pubs.acs.org/Langmuir © 2009 American Chemical Society
Hybrid Cellular-Inorganic Core-Shell Microparticles: Encapsulation of Individual Living Cells in Calcium Carbonate Microshells Rawil F. Fakhrullin* and Renata T. Minullina Department of Biochemistry, Kazan State University, Kreml uramı 18, Kazan, 420008 Tatarstan, Russian Federation Received April 1, 2009. Revised Manuscript Received May 4, 2009 We report the fabrication of hybrid cellular-inorganic core-shell microparticles obtained by encapsulation of individual living yeast cells Saccharomyces cerevisiae in calcium carbonate microshells and demonstrate the viability of the encapsulated cells. Our method is based on the direct precipitation of calcium carbonate on the cell walls of yeast cells. Resulting hybrid microparticles consist of single yeast cells coated with semipermeable inorganic microshells, which resemble the original ellipsoid shapes of yeast cells, exhibit negative zeta-potential, and have micrometer-thick calcium carbonate walls. The combination of the functional properties of living cells and calcium carbonate microshells promises a wide area of applications of these hybrid core-shell microparticles in the development of novel materials.
Nature provides several species of unicellular microorganisms, such as foraminifera1 and coccoliths,2 with protective shells built from calcium carbonate in various crystalline forms. These shells are helpful for survival of these miniature creatures in harsh environmental conditions. Acting as a barrier, natural inorganic microshells protect microorganisms from predators while allowing them spatial movements and the uptake of nutrients. It is not clear yet how this process has been evolutionary developed; therefore, the influence of proteins,3,4 vitamins,5 and living cells6 on the growth of CaCO3 amorphous and crystalline microparticles has been extensively studied. In addition, the attention toward CaCO3 microparticles is stimulated by their use in materials science as versatile templates and sorbents, basically due to their nontoxicity, stability, and biocompatibility. Recently, CaCO3 microparticles of various morphologies have been utilized as core-particles in the fabrication of polyelectrolyte microcapsules7,8 and protein encapsulation9 and as threedimensional templates for the fabrication of living multicellular assemblies.10 The special interest was given to the development of hybrid CaCO3 microparticles which combine inorganic cores and organic functional moieties11 and to the fabrication of core-shell12 and hollow microparticles.5,13 *Corresponding author. Telephone: +78432315246. E-mail:
[email protected]. (1) Langer, M. R.; Silk, M. T.; Lipps, J. H. J. Foraminiferal Res. 1997, 27, 271–277. (2) Henderiks, J. Mar. Micropaleontol. 2008, 67, 143–154. (3) Sondi, I.; Salopek-Sondi, B. Langmuir 2005, 21, 8876–8882. (4) Voinescu, A. E.; Touraud, D.; Lecker, A.; Pfitzner, A.; Kunz, W.; Ninham, B. W. Langmuir 2007, 23, 12269–12274. (5) Butler, M. F.; Frith, W. J.; Rawlins, C.; Weaver, A. C.; Heppenstall-Butler, M. Cryst. Growth Des. 2009, 9, 534–545. (6) Lian, B.; Hu, Q.; Chen, J.; Ji, J.; Tengon, H. H. Geochim. Cosmochim. Acta 2006, 70, 5522–5535. (7) Holt, B.; Lam, R.; Meldrum, F. C.; Stoyanov, S. D.; Paunov, V. N. Soft Matter. 2007, 3, 188–190. (8) Andreeva, D. V.; Gorin, D. A.; Mohwald, H.; Sukhorukov, G. B. Langmuir 2007, 23, 9031–9036. (9) Volodkin, D. V.; Larionova, N. I.; Sukhorukov, G. B. Biomacromolecules 2004, 5, 1962–1972. (10) Fakhrullin, R. F.; Paunov, V. N. Chem. Commun. 2009, 2511–2513. (11) Li, X.; Hu, Q.; Yue, L.; Shen, J. Chem. Eur. J. 2006, 12, 5770–5778. (12) Thomas, J. A.; Seton, L.; Davey, R. J.; De Wolf, C. E. Chem. Commun. 2002, 1072–1073. (13) Hirai, T.; Hariguchi, S.; Komasawa, I. Langmuir 1997, 13, 6650–6653.
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In this Letter, we report the development of hybrid cellularinorganic core-shell microparticles by the encapsulation of individual living yeast cells into the CaCO3 microshells. We demonstrate that the resulting hybrid microparticles consist of single living cells being core-particles coated with a uniform layer of crystalline calcium carbonate. Our method utilizes the use of living yeast cells which are introduced into supersaturated solutions of Ca2+ and CO32- ions and facilitate the formation of microshells templated over the individual cells instead of the growth of calcium carbonate microparticles which typically occurs in absence of cells.8 We found that the yeast cells encapsulated in the CaCO3 microshells remain viable even after several weeks of storage, which proves the noninvasive way of encapsulation, and therefore, our method can be regarded as an artificial approach mimicking biomineralization processes in unicellular microorganisms.1,2 We suppose that these hybrid core-shell microparticles are potentially interesting as novel templates for polyelectrolyte deposition, microreactors, and drug carriers. Here, we describe our approach in more detail. We followed the previously published method to produce spherical CaCO3 microparticles8 and used baker’s yeast cells (Saccharomyces cerevisiae) as living templates. Prior to encapsulation, yeast cells (at wet weight concentration 100 mg mL-1) were resuspended in 0.33 M aqueous CaCl2 and incubated at room temperature for 20 min while stirring. Next, an equal volume of 0.33 M aqueous Na2CO3 was added into the beaker containing the suspension of yeast cells in aqueous CaCl2, and the mixture was stirred vigorously during 5 min. As a result, the immediate formation of a white precipitate was observed. The precipitate was collected, filtered, and washed extensively with water in order to remove the excess of Na2CO3 and CaCl2 and thus prevent recrystallization, which occurs if the aging of the precipitate is allowed for more than 2 h (optical and scanning electron microscopy images of the recrystallized particles are given in Figure S1 in the Supporting Information). The precipitate was then dried at room temperature overnight and stored at +4 °C. We examined bare yeast cells and microparticles obtained using bright field and polarized microscopy. It is well-known that yeasts are fungi cells which have a thick cell wall preserving an
Published on Web 05/12/2009
DOI: 10.1021/la901395z
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almost ellipsoid shape of the cells. Figure 1a demonstrates that the yeast cells have sizes from 4 to 7 μm. The corresponding polarized microscopy image of bare yeast cells (Figure 1b) shows no birefringence from optically isotropic yeasts, indicating that the native yeast surfaces are free from artificial anisotropic shells. Next, we examined the microparticles obtained by the precipitation of CaCO3 in presence of yeast cells. As shown in Figure 1c and d, we observed the ellipsoidal and spherical microparticles with shapes and sizes similar to those of yeast cells (in addition, several merged particles of complex rhombohedral morphologies, apparently consisting of a number of aggregated microparticles were found; data not shown). Unlike the native yeast cells, the microparticles obtained are birefringent if observed in polarized light, which is characteristic of CaCO3 microcrystals produced using the same method but in the absence of yeast cells (Figure S2, Supporting Information). However, if these microparticles are treated with 0.1 M HCl for 30 min, which leads to the complete decomposition of CaCO3, we could observe only bare cells and no birefringent microparticles were detected (Figure S3, Supporting Information). Furthermore, the crystalline structure of the CaCO3 microparticles obtained was analyzed by X-ray powder diffraction (XRD) technique, and the XRD pattern is demonstrated in Figure 1e. The XRD spectrum is characteristic to vaterite, the unstable crystalline CaCO3 polymorph, although some peaks from the more stable calcite polymorph can be seen as well.4,14-17 We suppose that the spherical microparticles are built from vaterite which normally forms spherical microcrystals,14 whereas calcite is apparently formed as a result of the subsequent vaterite recrystallization (Figure S1, Supporting Information), or, perhaps, rhombohedral calcite microparticles are produced along with spherical CaCO3 microparticles.14 Next, we found that the zeta-potential of CaCO3 microshells dispersed in water (-27 mV) is similar to that of the aqueous dispersions of spherical CaCO3 microparticles obtained following the same technique in absence of yeast cells (-28 mV), whereas the zeta-potential of native yeast cells in water is around -12 mV. The apparent absence of free yeast cells in the CaCO3 precipitate along with the presence of ellipsoid CaCO3 microparticles suggests that the cells might be encapsulated by the inorganic microshells. If the cells are inside the CaCO3 microparticles and maintain their viability, the enzymatic activity can be reliable evidence of successful encapsulation. Therefore, in order to demonstrate the encapsulation and to check the viability of the yeast cells, we incubated the suspensions of bare yeast cells, the CaCO3 microparticles synthesized in the absence of yeast cells, and the CaCO3 microparticles obtained in the presence of yeast cells as described above and supposedly encapsulating yeast cells in 0.001% aqueous fluorescein diacetate (FDA) at wet weight concentration 10 mg mL-1 during 30 min at room temperature, washed thrice with water and investigated with a fluorescent microscope. It is known that intact nonfluorescent FDA passes through cellular membranes of living cells and undergoes hydrolysis by intracellular esterases, yielding the formation of the fluorescent substance, fluorescein.18 Oppositely, if the CaCO3 (14) Wang, L.; Sondi, I.; Matijevic, E. J. Colloid Interface Sci. 1999, 218, 545–553. (15) Nassrallah-Aboukais, N.; Boughriet, A.; Laureyns, J.; Aboukais, A.; Fischer, J. C.; Langelin, H. R.; Wartel, M. Chem. Mater. 1998, 10, 238–243. (16) Rautaray, D.; Ahmad, A.; Sastry, M. J. Am. Chem. Soc. 2003, 125, 14656–14657. (17) Rautaray, D.; Ahmad, A.; Sastry, M. J . Mater. Chem. 2004, 14, 2333–2340. (18) Bell, R. S.; Bourret, L. A.; Bell, D. F.; Gebhardt, M. C.; Rosenberg, A.; Berrey, H. B.; Treadwell, B. V.; Tomford, W. W.; Mankin, H. J. J. Orthop. Res. 1988, 6, 467–474.
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Figure 1. (a) Bright field optical microscopy image of the yeast cells; (b) corresponding polarized microscopy image of the yeast cells; (c) bright field optical microscopy image of the CaCO3 microshells templated on yeast cells; (d) corresponding polarized microscopy image of the same sample; and (e) X-ray diffraction pattern of CaCO3 microshells templated on yeast cells.
microparticles do not encapsulate the cells or the encapsulated cells are not viable, no fluorescent product is produced. In Figure 2, where the representative bright field, polarized, and fluorescent images of cells and CaCO3 microparticles treated with FDA are given, we demonstrate that the cells are indeed encapsulated in CaCO3 microshells and viable. As one can see in Figure 2a-c, living yeast cells treated with FDA are not birefringent and the green fluorescence indicates the viability of the cells. As expected, cell-free CaCO3 microparticles (Figure 2d-f) are birefringent and no fluorescence is observed because the initially nonfluorescent FDA was not hydrolyzed as in case of the native yeast cells. In contrast, the CaCO3 microparticles obtained by the precipitation of calcium carbonate in presence of native yeast cells (Figure 2g-i) are both fluorescent and birefringent, thus indicating that the individual living cells are encapsulated in the inorganic CaCO3 microshells. Indeed, as it can be concluded from the previous images of the native cells and the cell-free CaCO3 microparticles, only the combination of both may produce such results. This data clearly confirms that the inorganic microparticles exhibit esterase-mediated fluorescence, which proves the development of hybrid microparticles with the living cells encapsulated in the CaCO3 microshells. Noteworthy, the fluorescence intensity of encapsulated cells is relatively weaker than of the native yeast cells, which might be explained by the influence of vaterite shells. Langmuir 2009, 25(12), 6617–6621
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Figure 2. Optical microscopy images (bright field, polarized, and fluorescent from left to right in each row) of (a-c) native yeast cells; (d-f) cell-free CaCO3 spherical microparticles; and (g-i) CaCO3 microshells encapsulating living yeast cells. All samples were treated with 0.001% aqueous FDA during 30 min at room temperature. (j-l) Bright field, polarized, and fluorescent optical microscopy images (left to right) of free yeast cells released from CaCO3 microshells treated with FDA as described above after incubation in 0.1 M EDTA during 1 h.
Furthermore, after demonstration of the enzymatic activity of the encapsulated cells inside the CaCO3 microshells, we tested the viability of the originally encapsulated yeast cells which were released from the CaCO3 microshells after 2 months of storage at room temperature. We realized that the decomposition of the CaCO3 using HCl may kill the encapsulated cells; therefore, we used ethylenediaminetetraacetic acid disodium salt (EDTA), which can be used to dissolve CaCO3 microcrystals and does not affect the viability of yeast cells.10 After the treatment of the resulting suspension of yeast cells with FDA (as described above), we incubated the hybrid cellular-calcium carbonate microparticles in aqueous 0.1 M EDTA for 1 h and examined them using fluorescence microscopy. Figure 2j-l shows the bright field, polarized, and fluorescent optical microscopy images of the released cells, which are not birefringent and express FDAmediated fluorescence approximately at the same intensity as the native cells, thus suggesting that even after the release from the inorganic microshells the viability of the cells is not jeopardized (the same effect was observed if the hybrid cellular-CaCO3 microparticles were first released from the inorganic microshells by EDTA and then stained with FDA; data not shown). The FDA test demonstrates that the encapsulation itself does not affect the cellular membranes and the enzymatic activity. This also indicates that the inorganic walls are permeable for such substances as FDA, which diffuses through the nanosized pores of CaCO3 microshells which are known to allow the diffusion of relatively large proteins.8 Moreover, budding of the released cells was observed after incubation in 0.1% aqueous sucrose for 12 h (Figure S4, Supporting Information), further indicating that the encapsulated yeast cells maintain vitality along with viability. Langmuir 2009, 25(12), 6617–6621
We counted the percentage of encapsulated and nonencapsulated yeast cells in order to investigate the yield of the fabrication of hybrid microparticles. To do this, we stained the microparticles with aqueous 0.01 mg mL-1 ethidium bromide (EB) solution, which is known to stain yeast cells.19 We noticed that EB effectively stained only bare yeast cells while the cells encapsulated in CaCO3 microshells were stained considerably weaker, apparently due to reduction of fluorescence intensity caused by the relatively thick inorganic shells (the same effect was observed in case of FDA staining). It is shown in Figure 3a that a bare yeast cell (indicated by an arrow) is almost undistinguishable from the hybrid cellular-inorganic microparticles if observed in bright field, but it can be easily determined under crossed-polarizers because of no birefringence, whereas all hybrid cellularinorganic microparticles are optically anisotropic (Figure 3b). As shown in Figure 3c, the same cell stained with EB exhibits intensive fluorescence. The typical image of the distribution of bare and encapsulated cells stained with EB is given in Figure 3d. We found that ∼25% of the cells were free in the samples examined, whereas the majority of the particles were the hybrid cell-templated microshells and the total yield of the encapsulation was ∼75%. To further characterize the hybrid microparticles obtained, we applied scanning electron microscopy (SEM). SEM images of CaCO3 microshells encapsulating living yeast cells are presented in Figure 4. As shown in Figure 4a, the microparticles are relatively monodisperse and well-separated, although several aggregated particles are present. The magnified SEM image of (19) Corliss, D. A.; White, W. E. J. Histochem. Cytochem. 1981, 29, 45–48.
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Figure 3. Hybrid cellular-inorganic microparticles and free yeast cells stained with EB: (a) bright field microscopy; (b) polarized microscopy; and (c) fluorescence microscopy. The arrow indicates the bare cell. (d) Typical image of the distribution of bare and CaCO3 encapsulated cells stained with EB. Table 1. Size Distribution of Bare Yeast Cells and Microshells object
length (μm)
width (μm)
length/width ratio
yeast cells hybrid microcapsules
5.8 ( 0.7 7.4 ( 1.4
4.8 ( 0.8 6.3 ( 1.2
1.21 1.17
microshells templated on yeast cells; (c) a hollow microcapsule as produced after the spontaneous removal of the yeast cell; and (d) a single yeast cell apparently partially encapsulated in a CaCO3 microshell.
removed, probably due to a mechanical impact. Interestingly, as it is shown in Table 1, the length/width ratio in bare yeast cells and CaCO3 encapsulated yeast cells is nearly the same, thus confirming that the microshells retain the original ellipsoid shapes of yeast cells. A peculiar image of an individual yeast cell which seems to be only partially encapsulated by a microshell is given in Figure 4d; such semiencapsulated cells were frequently found in the samples observed, and we suppose that these are the free cells which were detected using EB staining. It is clear that further experiments are needed to find out the exact mechanisms of the formation of such unusual structures. This work is currently being performed by our group and will be reported in a follow-up paper. So far, we believe that (i) living cells act as templates for crystal growth, (ii) apparently positive Ca2+ is accumulated on the slightly negative cell walls, and (iii) upon introduction of CO32- the following nucleation and growth of CaCO3 microshells occurs on these preadsorbed sites. Therefore, the cells are completely coated with a thick inorganic layer, which is still permeable for nutrients and low-weight organic molecules, thus providing cellular viability. A similar mechanism was proposed by Morita20 to explain the calcite precipitation by marine bacteria in anaerobic conditions. The proposed mechanism involves the metabolic CO32- ions along with the external CO32- ions. The presence of the remaining bare cells along with the completely encapsulated cells can be explained by the spontaneous recrystallization which causes the release of the cells from the microshells. We suppose that the formation of such hybrid core-shell microparticles is facilitated not only by yeast cells but by other microorganisms as well. Some previous reports describe the formation of biogenic CaCO3 in the presence of various microorganisms; however, most likely due to the long incubation times (generally exceeding several days), the authors of these reports observed only relatively large microcrystals presumably produced in the presence of cells and no individual encapsulated cells were found.6,16,17,20-25 Recently, large populations of human cells were encapsulated in mineralized polysaccharide millimeter-sized capsules in order to produce multifunctional scaffolds and delivery vehicles in tissue engineering.26 The current paper shows that at the early stages
a typical microshell (Figure 4b) shows the ellipsoid shape which is characteristic of yeast cells. Further, we measured the length and width distribution of the microshells obtained, and the results are given in Table 1. It can be concluded that the mean length and width of yeast cells encapsulated in CaCO3 microshells are approximately 1.5 μm larger than the corresponding values of bare cells, which suggests that the thickness of the shells must be around 1.5 μm. This suggestion corresponds well with the SEM images of hollow and partially open microshells (Figure 4c), where the core cell is
(20) Morita, R. Y. Geomicrobiol. J. 1980, 2, 63–82. (21) Novitsky, J. A. Geomicrobiol. J. 1981, 2, 375–388. (22) Ferrer, M. R.; Quevedo-Sarmiento, J.; Bejar, V.; Delgado, R.; Ramos-Cormenzana, A.; Rivadeneyra, M. A. Geomicrobiol. J. 1988, 6, 49–57. (23) Ferrer, M. R.; Quevedo-Sarmiento, J.; Rivadeneyra, M. A.; Bejar, V.; Delgado, R.; Ramos-Cormenzana, A. Curr. Microbiol. 1988, 17, 221–227. (24) Chen, L.; Shen, Y.; Xie, A.; Huang, B.; Jia, R.; Guo, R.; Tang, W. Cryst. Growth Des. 2009, 9, 743–754. (25) Rautaray, D.; Sanyal, A.; Adyanthaya, S. D.; Ahmad, A.; Sastry, M. Langmuir 2004, 20, 6827–6833. (26) Green, W. D.; Leveque, I.; Walsh, D.; Howard, D.; Yang, X.; Patridge, K.; Mann, S.; Oreffo, R. O. C. Adv. Funct. Mater. 2007, 19, 2236–2240.
Figure 4. Scanning electron microscopy images of (a,b) CaCO3
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of CaCO3 precipitation individual cells act as nucleation sites and are completely and uniformly coated with the CaCO3 micrometer-thick layer. Such hybrid microparticles can be easily fabricated and, as long as they combine the properties of CaCO3 and living cells, can be used in a number of practical applications. To conclude, we have fabricated hybrid cellular-inorganic core-shell microparticles by encapsulating individual living yeast cells in CaCO3 microshells and demonstrated the viability of the cells embedded inside the microshells. The developed hybrid microparticles may find potential application in materials science. Acknowledgment. We thank Dr. V. N. Paunov (University of Hull, England) and Prof. Dr. F. S-ahin (Yeditepe University,
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Istanbul, Turkey) for their generous support. The work was partially supported by the Government of Tatarstan (AlgarısGrantlar Programması). Supporting Information Available: Optical microscopy and SEM images of CaCO3 microcrystals after recrystallization; optical microscopy images of CaCO3 microparticles; optical microscopy images of CaCO3 microshells encapsulating yeast cells before and after treatment with HCl; and optical microscopy image of the budding yeast cells released from the calcium carbonate microcapsules by dissolving the inorganic microshells with EDTA. This material is available free of charge via the Internet at http://pubs.acs.org.
DOI: 10.1021/la901395z
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