsulate, and should be minimized. It appears that their volume fraction diminishes by increasing silica concentration: for nanoparticles with 8 wt.% of silica, the droplets account for a substantive part of the total water phase volume (40–50%), while
for 20 wt.% silica nanoparticles (the highest concentration compatible with our method) this represented a very minor fraction, around 7%, which we estimate acceptable for most applications. 3.4. Purification of silica gel nanoparticles The main target of this phase is the purification of the nanoparticles from solvents and emulsifiers used in their synthesis and deliver them in water dispersion in the least denaturing and most stable conditions. In commonly adopted procedures, nanoparticles are produced under basic conditions (pH >9) and their suspensions are precipitated by the addition of a fairly polar organic sol-
Fig. 3. Left: decrease of the Z-average size of the 20% SiO2 nanoparticles synthesized in W/O microemulsion by progressive dilution with fresh hexane–Berol solution. Dilution factors on the abscissa are reported as ratio volume diluent added/ volume microemulsion. Right: size distribution of the same nanoparticle suspension before and after increasing dilutions. Initially monomodal, the distribution splits into two distinct peaks, respectively at 10 nm and at the initial size of 50 nm. Note that the nanoparticles size axis is reported in logarithmic scale.
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vent (e.g. acetone, ethanol) where both the hydrophobic phase (hexane) and the emulsifier are soluble [26]. After washing the precipitate with fresh solvent to remove all the surfactant, the nanoparticles can be re-dispersed in a basic water solution. The high pH is instrumental to maintain the ζ-potential of the silica nanoparticles sufficiently negative to prevent strong aggregation, and thus to sufficiently slow down the interparticle condensation of superficial Si–OH groups to avoid formation of irreversible gels or precipitates. These procedures, however, are unsuitable in the presence of sensitive biomolecules, since they require at the very least neutral pH, where the ζ-potential value of silica is too low for avoiding irreversible agglomeration in the precipitate, and, as already mentioned we seek to minimize the use of organic solvents in high concentrations. We have therefore developed a process, which (a) is based on a smooth change of the nanoparticle environment, from a hydrophobic- to a waterdispersed state, (b) keeps the nanoparticles always in a stable colloidal suspension throughout the procedure. The crucial point of this procedure is that Berols are difficult to remove from the silica surface in a water environment: they bear oligo(ethylene glycol) structures, which strongly interact with silanols through hydrogen bonding (as generally for amphiphilic derivatives of PEG, such as Pluronics). Even more important, they have a high HLB value, which means that they tend to disperse water instead of being dispersed in water. The addition of fairly limited quantities of other organic components improves the solubility of Berols in the emulsion continuous phase and forces them to form dispersable aggregates (regular emulsions) (Fig. 4). The size of these aggregates can be controlled by varying the amount and the composition of the organic solvent (that is varying the interfacial energy of a Berol layer). Moreover, in some conditions the same behaviour
Fig. 4. DLS analysis of a 10 wt.% Berol water mixtures (Berol 26 and Berol 267 in 1:1 ratio) containing different amounts of ethanol and NMP. The use of ethanol alone is possible only above 20 wt.%, while below phase separation takes place; the size of the aggregates decreases by increasing the amount of ethanol. 18 wt.% EtOH and 7 wt.% NMP (total amount <26 wt.%) is a good compromise since it produces Berol aggregates smaller than silica nanoparticles (25 nm vs. 50–90 nm) while minimizing the quantity of organic solvents.
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can be observed in the presence of hexane, producing hexanecontaining emulsions. For example, 33 wt.% or more concentrated ethanol solutions succeed in dispersing Berol/hexane mixtures (10 and 25 wt.%, respectively) in water, while 20 wt.% ethanol is enough to disperse Berol alone (10 wt.%). Hexane can be removed from the dispersion by evaporation, generally reducing the size of the aggregates. Our target was to use the least denaturing solvents, at the lowest possible concentrations (and for the shortest time), and producing Berol-containing aggregates with the most different dimensions from those of the nanoparticles, for an easy purification via dialysis or ultrafiltration. We have found an optimal performance at 18 wt.% ethanol and 7 wt.% N-methyl pirrolidone, NMP; this solvent mixture offers the best compromise between emulsificability of Berol/hexane mixtures (other solvents, such as acetonitrile and methanol, were ineffective), the size of the aggregates after hexane evaporation and hydrophilicity of the organic components: in water mixtures the denaturing effect of an organic solvent on an enzyme (the cosolvent concentration yielding a 50% reduction of catalytic activity) is often proportional to its octanol/water partition coefficient. Experimentally, we have evaporated part of the hexane from the nanoparticles/hexane dispersions prior to introduce the NMP/ethanol water solution; screening different solvent formulations, we have recorded the formation of a clear and stable emulsion at a 1:2 hexane/water solution volume ratio. In the aggregates (we may imagine them as emulsified droplets) the core is likely composed by ethanol and Berol hydrophobic chains, while, since NMP and Berol are immiscible, we exclude the presence of NMP. The evaporation of hexane from this emulsion did not change its state, nor caused significant silica agglomeration. It is noteworthy that in the absence of NMP the emulsion could not be formed, while the presence of ethanol is not essential; on the other hand, without ethanol silica precipitates after hexane evaporation. At this stage, dynamic light scattering revealed objects having a size substantially identical to that of the nanoparticles in the W/O emulsion (Fig. 5) without any sign of agglomeration. This, however, does not exclude that Berol aggregates may dynamically and reversibly sit on the nanoparticles surface, similarly to what recorded in literature for Pluronics [7], interacting through Berol-silanol H-bonds (Scheme 2, B). Two findings could indeed suggest a similar phenomenon to involve Berol aggregates and silica nanoparticles in our system: (a) If hexane is completely evaporated after emulsification, precipitation occurs. This may be due to the formation of a Berol pseudo-bilayer that can spread better and adhere more firmly to the surface than hexane-rich emulsion droplets (Scheme 2, C ). (b) The dispersion precipitates also by replacing the dispersion organic components with water during ultrafiltration (the hydrophobic aggregates are supposedly smaller than the nanoparticles and can be separated from them through membrane with an appropriate size cut-off, see Fig. 4). In this case we believe this to be caused by agglomeration and
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direct interparticle H-bonding when the emulsion droplet concentration falls but the solvent is still rich in NMP and ethanol (Scheme 2, C ). These interpretations are clearly just hypothetical, but can be substantially confirmed if precipitation could be avoided by replacing the emulsion droplets with other components that are known to have surface interactions with colloidal silica, such as Pluronic F127. During ultrafiltration experiments we have constantly replaced the filtered emulsion with a 0.5%wt Pluronic water solution; the organic components (Berol aggregates, ethanol and NMP) were removed from the system, obtaining silica nanoparticles in a fairly diluted Pluronic solution at neutral pH, where the reduction in electrostatic repulsion is most likely compensated by the steric stabilization provided by the adsorption of Pluronic micelles (Scheme 2, part C). Within the experimental conditions used, the average particle size increased from 50 to 60 nm before ultrafiltration to about 110–120 nm (Fig. 5), a result qualitatively confirmed by transmission electron microscopy (Fig. 6). It must be pointed out that this increase is likely due not only to some aggregation in the process, but also to the presence of Pluronic on the nanoparticles surface. 3.5. Enzyme activity after nanoencapsulation Enzyme-containing silica gel nanoparticles have been prepared following the method described above; the enzyme (horseradish peroxidase) was introduced in the silicic acid solution immediately after buffering. After work-up, HRP activity in the Pluronic-stabilized nanoparticles was assessed with the pyrogallol assay, where hydrogen peroxide oxidizes the colorless pyrogallol to the yellow purpurogallin [12].
Fig. 5. Size distribution of silica gel nanoparticles from the preparation in W/O microemulsion to the final colloidal dispersion in water after workup. Data were obtained from DLS analysis. (- - -) Silica gel nanoparticles, 20 wt.% in SiO2 , synthesized in a W/O microemulsion with 10 mL hexane, 1 g Berol, 250 L of water phase. (. . .) Concentrated silica nanoparticle suspension (1 g Berol, ∼5 mL hexane and 300 L nanoparticles) re-dispersed in 10mL of NMP/ethanol/water solution during work-up. (—) Silica gel nanoparticles dispersed in water–Pluronic solution after work-up procedure. The peak at 20 nm represents Pluronic F127 micelles.
The comparison between the activity of free HRP (4.20 U/mL, measured at HRP concentration of 0.03 mg/mL) with that of the encapsulated one (0.023 U/mL in 8 wt.% silica nanoparticles and 0.320 U/mL in 20 wt.% silica nanoparticles) with the same overall enzyme concentration, shows a sharp decrease for both formulations, particularly dramatic for the 8% silica nanoparticles (Fig. 7).
Scheme 2. Sketch of the work-up process developed. The Berol-coated silicagel nanoparticles are originally dispersed in a hydrophobic phase (A); by evaporating a major part of it and adding a 7% NMP, 18% ethanol water solution an emulsion is generated, whose droplets contain hexane in the core and Berol and organic solvents at the interface (B). As described in the text, complete evaporation of hexane (C ) or complete removal of the organic components through ultrafiltration (C ) cause aggregation of the nanoparticles and precipitation. On the contrary, the replacement of the organic emulsion with a Pluronic micellar dispersion (C) keeps the nanoparticles in a dispersed state.
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Fig. 6. TEM images of silica gel nanoparticles after work-up. Most nanoparticles show a diameter in the range 60–100 nm, but some clusters are visible. The white spots visible in the right image are likely Pluronic micelles.
Three phenomena can possibly determine this decrease of activity: (a) partial loss during the purification, since the relatively small enzyme (MW at about 4 × 104 g/mol, hydrody˚ [27] can permeate through the pores of namic radius ∼30 A) the ultrafiltration membranes with molecular weight cut-off of 10 × 104 g/mol, (b) partial denaturation of the enzyme that may be caused by the sol–gel process in W/O microemulsion, or by the organic solvents used in the purification, (c) intrinsic reduction of activity, because of the immobilization in a matrix.
By measuring HRP activity throughout the process, it is possible to see that the activity of the 8% silica nanoparticles is initially very similar to that of the 20% wt. nanoparticles and drops only during the last stage of purification (Fig. 8); 20 wt.% silica nanoparticles show a negligible decrease. This finding suggests that the purification conditions, common to the two nanoparticles, produce negligible denaturation to the enzyme; on the contrary, the enzyme seems to leak out of the less concentrated nanoparticles. This can happen because either HRP is present in non-gelled water droplets, or it permeates through the
Fig. 7. Left, top: reaction scheme of the oxidation of pyrogallol to purpurogallin catalysed by HRP. Left, bottom: pyrogallol and HRP-containing silica gel nanoparticles in water before and after the addition of hydrogen peroxide, that allows HRP to produce the yellow-colored purpurogallin. Right: spectrophotometric analysis of HRP activity: comparison between free and encapsulated HRP in 8 and 20 wt.% silica nanoparticles (same theoretical). The enzyme concentration is kept constant at 0.03 mg/mL with nanoparticles (if present) at 0.08% (v/v) nanoparticles, 0.5 wt.% pyrogallol and 0.03 wt.% H2 O2 .
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solvents. High solid nanoparticles have shown the best results both in terms of encapsulation efficiency and enzyme activity, using HRP as a model enzyme. A number of questions are now open and are currently under investigation: - How does the enzyme activity depend on the presence of Pluronic on the surface of the nanoparticles and on the presence of aggregates? We are indeed studying the possibility to replace Pluronic steric stabilization with the electrostatic one arising from polyelectrolyte adsorption, with the hope also to reduce particle aggregation during the work-up. - How applicable is this method to the encapsulation of other and possible less stable enzymes? We are expanding these studies to the incorporation of other bacterial and mammalian enzymes. Fig. 8. Spectrophotometric analysis of HRP activity encapsulated in 8 wt.% silica nanoparticles at different stages of the purification process. It is apparent how the enzymatic activity is substantially unaffected during the first stages.
gel structure of the nanoparticles: both phenomena are strongly reduced by increasing the solid content of the nanoparticles and this explains the remarkable difference between 8 and 20 wt.% silica nanoparticles. The reduction in HRP activity observed for the high solid content 20 wt.% silica nanoparticles (roughly one order of magnitude lower than the free enzyme) could be ascribed to denaturation in the sol–gel process (in the purification this has been previously excluded), or to the more difficult access of the substrates to the catalytic centre/lower diffusion coefficient of the enzyme. In particular, the second option would be a result of lower diffusion coefficient of substrates and products in the matrix, slower motions of the enzyme and possibly also difficult permeation of the slightly anionic pyrogallol in an anionic matrix. The first option is less likely, since, opposite to what recorded, one should observe less activity (=more denaturation) in the high solid samples, where the changes induced by the sol–gel process are more dramatic. Moreover, literature about enzyme encapsulation in silica gel matrices does not report significant denaturation caused by the sol–gel process [18,28]. In summary, we are inclined to exclude significant denaturation effects of both sol–gel and work-up procedures and to explain the difference of HRP activity between high and low solid content nanoparticles in terms of enzyme leakage or poor encapsulation efficiency of the immobilization process. 4. Conclusions We have here presented the successful proof of principle of a modified sol–gel process in W/O microemulsion, which minimizes harsh conditions that can possibly cause denaturation of encapsulated enzymes. It has been shown that it is possible to produce and purify silica gel nanoparticles with controlled diameter in conditions of physiological pH and minimizing the presence of organic
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