Insight into Cellular Response of Plant Cells Confined within Silica

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Insight into Cellular Response of Plant Cells Confined within Silica-Based Matrices Christophe F. Meunier,†, Joanna C. Rooke,† Kata Hajdu,† Pierre Van Cutsem,‡ Pierre Cambier,‡ Alexandre L
eonard,†,^ and Bao-Lian Su*,†,§

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† Laboratory of Inorganic Materials Chemistry (CMI), The University of Namur (FUNDP), 61 Rue de Bruxelles, B-5000 Namur, Belgium, ‡Unit of Research in Plant Cellular and Molecular Biology (URBV), The University of Namur (FUNDP), 61 Rue de Bruxelles, B-5000 Namur, Belgium, and §State Key Laboratory of Advanced Technology for Materials Synthesis and Processing, Wuhan University of Technology, 122 Hongshan Luoshi Road, 430070 Wuhan, China. Research Fellow Position, Fonds National de la Recherche Scientifique, 5 rue d’Egmont, 1050 Bruxelles, Belgium. ^Postdoctoral Researcher Position, Fonds National de la Recherche Scientifique, 5 rue d’Egmont, 1050 Bruxelles, Belgium

Received October 16, 2009. Revised Manuscript Received December 2, 2009 The encapsulation of living plant cells into materials could offer the possibility to develop new green biochemical technologies. With the view to designing new functional materials, the physiological activity and cellular response of entrapped cells within different silica-based matrices have been assessed. A fine-tuning of the surface chemistry of the matrix has been achieved by the in situ copolymerization of an aqueous silica precursor and a biocompatible trifunctional silane bearing covalently bound neutral sugars. This method allows a facile control of chemical and physical interactions between the entrapped plant cells and the scaffold. The results show that the cell-matrix interaction has to be carefully controlled in order to avoid the mineralization of the cell wall which typically reduces the bioavailability of nutrients. Under appropriate conditions, the introduction of a trifunctional silane (ca. 10%) during the preparation of hybrid gels has shown to prolong the biological activity as well as the cellular viability of plant cells. The relations of cell behavior with some other key factors such as the porosity and the contraction of the matrix are also discussed.

Introduction Nature is composed of a wide collection of complex systems working together in harmony such that interactions between many different components produce patterns or behaviors that are not obtained by combining the discrete actions of each individual part. In particular, living cells can be considered as a powerful tool in the development of novel “green” technologies owing to their sophisticated organization, complex information systems, and elaborate processes. Recently, numerous studies have been devoted to harnessing the benefits of whole cells in the construction of new devices such as bioreactors,1 biosensors,2,3 and artificial organs.4-6 These applications generally require the integration of cells into an abiotic material. Cells, isolated from their native environment, are very fragile. By a smart combination of these biospecies with advanced materials, they can be protected from harsh environments. In addition, this ingenious strategy allows the control of the three-dimensional cellular environment. Exploiting the capability of cells to sense their environment via signal transduction pathways, cells could thus be oriented to achieve specific tasks. *Corresponding author. E-mail: [email protected] or baoliansu@ whut.edu.cn. (1) Schmid, A.; Dordick, J. S.; Hauer, B.; Kiener, A.; Witholt, B. Nature 2001, 409, 258. (2) Lei, Y.; Chen, W.; Mulchandoni, A. Anal. Chim. Acta 2006, 568, 200. (3) Daunert, S.; Barret, G.; Feliciano, J. S.; Shetty, R. S.; Shrestha, S.; Smith-Spencer, W. Chem. Rev. 2000, 100, 2705. (4) Read, T.-A.; Sorensen, D. R.; Mahesparan, R.; Enger, P.; Timpl, R.; Olsen, B. R.; Hjelstuen, M. H. B.; Haraldseth, O.; Bjerkvig, R. Nat. Biotechnol. 2001, 19, 29. (5) Orive, G.; Hern
andez, R. M.; Gasc
on, A. R.; Calafiore, R.; Chang, T. M. S.; De Vos, P.; Hortelano, G.; Hunkeler, D.; Lacı´ k, I.; Shapiro, A. M. J.; Pedraz, J. L. Nat. Med. 2003, 9, 104. (6) Carturan, G.; Dal Toso, R.; Boninsegna, S.; Dal Monte, R. J. Mater. Chem. 2004, 14, 2087.

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Particularly, plant cell systems have a bright future in the development of sustainable technologies. Plant cells have some intrinsic properties that make them suitable to designing biosensors7 and eco-friendly bioreactors.8-10 They can also lend themselves to decontamination applications to clean up polluted soil, water, and air. For instance, their cellular metabolism can be exploited advantageously to decompose toxic chemicals.11,12 Moreover, the extraction of heavy metals13,14 by living cells can be combined with the biosynthesis of valuable nanomaterials.15,16 The main challenge in designing such kinds of living functional materials is the preservation of cell viability inside the abiotic structures. Silica sol-gel chemistry has emerged as a biocompatible approach to entrap proteins,17-20 organelles,21 and whole (7) Nguyen-Ngoc, H.; Tran-Minh, C. Anal. Chim. Acta 2007, 583, 161. (8) Hellwig, S.; Drossard, J.; Twyman, M. R.; Fischer, R. Nat. Biotechnol. 2004, 22, 1415. (9) Roberts, S. C. Nat. Chem. Biol. 2007, 3, 387. (10) Rao, S. R.; Ravishankar, G. A. Biotechnol. Adv. 2002, 20, 101. (11) Yoon, J. M.; Olivier, D. J.; Shanks, J. V. Chemosphere 2007, 68, 1050. (12) Vila, M.; Pascal-Lorber, S.; Rathahao, E.; Debrauwer, L.; Canlet, C.; Laurent, F. Environ. Sci. Technol. 2005, 39, 663. (13) McGrath, S. P.; Lombi, E.; Gray, C. W.; Caille, N.; Dunhan, S. J.; Zhao, F. J. Environ. Pollut. 2006, 141, 115. (14) Meagher, R. B.; Heaton, A. C. P. J. Ind. Microbiol. Biotechnol. 2005, 32, 502. (15) Sharma, N. C.; Shahi, S. V.; Nath, S.; Parson, J. G.; Gardea-Torresdey, J. L.; Pal, T. Environ. Sci. Technol. 2007, 41, 5137. (16) Gardea-Torresdey, J. L.; Parsons, J. G.; Gomer, E.; Peralta-Videa, J.; Troiani, H. E.; Santiago, P.; Yacaman, M. J. Nano Lett. 2002, 2, 397. (17) Luo, T.-J. M.; Soong, R.; Lan, E.; Dunn, B.; Montemagno, C. Nat. Mater. 2005, 4, 220. (18) Besanger, T. R.; Brennan, J. D. J. Sol-Gel Sci. Technol. 2006, 40, 209. (19) Jin, W.; Brennan, J. D. Anal. Chim. Acta 2002, 461, 1. (20) Gill, I.; Ballesteros, A. TIBTECH 2000, 18, 282. (21) Meunier, C. F.; Van Cutsem, P.; Kwon, Y.-U.; Su, B.-L. J. Mater. Chem. 2009, 19, 1535.

Published on Web 02/10/2010

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cells22-26 within silica matrices. Pioneering work on prokaryotic cell encapsulation has shown that bacteria can remain alive for many weeks within a porous silica matrix.27 However, the immobilization of plant and animal cells is more delicate and usually requires a two-step encapsulation procedure in order to avoid deleterious contact of cells with sol-gel byproduct. Cells are typically immobilized into alginate beads which are subsequently coated or entrapped by adding silica precursors.6,28 Nevertheless, alginate oligomers can be recognized by plant cells as stress messengers (i.e., an elicitor).29,30 Thus, for some applications this immobilization approach is not suitable since high cellular stress can result in unproductive cells. The present work describes the recent efforts to maintain living eukaryotic plant cells within a biocompatible porous silica matrix. Carturan was the first to report the encapsulation of plant cells in silica, achieved by covering them with a porous silica layer. Even though the enzymatic activity of cells was preserved, no viability data are available.31,32 Here, we investigate the cellular response of cells which are confined within porous silica cages. Considering that the cells from mesophyll tissue grow by expansion (and not by division)33 during the last stage of leaf development, it seems entirely possible that cells can be kept alive within a restricted space. Using a biocompatible synthesis pathway, a robust hybrid silica gel can directly be formed around the plant cells without releasing any byproduct during its construction. As a consequence, the real interactions between cells and matrices can be studied. The results obtained show that the fine-tuning of the host matrix properties based upon the information gathered on cell-matrix interactions is essential when designing living and active functional materials.

Experimental Section Materials. Protease assay kit, Murashige and Skoog medium, kinetin, 1-naphthaleneacetic acid (NAA), sodium silicate solution, aminopropyltrimethoxysilane, and D-gluconolactone were purchased from Sigma-Aldrich. Amplex Red Hydrogen Peroxide assay kit was obtained from Molecular Probes Co. N-(3-Trimethoxysilylpropyl)gluconamide (GLTMS) was synthesized from D-gluconolactone and aminopropyltrimethoxysilane as reported elsewhere.34 Cell Culture. Suspension-cultured cells derived from leaves of A. thaliana strain L-MM1 ecotype Landberg erecta were cultivated in Murashige and Skoog medium (4.43 g L-1, pH 5.7) supplemented with sucrose (30 g L-1), 0.05 μg mL-1 of kinetin, and 0.5 μg mL-1 of NAA. Plant cell cultures were maintained under a 16/8 h light/dark photoperiod, at 25 C, on a rotary (22) Baca, H. K.; Carnes, E.; Singh, S.; Ashley, C.; Lopez, D.; Brinker, J. F. Acc. Chem. Res. 2007, 40, 836. (23) Avnir, D.; Coradin, T.; Lev, O.; Livage, J. J. Mater. Chem. 2006, 16, 1013. (24) B€ottcher, H.; Soltmann, U.; Mertig, M.; Pompe, W. J. Mater. Chem. 2004, 14, 2176. (25) Rooke, J. C.; Meunier, C. F.; L
eonard, A.; Su, B.-L. Pure Appl. Chem. 2008, 80, 2345. (26) Rooke, J. C.; L
eonard, A.; Su, B.-L. J. Mater. Chem. 2008, 18, 1333. (27) Fennouh, S.; Guyon, S.; Jourdat, C.; Livage, J.; Roux, C. C. R. Acad. Sci. II 1999, 2, 625. (28) Perullini, M.; Jobbagy, M.; Soler-Illia, G. J. A. A.; Bilmes, S. A. Chem. Mater. 2005, 17, 3806. (29) Akimoto, C.; Aoyagi, H.; Tanaka, H. Appl. Microbiol. Biotechnol. 1999, 52, 429. (30) Chandia, N. P.; Matsuhiro, B.; Mejias, E.; Moenne, A. J. Appl. Phycol. 2004, 16, 127. (31) Campostrini, R.; Carturan, G.; Caniato, R.; Piovan, A.; Filippini, R.; Innocenti, G.; Cappelletti, E. M. J. Sol-Gel Sci. Technol. 1996, 7, 87. (32) Carturan, G.; Dal Monte, R.; Pressi, G.; Secondin, S.; Verza, P. J. Sol-Gel Sci. Technol. 1998, 13, 273. (33) Koehler, P. G. Ann. Bot. (London, U. K.) 1973, 37, 65. (34) Haupt, M.; Knaus, S.; Rohr, T.; Gruber, H. J. Macromol. Sci., Pure Appl. Chem. 2000, A37, 323.

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shaker at 115 rpm. Cells were diluted 8-fold in fresh medium every 7 days. Plant Cell Encapsulation. An aqueous sol-gel route was used to encapsulate fragile plant cells into pure (HG0) and organically modified silica matrices (HG10, HG15, and HG20). A sodium free silica sol stock solution was prepared from a sodium silicate solution (1.5 M) as previously described.35 Hybrid silica gel (HG0) was synthesized by mixing 8 mL of silica stock with 800 μL of 10-fold concentrated biological medium. The pH value was then adjusted to 5.7 with 0.2 M KOH. Finally, the cell suspension was gently added in order to provide a final cell density of 25 g of fresh weight per liter of gel. Gelation occurred within a few minutes at 25 C. Organically modified silica matrices were obtained using the same sodium free silica stock solution combined with a GLTMS solution. This organically modified silane stock solution was obtained by mixing 4.2 g of GLTMS into 30 μL of 1 M HCl and 10 mL of bidistilled water. After 16 h, the byproduct methanol was efficiently removed by rotary evaporation (up to 95%). To study the effect of this trifunctional silane on the cellular response, different amounts of GLTMS were added into the free sodium silica sol before the addition of cells. Hybrid gels HG10, HG15, and HG20 correspond to molar percentages of 10, 15, and 20%, respectively. All the hybrid gels were aged in the Murashige and Skoog medium (replaced every week) under the same conditions as for the cell suspensions. Characterization Techniques. Matrix Properties. The morphological and textural properties of the silica-based matrices were acquired using aerogels obtained from the hybrid gels through a process of ethanol dehydration and subsequent critical point drying with liquid carbon dioxide. This procedure prevents shrinkage and preserves the structure yet removes the liquid component from the porous network. Scanning electron microscopy (SEM, JEOL JSM-7500F) observations were made on aerogels sputter-coated with gold. Nitrogen adsorption-desorption experiments were recorded at -196 C with a volumetric analyzer (Tristar 3000 from Micromeretics). Prior to analysis, the aerogels were degassed under vacuum (70 mTorr) for 24 h at 60 C. The efficiency of the incorporation of the trifunctional silane (GLTMS) into the silica structure was evaluated by 29Si MAS NMR spectroscopy. Hybrid gels (5 mL) were aged for 2 days and washed 3 times with bidistilled water (20 mL) for about 3 h under vigorous stirring. The gels were then dehydrated with ethanol and dried at 60 C. The obtained white powders were subsequently analyzed on a Bruker AVANCE 500 MHz NMR spectrometer. The kinetics of gel shrinkage was also investigated by following the dimensional changes of bulk samples (aged at 25 C) using a micrometer. The v/v data, compared to the initial volume of hybrid gel, provide the magnitude of shrinkage. It has to be noticed that all measurements were carried out by the same experimenter in order to avoid operator variability. Biological Activity and Viability of Cells. The physiological functions of entrapped cells were assessed by monitoring the oxygen consumption in a Clark cell vessel (Oxy-lab manufactured by Hansatech Instruments). Typically, 500 mg of monolithic gel pieces (3 mm3) were added to 1.0 mL of Murashige and Skoog medium (MS medium). The activity of the plant cell suspension was measured just before immobilization and was taken as the reference (100%). Cell viability was determined by gently crushing hybrid gels on the surface of a MS agar medium. The ability of encapsulated cells to grow and form so-called callus tissues (unorganized plant tissues) was used as an indicator of cell viability. Investigation of Biochemical Interactions. Transmission electron microscopy (TEM, Philips Tecnai 10) micrographs of hybrid gels were taken with an accelerating voltage of 80 kV. Prior to analysis, hybrid gels were cut into small cubes (2 mm3) and fixed with 2.5% glutaraldehyde in a sodium cacodylate buffer (0.1 M, pH 7.4) for 2.5 h. The samples were rinsed in 0.2 M cacodylate (35) Meunier, C. F.; Van Cutsem, P.; Kwon, Y.-U.; Su, B.-L. J. Mater. Chem. 2009, 19, 4131.

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buffer and postfixed in a 1% OsO4 buffered solution overnight. The gels were washed with the same buffer and then dehydrated with increasing concentrations of ethanol. The dehydrated materials were set in propylene oxide before being embedded into an epoxy resin LX112. Ultrathin sections were made with an ultramicrotome and were contrasted with lead citrate and uranyl acetate. All experiments were carried out in triplicate. Hydrogen peroxide produced by the entrapped plant cells was also measured. Aliquots of gel supernatants (200 μL) were removed regularly over a period of 24 days. The H2O2 assay was performed using the Amplex Red Hydrogen Peroxide Assay Kit according to the supplier’s instructions. The absorbance of samples and standards was measured at 570 nm with a microplate reader (Bio-Rad Instruments). Protease activity in samples was measured using a Protease Fluorescent Detection Kit. Briefly, hybrid gels (or free cells) were ground to a fine powder in liquid nitrogen. Proteins were then extracted with 0.1 M Tris-HCl (pH 7.5) at 4 C.36 The samples were centrifuged at 20000g for 5 min. The protein concentration of the recovered supernatants was measured using the Bradford method. The protease assays were carried out in 200 μL microcentrifuge tubes containing the FITC-casein substrate, a phosphate buffer (pH 7.6), and the cell extract. The tubes were gently mixed and incubated at 37 C for 24 h. The reaction was stopped by the addition of 150 μL of 0.6 N trichloroacetic acid, with a subsequent incubation at 37 C for 30 min. Precipitated proteins were pelleted by centrifugation, 15000g for 10 min. 2 μL of the supernatant was added to 200 μL of 0.5 M Tris-HCl (pH 8.5). The samples were mixed, and the fluorescence intensity was recorded at 538 nm with an excitation at 485 nm. The protease activity was determined as the change in fluorescence intensity per milligram of protein. All experiments were carried out in triplicate.

Results and Discussion Matrix Properties: Chemical Environment and Textural Features. Arabidopsis thaliana plant cells were encapsulated within different silica-based matrices. Since biological entities (organelles, whole cells) are very sensitive to modifications of their physiological environment, a biocompatible synthesis pathway was previously designed to allow the formation of a robust silica gel around fragile thylakoids, without releasing any byproduct during the synthesis.35 This method was adapted and used to encapsulate whole plant cells. A trifunctional silane bearing covalently bound neutral sugars (GLTMS) was added to the silica sol in order to study the biochemical properties of the resulting organo-modified hybrid material. The incorporation of GLTMS can tailor the chemical and physical properties of the porous silica cages that formed around the cells. 29Si MAS NMR spectra of thoroughly washed samples give information about the effective modifications of the silica walls. All the spectra of the dry gels possess peaks at -111 (Q4), -100 (Q3), and -90 ppm (Q2) assigned to SiO4, SiO3(OH), and SiO2(OH)2 units, respectively (Figure 1). These well-known resonance signals of the silica scaffold are accompanied by two additional signals in the case of hybrid gels HG10, HG15, and HG20 which contained different amounts of silyl-modified gluconamide (10, 15, and 20 mol %). The signals at -66 and -55 ppm are characteristic of the formation of Si-O-Si linkages between the trifunctional silane and the silica scaffold since T3 and T2 correspond to C-SiO3 and C-SiO2(OH), respectively. Brook and co-workers reported that trifunctional silanes bearing polyol moieties (GLTMS) present a lower reactivity compared to tetrafunctional silanes.37 As a (36) Swidzinski, J. A.; Leaver, C. J.; Sweetlove, L. J. Phytochemistry 2004, 65, 1829. (37) Chen, Y.; Zhang, Z.; Sui, X.; Brennan, J. D.; Brook, M. A. J. Mater. Chem. 2005, 15, 3132.

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Figure 1. Solid-state 29Si MAS NMR spectra of hybrid gels. Table 1. Relative Amounts of T2, T3, Q2, Q3, and Q4 Units Observed by 29Si MAS NMR of Hybrid Gelsa gels

T2 (%)

T3 (%)

Q2 (%)

Q3 (%)

Q4 (%)

HG0 8 22 70 HG10 3 6 8 20 63 HG15 3 9 8 18 62 HG20 4 11 5 25 55 a The percentages of Tn and Qn are given with a 3% error range.

consequence, the addition of GLTMS to the starting silica sol preferentially affects the surface chemistry of the resulting hybrid gel. Table 1 shows that the actual amount of T3 and T2 groups, covalently linked to the silica material, increases with the amount of GLTMS added to the silica sol. By modifying the GLTMS/ SiO2 ratio, the quantity of organic groups at the surface of the silica scaffold can be tailored. It is known that the presence of such organic moieties decreases the surface acidity of silica. Moreover, the introduction of sugar alcohols increases the water retention of the matrices by modifying their hygroscopic properties. By controlling the amount of organic groups on the surface of the matrix pores, it is thus possible to control the interactions between the cells and the material. In order to determine and understand the effect the pore structure of hybrid gels has on the viability of plant cells, SEM and nitrogen sorption experiments were carried out on aerogels obtained by treating hybrid gels under critical conditions of CO2. This method can keep the silica scaffold intact after removal of the solvent. Figure 2 shows that the texture of all the gels is formed by the aggregation of silica particles. Hybrid gels HG0, HG10, and HG15 present macropores with a large pore size distribution centered at around 150 nm (Figure 2). In the case of hybrid gel HG20, the micrograph highlights a different structure. Whereas hybrid gels HG0, HG10, and HG15 exhibit a continuous tridimensional silica network, hybrid gel HG20 is formed by the packing of individual submicrometric particles (ca. 150 nm). The voids left between these large particles create the macroporous structure. These observations reveal that different morphologies can be obtained depending on the initial GLTMS/SiO2 ratio. The addition of GLTMS seems to alter the porous properties of the matrix. For example, a molar ratio higher than 15% GLTMS (HG20) causes the collapse of the well-defined macropores observed for the pure silica gel (HG0). At low concentration, the porous structure is well preserved (HG10). This phenomenon could be explained by an increase in the solubility of the sugar-coated colloidal particles, which altered the phase Langmuir 2010, 26(9), 6568–6575

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Figure 2. Scanning electron microscopy images from hybrid gels HG0, HG10, HG15, and HG20. Table 2. Plant Cell Viability and Textural Properties of Porous Hybrid Silica Gels: Specific Surface Area (SBET), Porous Volume (Vp), and Average Pore Size Diameter (Dp) gels

SBET (m2 g-1)

HG0 HG10

990 354

HG15 HG20

159 113

Dp (nm)

Vp (cm3 g-1)

viability (days)

3.4 1.6

3 5

>50

Figure 3. Nitrogen adsorption-desorption isotherms of aerogels derived from hybrid gels.

separation process occurring during sol-gel phase transition, especially for high content of GLTMS. The adsorption-desorption data of all materials revealed type II isotherms which confirmed the macroporous nature of the hybrid gels (Figure 3). Table 2 shows that the addition of trifunctional silanes reduces the surface area, which could be explained by a possible loss of micropores. Indeed, small pores (