tional methods [1]. The approach is based on the hydrolysis and polycondensation of a molecular precursor such as a silicium alkoxide (tetramethylorthosilicate, ...
Reprint from : Progress in Condensed Matter Physics ’Festschrift in honour of Vincenzo Grasso’ 139-152; Editors G. Mondio and L. Silipigni; Published by Societ´a Italiana di Fisica; ISBN 88-7438-010-0
WATER AND PROTEINS CONFINED IN SILICA HYDROGELS AND SILICA NANOPARTICLES: STRUCTURAL, DYNAMIC AND FUNCTIONAL STUDIES ´ , E. M. Cammarata, C. Caronna, G. Fiandaca, M. Levantino, G. Schir o Vitrano and A. Cupane
Istituto Nazionale per la Fisica della Materia and Dipartimento di Scienze Fisiche ed Astronomiche dell’Universit´a di Palermo, via Archirafi 36, I-90123 Palermo, Italy
Abstract In this paper we review some recent experimental work performed at our laboratory on the structural and dynamic properties of water and proteins encapsulated in macroscopic silica hydrogels and in silica nanoparticles obtained with the sol-gel method. For encapsulated proteins, studies have been extended to monitor also the spectral and conformational properties, the structural stability and the functionality. Several spectroscopic techniques have been employed; these include small angle X-ray scattering, dynamic light scattering, broad band dielectric spectroscopy, low temperature absorption spectroscopy in the mid and near infrared, room temperature circular dichroism and stopped flow spectroscopy. The reported data highlight the interest of silica hydrogels and silica nanoparticles as confining systems for liquids and proteins both for basic and applied research.
1
Introduction
The sol-gel process is a chemical synthesis technique used to prepare silica gels, glasses and ceramics at much milder conditions (e.g. temperature) than is possible by conventional methods [1]. The approach is based on the hydrolysis and polycondensation of a molecular precursor such as a silicium alkoxide (tetramethylorthosilicate, TMOS, and 1
tetraethylorthosilicate, TEOS, are the most commonly used): by controlling the external conditions (such as temperature, pH, ionic strength, precursor/water/protein ratio) it is possible to prepare not only bulk materials, like macroscopic hydrogels, but also silica nanoparticles. In view of the mild, ambient, conditions of the sol-gel process, it is possible to encapsulate within the silica matrix numerous organic molecules such as dyes, biomolecules, proteins or even cells. This feature makes silica hydrogels and/or silica nanoparticles obtained from the sol-gel approach very appealing new materials in the areas of photonics and (bio)chemical sensors [2],[3]. On the other hand, silica hydrogels even in the absence of any dopant molecules contain water, which is confined inside the pores of the vitreous silica matrix; they are therefore suitable systems to study the structural and dynamic properties of water in restricted geometry. Studies on water in confined geometry are relevant not only to understand the physical properties of glassforming liquids and to test the predictions of current theories [4],[5], but also for their biophysical implications [6],[7]: indeed, confinement in silica hydrogels may closely mimic the conditions of water at protein surfaces in highly crowded biomolecular systems, like e.g. inside the cell. Moreover, the structure of the silica matrix (pores dimensions, fractal dimensionality etc..) can be varied and controlled by using different sol-gel protocols [1] (acid vs. basic catalysis, alkoxide precursor/water ratio, ionic strength, temperature etc.); for each sample the matrix and water structural properties can be characterized by X-ray scattering measurements. As far as proteins encapsulated within silica hydrogels are concerned [8], they usually show increased resistance to denaturation induced either by chemical agents or by heat [9], and reduced rates of conformational transitions [10]; almost no information is available up to now concerning the dynamics of encapsulated proteins and their relations with the dynamic properties of the embedding matrix. Silica hydrogels are particularly useful in this respect, since the hydration level h (defined as the ratio grH2 O/grSiO2 ) of the hydrogel can be varied over a wide range, from h values larger than 1 to h values of a few percent. In this work we review some recent experimental results obtained by our group using a variety of spectroscopic techniques on the structure of silica hydrogels and silica nanoparticles, on the relaxation dynamics of confined water, and on the structural, dynamic and functional properties of encapsulated proteins.
2 2.1
Experimental Methods Samples
Preparation of silica hydrogels. A solution containing 75% v/v TMOS, 25% H 2 O (Millipore purified, resistivity ' 18M Ω· cm) and 2 · 10 −3 M HCl is sonicated for 20 min and diluted with an equal quantity of water. After gentle mixing the resulting sol is poured into semimicro polystyrene cuvettes; in these conditions sample gelification occurs in about 1 h at room temperature. After 4 days the sample is extracted from the cuvette and left to age at room temperature: it progresssively loses weight and reduces its volume until it reaches, after about 20 days, approximately one seventh of
its initial volume; no further relevant volume contraction is observed upon prolonged aging. Typical samples for spectroscopic investigations are slabs of transparent vitreous material having dimensions of 1.7x0.5x0.2 cm 3 and hydration level h = 0.35. Protein encapsulation. To encapsulate proteins within silica hydrogels, the above TMOS/H2 O/HCl solution, after sonication, is mixed with a solution containing protein at the desired concentration, phosphate buffer pH 7, and 60% v/v glycerol. In these conditions sample gelification, even at 7 ◦ C, occurs in a few minutes. To avoid any damage to the protein, immediately after gelification the gel is covered with a “protectant” solution [11],[12] containing buffer phosphate pH 7 and the desired amount of glycerol, and left to age at 7 ◦ C for one day. Proteins encapsulated following the above procedure exhibit intact conformational and functional properties. Preparation of silica nanoparticles. To prepare silica+proteins aggregates, a water solution containing 10−4 M TMOS and 2 · 10−3 M HCl, after sonication, is mixed with an equal quantity of a 8 · 10−5 M protein solution in phosphate buffer 4 · 10 −2 M, pH 7. The solution obtained is stored at 7 ◦ C for about two days. In the first 30 minutes after mixing the sample becomes turbid, indicating formation of micron-sized aggregates; as time elapses these aggregates precipitate and a clear coloured “solution” is obtained, in which the remaining protein concentration is about 1/3 of the initial one. After two days the solution is centrifuged at 2500 rpm for about 20 min and filtered through a 1.2 µm filter. If, after centrifugation and filtration, the final “solution” is filtered through a 0.2 µm filter, a totally colourless filtrate is obtained : this indicates that the remaining protein is not free in solution but encapsulated within silica aggregates. Data reported in section 3.1 will show that our “solution” is rather a suspension of nanometer-sized silica particles in which proteins are encapsulated.
2.2
Instrumentation.
X-ray scattering. Small Angle X-ray Scattering (SAXS) data in the scattering vector, q, range 0.008 - 0.6 ˚ A−1 were collected with an Anton-Paar compact Kratky camera equipped with a step-scanning motor and a scintillator counter; the X-ray source was a Ni-filtered Cu Kα radiation (wavelength 1.5418 ˚ A). Measurements were collected at room temperature and, unless otherwise stated, at atmospheric pressure. Scattering contributions from air and camera were measured in separate experiments and suitably subtracted. Smearing effects due to the linear beam focusing were also taken into account. Dynamic light scattering. Dynamic light scattering measurements were performed with a Brookhaven apparatus. We wish here to express our gratitude to prof. M.U. Palma and prof. M.B. Palma-Vittorelli for use of the instrument and to Dr. A. Emanuele and Dr. F. Pullara for their skilful scientific help. All the experiments reported in this paper were performed at a temperature of 20 ◦ C. Absorption spectroscopy. Absorption spectra in the near infrared were measured with a Jasco V-570 spectrophotometer equipped with an Oxford Instruments Optistat cryostat. The temperature was measured in the copper sample holder and was controlled within
0.2 K with an Oxford Instruments ITC 503 temperature controller. Spectra in the mid infrared were measured with a Jasco FTIR 140 spectrophotometer equipped with a PbS detector; the cryostat and temperature control apparatus were the same as those used for measurements in the near infrared. Samples were placed in a cell mounting CaF 2 windows and a 0.050 mm spacer. The single beam spectrum in the 1000 - 4000 cm −1 wavenumber range was measured with 300 scans at 1 cm −1 resolution. The sample absorption spectrum was calculated with respect to the empty cell; the spectral contributions of the silica matrix and of the solvent were measured in a separate experiment and suitably subtracted from the sample spectrum. A cubic baseline was also subtracted from each spectrum, using the Peakfit package. Dielectric spectroscopy. Broad band dielectric measurements were performed using two different systems: between 10−3 and 6 · 104 Hz frequency response analysis was carried out (Solartron frequency response analyzer FRA 1250 combined with a Solartron 1296 dielectric interface); from 5 · 102 to 106 Hz a Hewlett Packard HP4284A LCR bridge was employed. Isothermal frequency scans of the complex admittance were performed in the temperature interval 130 − 300 K; the complete thermalization of the sample was checked at each temperature verifying that successive scans were indistinguishable within the experimental error. Circular dichroism. Circular dichroism (CD) measurements were performed at 20 ◦ C with a Jasco J-715 spectropolarimeter. All samples were equilibrated with the desired denaturant concentration for about 15 min prior to the measurement of the CD spectrum. For each denaturant concentration a baseline measured in separate experiments was subtracted from the measured spectrum. Stopped-flow measurements. Kinetics of carbonmonoxide (CO) binding to deoxymyoglobin were measured with a Biologic Science SMF-300/S stopped flow accessory used in conjunction with a Jasco J-715 spectropolarimeter. A solution of deoxymyoglobin (either free in solution or encapsulated in silica nanoparticles) was rapidly mixed with a solution containing the same solvent and the desired quantity of CO; the fraction of deoxygenated protein as a function of time was determined from the absorption change at 439 nm. We are indebted to Prof. L Cordone for use of the spectropolarimeter and of the stopped flow accessory.
3 3.1
Results Structural properties of silica hydrogels and nanoparticles
Fig.1 shows typical SAXS patterns obtained from silica hydrogels. The first intensity decay observed by increasing the scattering vector q is due to the presence of structural inhomogeneities (“pores”) in the silica matrix [13]. From a Guinier analysis [14] we obtain the radius of gyration of the structural inhomogeneities: values between 15 and 35 ˚ A are obtained, depending on hydrogel aging and hydration level. Taking into account the fact that the acid-catalyzed hydrolysis step used in our sol-gel protocol tends to produce highly branched silica wires that, upon dehydration, tend to merge to form
scattered intensity (a.u.)
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Figure 1: Normalized SAXS patterns obtained from silica hydrogels before (•) and after (◦) vacuum drying. The arrow indicates the interference peak at q ∼ 0.35 ˚ A−1 .
a more compact solid, it is likely that the matrix inhomogeneities are pores filled by loosely entangled silica wires and by water. The coefficient of the power-law decay observed at intermediate q values (although determined over a q range somewhat smaller than a decade and therefore subject to some incertitude) is definitely less than 4; values between 2.5 and 3 are observed, suggesting a fractal structure of the pores [15]. The most interesting feature of the SAXS patterns obtained from our hydrogels is the interference peak observed at q ∼ 0.35˚ A−1 . The Bragg value (d = 2π/qmax ) corresponding to −1 ˚ qmax = 0.35A is d = 18˚ A; this means that the interfering objects cannot be the pores. We propose that the peak at q ∼ 0.35 ˚ A−1 could be due to the interference between intra-pore structures, likely related to a non uniform water distribution inside the pores [16]. Experimental support to this hypothesis is given by the SAXS pattern obtained from a vacuum-dried sample. During the vacuum drying treatment about 80% of water trapped in the hydrogel is lost and the sample cracks. As shown in Fig. 1, the SAXS pattern relative to this sample is shifted to higher q values (indicating smaller pore size) and shows little, if any, presence of the peak at q = 0.35 ˚ A−1 , thus confirming its attribution to a non uniform water grouping ( “droplets” ) inside the pores of the silica matrix. A quantitative analysis of the entire SAXS pattern (see ref. [17]) enables to determine the average droplet radius R = 7.5 ˚ A. Dimensions of silica nanoparticles have been investigated with dynamic light scattering as reported in Fig. 2. The correlogram in fig.2a can be fit with a single exponential; moreover, the inverse of the decay time exhibits a linear dependence upon q 2 (fig. 2b). From data in fig. 2, using Stokes law, we obtain an hydrodynamic radius R H ' 100 nm; moreover data in fig. 2b indicate that the motion of the silica nanoparticles in solution is almost purely diffusive and that particle-particle interactions can be neglected. If the sample is stored in the cold, such a behavior is conserved for more than 1 week, although a slow radius increase of the aggregates is observed.
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Figure 2: Panel a): scattered light correlogram relative to a suspension of silica+hemoglobin aggregates; dots are experimental points, continous line is a fit in terms of a single exponential; panel b): the inverse of the decay time as a function of q 2 .
3.2
Structural and dynamic properties of water confined in silica hydrogels
The structural and dynamic properties of water trapped within the pores of silica hydrogels have been investigated with near infrared (NIR) absorption spectroscopy and with broad band dielectric spectroscopy, respectively. Fig. 3a shows absorption spectra
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Figure 3: Panel a): absorption spectra (1.45 µm water overtone band) of a silica hydrogel at selected temperatures in the range 325-5K. The spectrum of ice Ih at 233K (dashed line) is also reported for comparison. Panel b): Arrhenius plots for the fraction of “weakly bonded” water molecules. Open symbols refer to water trapped within a silica hydrogel; filled symbols refer to bulk water. The continuous line is a fit of the bulk water data in terms of a power law (critical exponent 1.5; critical temperature 228 K).
in the NIR overtone region of water confined in a suitably aged silica hydrogel (aging time 3 months; hydration level h = 0.35) at various temperatures between 5 and 325 K. Relevant information can be obtained from inspection of the raw spectra in fig 3a: i) the sample remains homogeneous and transparent down to 5 K and water crystallization does not occur: indeed, no baseline increase due to scattering from a frozen sample is observed, even at 400 nm; ii) as the temperature is lowered, the spectra monotonically move toward an a “ice-like” spectrum, with however one relevant difference: in fact, the
absorption band at 1.41 mµ, characteristic of “weakly bonded” water molecules [18] and totally absent in the spectrum of ice I h , is detected at all temperatures, even at 5 K. Results of a quantitative analysis performed by deconvoluting the spectra in terms of Gaussian components [19] are shown in Fig. 3b, where the fractional intensity of the 1.41 mµ band is reported, on a logarithmic scale, as a function of the inverse temperature. Fig. 3b shows that the behavior of water trapped in the silica hydrogel is profoundly different from that in the bulk. Indeed, data for bulk water indicate that the fraction of “weekly bonded” water molecules tends to zero as the temperature is lowered deep into the supercooled region and are consistent with a critical divergence at T = 228 K [20]. On the contrary, data for the hydrogel show no evidence of a divergence at 228 K and indicate that a fraction ('5%) of weakly bonded water molecules is present even at 5 K. Moreover, at temperatures lower than 130 K, the equilibrium between components is frozen, suggesting that the sample has reached a glassy state. We attribute this behavior to the fact that in the hydrogel the geometric constraints imposed by the silica matrix to the trapped water molecules are not compatible with the presence of an extended hydrogen bonded ice-like network; this also rationalizes the fact that water does not crystallize even at cryogenic temperatures.
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Figure 4: Panel a): 3D plot of the imaginary part of the complex permittivity of water trapped in silica hydrogel as a function of frequency and inverse temperature. Panel b): Arrhenius plot of the two relaxation times obtained from the spectra in panel a). Different symbols refer to different experiments and illustrate the data reproducibility.
Information on the dynamic properties of water confined in silica hydrogels has been obtained with broad band dielectric spectroscopy in the temperature interval 130−290 K [16]. Fig. 4a is a 3D plot of the imaginary part of water in hydrogel complex permittivity as a function of frequency and inverse temperature. Two relaxations of approximately the same intensity are detected: in this work we will focus on the low frequency one, in view of its peculiar non-Arrhenius cooperative behavior. This is clearly shown in fig. 4b, where the relaxation times are reported as a function of the inverse temperature. The temperature dependence of the “slow” relaxation can be analyzed with a Vogel-FulcherTamman (VFT) expression τ = τ0 exp[DT0 /(T − T0 )], giving τ0 = 10−11 s, T0 = 100 K and D = 19, which corresponds to a fragility parameter [21] m = 37. The characteristic slowing down of the dielectric relaxation time shown by our VFT behavior is typical
of fragile glass-forming systems and of supercooled liquids, whose cooperative nature is enhanced by confinement. In view also of the SAXS results, we attribute this relaxation to the collective behavior of clusters of water molecules inside the pores (“droplets”). 1
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Figure 5: Panel a): stretching band of the bound CO molecule of carbonmonoxy hemoglobin in R (continuous line) and T (dashed line) quaternary conformation, at T=20K. Panel b): near infrared spectra in the band III region of deoxy hemoglobin in R (continuous line) and T (dashed line) quaternary conformation, at T=20K.
3.3
Spectral properties of proteins encapsulated in silica hydrogels
We have succeded in encapsulating proteins within silica hydrogels showing good optical properties; the protein concentration within the hydrogel can be varied from 10 −6 to 10−2 M, thus opening the possibility of using several spectroscopic techniques (Fourier Transform Infrared spectroscopy, Near Infrared and visible absorption spectroscopy [22], Circular Dichroism, etc..) to investigate the structural, dynamic and functional properties of encapsulated proteins. This is of paramount relevance, since gel encapsulation may enable to stabilize functionally relevant protein intermediate species, otherwise barely accessible to experimental investigation [23]. As an example, we report in Fig. 5a the 20 K FTIR spectra of carbonmonoxy hemoglobin (HbCO) encapsulated in T and R quaternary conformation: the band observed at 1952 cm −1 arises from the stretching of the bound CO molecule and is a fine probe of the structural and dynamic properties of the distal part of the heme pocket [24], [25]. Fig. 5b reports the 20 K near infrared absorption spectra of deoxy hemoglobin (Hb) encapsulated in T and R quaternary conformation: the band at 13200 cm −1 (usually called “band III” [26]) arises from a charge transfer electronic transition involving molecular orbitals mainly localized on the iron atom and on the porphyrin ring respectively, and is therefore a fine probe of the structural and dynamic properties of the proximal part of the heme pocket. It has to be stressed that while HbCO in R quaternary conformation and Hb in T quaternary conformation are equilibrium species, their partners (i.e. HbCO in T conformation and Hb in R conformation) are non-equilibrium intermediates that are trapped through our sol-gel encapsulation protocol: spectra of such intermediates are reported here for the first time. Data in Fig. 5 show that the T ⇔ R transition induces structural alterations in the heme pocket of hemoglobin that can be monitored
by conformation sensitive marker bands in the mid and near infrared. These structural changes appear more relevant in the proximal part of the heme pocket: in fact, a 35 cm−1 spectral shift, corresponding to 2.6%, is observed for band III as compared to 1 cm−1 spectral shift - 0.5% - for the CO stretching band. Moreover, the asymmetric red shift observed for band III suggests that this band is inhomogeneously broadened and that the T⇔R transition alters the energy landscape and therefore the conformational substates distribution of the hemoglobin molecule.
3.4
Conformational stability and functional properties of proteins encapsulated in silica nanoparticles
Protocols for encapsulating proteins within macroscopic silica hydrogels produced with the sol-gel approach are nowadays well established and widely used. Encapsulated proteins exhibit increased resistance against denaturation and retain almost intact functional properties: these features make the sol-gel approach particularly interesting in the field of biosensors [2],[3].
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Figure 6: Circular dichroism spectra in the Soret region of ferric cytochrome c in solution (panel a) and encapsulated in silica nanoparticles (panel b). The fraction of denatured protein is reported in panel c as a function of denaturant concentration; closed symbols: protein in solution; open symbols: protein encapsulated in silica nanoparticles.
Functional studies on proteins encapsulated within standard macroscopic hydrogels are however limited by the diffusion time (usually from minutes to hours, depending on gel thickness) of ligands (either gaseous or salts) through the macroscopic silica matrix. To circumvent this difficulty we have recently developed a new sol-gel protocol [27] which enables to obtain clear suspensions of nanometer-sized silica aggregates (silica nanoparticles) in which proteins are embedded. As shown is section 3.1, typical dimensions of the aggregates are of the order of 100 nm (hydrodynamic radius), each nanoparticle encapsulating 102 − 103 protein molecules. The resistance of proteins encapsulated within our silica nanoparticles against denaturation induced by the chemical denaturant guanidinium hydrochloride has been investigated with circular dichroism in the Soret region (350 - 500 nm). Data reported in Fig. 6 refer to ferric cytochrome c; they show that protein conformational stability is substantially enhanced by encapsulation: in fact,
even at 6 M denaturant concentration (where 100% of the protein in solution is denatured) about 90% of the nanoparticle-encapsulated proteins are found in their native conformation. Analogous results are found also for other proteins such as myoglobin or hemoglobin. 1
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Figure 7: Kinetics of CO binding to deoxymyoglobin. (O): protein in solution; (+): protein encapsulated in silica nanoparticles.
To investigate the functional properties of encapsulated proteins we have followed, using standard stopped-flow techniques, the kinetics of CO binding to myoglobin. In this classic experiment, a sample of deoxygenated protein is rapidly mixed with a solution containing a known amount of CO; the fraction of ligand bound proteins is then monitored as a function of time by looking at the absorbance change at a suitable wavelength. Results are reported in Fig. 7, where the kinetics relative to encapsulated myoglobin are compared to those relative to the protein free in solution; strikingly, no difference is observed in the millisecond time range. This relevant result indicates that encapsulation of proteins into silica nanoparticles, while substantially increasing their conformational stability (see Fig. 6), does not alter their intrinsic functional properties.
4
Conclusions
From the reported data we derive the following picture of our systems. Macroscopic silica hydrogels appear as porous materials: the pores have average radius of about 15 − 30 ˚ A, depending on aging, and likely fractal geometry. Water and proteins can be encapsulated inside the pores. Internal water distribution is not uniform; water molecules appear grouped into “droplets” of about 7 ˚ A radius: interference between droplets gives rise to A−1 observed in the SAXS pattern. Trapped water the characteristic peak at about 0.35 ˚ (or at least a substantial fraction of it) has peculiar structural and dynamic properties, in that it does not crystallize even at 5K and its dielectric relaxation time exhibits a collective Vogel-Fulcher-Tamman behavior in the temperature range 300 − 160K. Concerning proteins, encapsulation within silica hydrogels hinders the protein quaternary conformational transitions and is therefore effective in trapping functionally relevant intermediate conformations of the protein; these, in turn, have been studied and characterized for the
first time in this work with spectroscopic techniques. Concerning protein/silica aggregates obtained with our modified sol-gel protocol, these appear from light scattering experiments as nanometer-sized particles having hydrodynamic radius of about 100 nm and diffusing almost freely in solution. Proteins encapsulated within these nanoparticles exhibit enhanced stability against chemically induced denaturation. However, their ligand binding kinetics is not limited and slowed down by ligand diffusion through a macroscopic silica matrix: indeed, stopped flow experiments indicate unaltered kinetics (with respect to the solution) even in the millisecond time range. In conclusion, the reported data show that silica hydrogels and nanoparticles are extremely interesting systems from several points of view: i) for basic research since the structure and dynamics of water in confinement and the relationships between the dynamic properties of encapsulated proteins and embedding matrix can be systematically studied, also at various hydration levels; ii) for biophysical studies on proteins, to stabilize functionally relevant protein conformational intermediates otherwise barely detectable; iii) for applied research in the field of biosensors, since encapsulated proteins exhibit increased conformational stability and fully intact functional properties.
Acknowledgements The authors gratefully acknowledge the scientific collaboration with Prof. A. Martorana and Dr. A. Longo on X-Ray scattering measurements and with Dr. F. Bruni on dielectric spectroscopy. We are also indebted to Mr. G. Lapis and G. Napoli of the cryogenic laboratory for technical help.
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