Cytocompatible Encapsulation of Individual Chlorella Cells within

Article pubs.acs.org/Langmuir

Cytocompatible Encapsulation of Individual Chlorella Cells within Titanium Dioxide Shells by a Designed Catalytic Peptide Sung Ho Yang,† Eun Hyea Ko,† and Insung S. Choi* Molecular-Level Interface Research Center, Department of Chemistry, KAIST, Daejeon 305-701, Korea S Supporting Information *

ABSTRACT: The individual encapsulation of living cells has a great impact on the areas of single cell-based sensors and devices as well as fundamental studies in single cell-based biology. In this work, living Chlorella cells were encapsulated individually with abiological, functionalizable TiO2, by a designed catalytic peptide that was inspired by biosilicification of diatoms in nature. The bioinspired cytocompatible reaction conditions allowed the encapsulated Chlorella cells to maintain their viability and original shapes. After formation of the TiO2 shells, the shells were postfunctionalized by using catechol chemistry. Our work suggests a bioinspired approach to the interfacing of individual living cells with abiological materials in a controlled manner.

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INTRODUCTION The individual encapsulation of living cells has recently attracted a great deal of attention, because it would contribute to the development of biosensor circuits, lab-on-a-chip systems, and bioreactors, as well as to fundamental studies in single cellbased biology.1 The coating and/or functionalization of living single-cells has been attempted on microorganisms and mammalian cells, mainly based on the layer-by-layer (LbL) self-assembly of polyelectrolytes.2 In addition, the surface of microbial cells was also decorated with nanoparticles,3 carbon nanotubes,4 and graphenes5 by the LbL method. On the other hand, individual yeast cells were encapsulated within robust inorganic shells such as silica6 and calcium phosphate7 by us and others, with LbL multilayers as a catalytic template for subsequent bioinspired mineralization processes. The inorganic calcium carbonate shell was also formed directly on yeast by using the cell wall as a nucleation site.8 The artificial shells were reported to control the cell division as well as enhancing its viability: these emerging properties had not been reported from the simple coating of cells with LbL multilayers, and therefore have potential in the generation of artificially formed, spore-like structures, i.e., “artificial spores”. Very recently, we also reported methods for postfunctionalizing the artificial shells that encapsulated a single yeast cell: the shell of polydopamine or thiol-containing silica was functionalized with ligands of interest, based on 1,4-conjugate addition and thiol-maleimide coupling reactions, respectively.9,10 Despite the development of the encapsulation methods, the materials for artificial shells have been limited to macromolecules or biogenic minerals, mainly because of the incompatibility of reaction conditions for nonbiogenic materials with chemically fragile living cells. Many inorganic materials are synthesized chemically under heterogeneous conditions, requiring high temperature and pressure as well as caustic chemicals, under which cells cannot survive.11 For example, © 2011 American Chemical Society

titanium dioxide (TiO2) has conventionally been prepared by various techniques, such as sol−gel processes, hydrothermal methods, chemical vapor deposition, ion beam-assisted processes, and electrodeposition, which are not suitable for biological applications. New interesting properties, however, would be realized with the use of nonbiogenic materials as a shell component, because the tunability of the shell in the aspect of structures and functions could be achieved more facilely. Interfacing of nonbiogenic materials with living cells requires biocompatible or biofriendly strategies, which would be intimated by recently reported, bioinspired approaches to the formation of inorganic materials, such as TiO2, Ga2O3, ZrO2, and BaTiOF4.12−18 In this paper, we report a bioinspired method for encapsulating individual living Chlorella cells with TiO2 by using a designed peptide, which is both cytocompatible and catalytically active for TiO2 formation.

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EXPERIMENTAL SECTION

Toxicity Test. The aqueous solution of various synthetic polyelectrolytes (concentration: 0.5 mg/mL) was prepared for toxicity test: poly(allylamine hydrochloride) (PAH, average Mw: ∼15 000, Aldrich), poly(diallyldimethylammonium chloride) (PDADMA, average Mw: 100 000−200 000, 20 wt % in H2O, Aldrich), poly(ethyleneimine) (PEI, average Mw: ∼750 000, 50 wt % in H2O, Aldrich), and poly(styrene sulfonate) (PSS, average Mw: ∼70000, powder, Aldrich). In addition, an aqueous solution of various peptides (concentration: 1 mg/mL) was prepared: protamine sulfate salt (protamine from salmon, grade X, Sigma), 12-mer peptide ((RKK)4, peptide sequence: RKKRKKRKKRKK), and 20-mer peptide ((RKK)4D8, peptide sequence: RKKRKKRKKRKKDDDDDDDD). We also prepared a 10-mM tris(hydroxymethyl)aminomethane Special Issue: Bioinspired Assemblies and Interfaces Received: September 19, 2011 Revised: November 29, 2011 Published: December 13, 2011 2151

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(TRIS) buffer (pH 7.4) solution of the TiO2 precursor, titanium bis(ammonium lactato)dihydroxide (TBALDH, 50 wt % in H2O, Sigma-Aldrich). The marine microalgae Chlorella sp. C-141 (sampling at coast; surface water, Jindong, Korea) cells19 were suspended in the f/2 medium and cultured in a shaking incubator at 23 °C under a halogen lamp for 72 h. The f/2 medium was purchased from the Korea Marine Microalgae Culture Center, and could be prepared with NaNO3 stock solution (1 mL), NaH 2 PO 4 ·9H 2 O stock solution (1 mL), Na2SiO3·9H2O stock solution (1 mL), trace metal solution (1 mL), vitamin solution (0.5 mL), and filtered seawater (950 mL). Each stock solution was prepared by following the previous report. A toxicity test was performed by immersing Chlorella sp. cells in each solution for 30 min. The viability information of Chlorella sp. cells was obtained by using fluorescein diacetate (FDA, Sigma). The stock solution of FDA (10 mg/mL) was prepared by dissolving FDA in acetone, because FDA was poorly soluble in water. The 2 μL of the stock solution was mixed with 1 mL of Chlorella sp. cell suspension (50-mM phosphate buffer solution, pH 6.5). The mixture was incubated for 30 min at 23 °C while shaking, and then the cells were collected by centrifugation, washed with aqueous 0.15 M NaCl solution, and characterized by confocal microscopy. TiO 2 Encapsulation and Postfunctionalization. The (RKK)4D8 (1 mg/mL) and TBALDH (10 mM) solutions were prepared in TRIS buffer (20 mM, pH 7.4), respectively. The Chlorella sp. cells were alternately immersed in the (RKK)4D8 solution and the TBALDH solution for 2 min for each step. Each cycle was repeated two times (for a total of three cycles). After LbL processes, the cells were removed and washed with aqueous 0.15 M NaCl solution. The same protocol was applied to the freshwater microalgae Chlorella vulgaris FC-16 cells and Saccharomyces cerevisiae (baker’s yeast). For the postfunctionalization, the stock solution (1 mg/mL) of pyrocatechol violet (Sigma) was prepared by dissolving it in TRIS buffer (20 mM, pH 7.4). The 10 μL of the stock solution was mixed with 1 mL of Chlorella sp. cell suspension (20-mM TRIS buffer solution, pH 7.4). After 20 min, the cells were removed and washed with aqueous 0.15 M NaCl solution. Characterizations. Scanning electron microscopy (SEM) imaging and energy-dispersive X-ray (EDX) spectroscopy elemental analysis were performed with a Sirion FEI XL FEG/SFEG microscope (FEI Co., The Netherlands) with an accelerating voltage of 15 kV after sputter-coating with platinum. The cells were observed with an LSM 510 META confocal microscope (Carl Zeiss, Germany).

positive-charge density in (RKK)4D8 was found to enhance the cytocompatibility of the (RKK)4 peptide and to reduce the cell aggregation (Figure 1a).25 The cell-viability was found to be

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Figure 1. Viability of Chlorella sp. cells in the presence of (a) (RKK)4 and (RKK)4D8 peptides (1 mg/mL) and (b) TBALDH (10 mM). Chlorella sp. cells in green were considered alive, and the other ones were considered dead. The scale bar is 20 μm.

RESULTS AND DISCUSSION With the inspiration from silaffins,12,13 silica-forming peptides found in diatoms, silica (SiO2)20−24 and titania (TiO2), have been formed under mild conditions by using several polyamines as a catalytic template: for example, poly(2-(dimethylamino)ethyl methacrylate) (PDMAEMA),15 PAH,12 PEI,16 protamines (arginine-rich nuclear proteins),17 and silaffin-related peptides12 for TiO2 formation. In particular, the arginine/lysine (R/K)rich peptides, including RKKRKKRKKRKK (denoted as (RKK)4), were identified as a bioinspired catalyst for TiO2 formation, based on the peptide library screening method.18 We selected Chlorella sp. C-141, unicellular marine green algae, as the unicellular organism for the TiO2 encapsulation, because the photocatalytic activity of TiO2, we thought, would be coupled synergistically with photosynthetic Chlorella sp. After systematic screening of cytotoxicity of various polyamines against Chlorella sp., we derivatized (RKK)4 with additional units of negatively charged aspartic acid (D): the cell-viability test showed that the positively charged (RKK)4 itself was cytotoxic against Chlorella sp., and the cells were aggregated in the presence of (RKK)4 (see the Supporting Information on the cell-viability test for five representative catalytic polymers, PDADMA, PAH, PEI, protamines, (RKK)4). The reduction of

similar to that of the native Chlorella sp. cells, while most of the cells were dead (or none was alive) and aggregated in the presence of (RKK)4. The titania precursor, TBALDH,12−18 was found to do no harm to Chlorella sp. cells (Figure 1b). After confirming the cytocompatibility of (RKK)4D8 and TBALDH, we encapsulated Chlorella sp. by the LbL technique. The LbL processes were started with (RKK)4D8, and TBALDH was subsequently deposited. In this stage, TBALDH was catalytically hydrolyzed to form titania by (RKK)4D8. The (RKK)4D8/ TBALDH deposition was repeated three times, leading to the formation of titania-encapsulated Chlorella sp. cells (Chlorella sp.@TiO2) (Scheme 1). The thickness of the TiO2 shell was estimated to be about 35 nm, based on a model experiment with carboxylic acid-terminated self-assembled monolayers on gold (see the Supporting Information). Confocal microscopy images showed that the Chlorella sp.@ TiO2 cells maintained their original shapes, and were separated individually from each other (Figure 2). We used two methods for the cell-viability test after titania formation: chlorophyll 2152

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Scheme 1. Procedure for TiO2 Encapsulation and Postfunctionalization of Chlorella Cellsa

a

The alternate LbL deposition of (RKK)4D8 and TBALDH onto the surface of Chlorella led to the bio-inspired formation of the TiO2 shell.

autofluorescence and FDA. The chlorophyll autofluorescence emission band at 650 nm has been used as a cell-viability test.26 The bright red fluorescence after TiO2 encapsulation indicated that no damage occurred to chloroplasts, the most important organelle in the Chlorella cells. FDA examines the activity of intracellular esterases and the membrane integrity, and emits green fluorescence by reactive oxygen species: Chlorella cells in green are considered alive, and the other ones are considered dead.26 The FDA test showed that the viability was about 69% after encapsulation. The decrease of viability could be explained by physical stress from centrifugation and/or chemical stress from positively charged parts of peptides. Our previous work on the silica-encapsulation also showed that the initial viability was about ∼70% after encapsulation processes,6 and we thought that the viability of above 50% would be high enough to use TiO2 as an encapsulation material. On the other hand, the FDA fluorescence implied that the TiO2 shell would be permeable to small molecules, such as water or nutrients, as inferred from the permeability of FDA. SEM micrographs further confirmed that the Chlorella cells were individually encapsulated with TiO2 (Figure 3). After 24 h of drying, Chlorella sp.@TiO2 maintained its original shape, while native Chlorella cells were noticeably shrunk because of dehydration. This mechanical hardness is one of the characteristics of the artificial shells for single-cell encapsulation. The highmagnification SEM micrograph (inset of Figure 3f) showed that the surface became rough after TiO2 shell formation. The surface was composed of TiO2 nanoparticles that had been

Figure 3. SEM micrographs of (a−c) native Chlorella sp. cells, and (d−f) Chlorella sp.@TiO2 with different magnifications after 24-h drying. Inset figures in c and f show the surface morphologies of each Chlorella sp. cell (scale bar: 100 nm).

observed in previous studies of biomimetic TiO2 formation.17,18 The successful polycondensation of TBALDH was confirmed by the elemental and line-scan analysis of EDX spectroscopy (Figure 4). In the elemental analysis profiles, the Ti peak newly appeared, and the O peak became greater, after encapsulation process. The Ti element line profile of Chlorella sp.@TiO2 also showed that polycondensation of TBALDH was successfully achieved on the cell surface. In the case of the native Chlorella cells, we did not observe a noticeable Ti peak. Taken together, the results indicated that TiO2 was formed successfully on the cell surface by (RKK)4D8. The TiO2 shells allowed for the introduction of various functional groups, without any significant disturbance of the viability of living cells, via bioinspired catechol chemistry.27 The introduction of functional groups onto the cell surface has generally involved complicated chemical and biological

Figure 2. Viability of Chlorella sp.@TiO2: (a,b) optical images, (c,d) red fluorescence images, and (e,f) green fluorescence images with different magnifications. (g) Merged image of a, c, and e. (h) Merged image of b, d, and f. The bright red fluorescence indicated that no damage occurred to chloroplasts, the most important organelle in the Chlorella cells. Chlorella sp. cells in green were considered alive, and the other ones were considered dead. 2153

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the sensor substrate by magnetic force, showing the applicability of the Chlorella cells to single cell-based sensors.29 We envisioned that the site-specific immobilization also could be chemically achievable by the catechol chemistry in our system; therefore, the direct postfunctionalization is an important step for various applications including cell-based sensors. To demonstrate the applicability of our method to the other living cells, the bioinspired encapsulation with TiO2 was also applied to Chlorella vulgaris FC-16 (freshwater Chlorella) and yeast (Saccharomyces cerevisiae; baker’s yeast). Chlorella vulgaris@TiO2 also maintained its original shape after 24 h of drying, and the morphological change of the surface confirmed the successful encapsulation of individual Chlorella vulgaris cells with TiO2 (see the Supporting Information). Yeasts can be a good candidate for single cell-based biosensors, because they exist individually and separately. In addition, they can be genetically engineered with ease for sensing applications: for example, genotoxicity was detected with genetically engineered yeast cells (GreenScreen genotoxicity assay).30 In this respect, yeast cells have been encapsulated within various materials, such as silica,6,10 polydopamine,9 calcium phosphate,7 calcium carbonate,8 and graphene,5 in addition to the coating approaches.2 Yeast was also alternately immerged in the (RKK)4D8 and TBALDH solutions (3-by-3 layers). However, yeast cells were highly aggregated and dead after TiO2 formation (see the Supporting Information). The cytotoxicity test indicated that TBALDH was toxic for yeast cells, while (RKK)4D8 was not. These results implied that the cytotoxicity of compounds depended critically upon the identity of cells. Although the yeast cells tolerated the positively charged polymers,6,9 they were labile to a small compound, TBALDH. In other words, the sophisticated combination of (RKK)4D8, TBALDH, and Chlorella cells led to the successful cytocompatible formation of Chlorella@TiO2.

Figure 4. EDX spectroscopy elemental analysis and Ti line profile of (a) native Chlorella sp. and (b) Chlorella sp.@TiO2.

processes, and the reported methods were limited because the chemical treatment was usually harmful to cells.25,28 However, the catechol group would easily self-assemble on the TiO2 surface under mild conditions. As a demonstration of the direct postfunctionalization, pyrocatechol violet was introduced to Chlorella sp.@TiO2. Native Chlorella cells were used as a control. After 20-min incubation, only Chlorella sp.@TiO2 showed the color change from green to violet, while native Chlorella cells did not (Figure 5). The color change indicated

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CONCLUSIONS In summary, living marine and freshwater Chlorella cells (Chlorella sp. C-141 and Chlorella vulgaris FC-16) were individually encapsulated with the nonbiogenic TiO2 shells by using a designed catalytic peptide, (RKK)4D8. In addition, the TiO2 shell was further functionalized via bioinspired catechol chemistry. This report is the first example of single-cell encapsulation with nonbiogenic inorganics, inspired by biomimetic mineralization, and could be applied to the synergistic combination of abiological materials and biological entities. Among other properties, it is durability against harsh environments, controllability in cell cycles, and reactivity for cell-surface modification that particularly characterize the artificial hard shells encapsulating single living cells, toward artificially formed, spore-like structures, “artificial spores”.9 The detailed investigation on these properties of Chlorella@TiO2, where biological Chlorella cells are interfaced with abiological TiO2, is our next research thrust.

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ASSOCIATED CONTENT

S Supporting Information * Additional information is available, including the viability of Chlorella sp. cells with several cationic polyamines, a graph of the average thickness of the TiO2 film versus the number of depositions, SEM micrographs of Chlorella vulgaris@TiO2, and SEM, EDX, and viability data of yeast@TiO2. This material is available free of charge via the Internet at http://pubs.acs.org.

Figure 5. Optical images of native Chlorella sp. cells and Chlorella sp.@ TiO2 (a) before and (b) after postfunctionalization of TiO2 shells with pyrocatechol violet.

the functionalizability of the TiO2 shells with the catechol moiety. There was a recent report that magnetically functionalized Chlorella cells were easily immobilized onto 2154

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(26) Nancharaiah, Y. V.; Rajadurai, M.; Venugopalan, V. P. Environ. Sci. Technol. 2007, 41, 2617. (27) Ye, Q.; Zhou, F.; Liu, W. Chem. Soc. Rev. 2011, 40, 4244. (28) Saxon, E.; Bertozzi, C. R. Science 2000, 287, 2007. (29) Zamaleeva, A. I.; Sharipova, I. R.; Shamagsumova, R. V.; Ivanov, A. N.; Evtugyn, G. A.; Ishmuchametova, D. G.; Fakhrullin, R. F. Anal. Methods 2011, 3, 509. (30) Cahill, P. A.; Knight, A. W.; Billinton, N.; Barker, M. G.; Walsh, L.; Keenan, P. O.; Williams, C. V.; Tweats, D. J.; Walmsley, R. M. Mutagenesis 2004, 19, 105.

AUTHOR INFORMATION Corresponding Author *Address: Molecular-Level Interface Research Center, Department of Chemistry, KAIST, Daejeon 305-701 (Korea). Fax: (+82) 42-350-2810. E-mail: [email protected]. Author Contributions † These authors contributed equally to this work.

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ACKNOWLEDGMENTS This work was supported by the Korea Research Foundation Grant funded by the Korean Government (MOEHRD, KRF2008-313-C00496) and the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2011-0001318). We thank M. S. Hyun and M. H. Kim at the National Nanofab Center for the SEM analyses.

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