pubs.acs.org/Langmuir © 2010 American Chemical Society
Autonomous Silica Encapsulation and Sustained Release of Anticancer Protein Ken-Ichi Sano,† Tamiko Minamisawa, and Kiyotaka Shiba* Division of Protein Engineering, Cancer Institute, Japanese Foundation for Cancer Research, Koto, Tokyo 1358550, Japan. †Present address: Molecular and System Life Science Unit, RIKEN Advances Science Institute, Wako, Saitama 351-0198, Japan. Received November 30, 2009. Revised Manuscript Received January 8, 2010 We present a novel method for preparing a silica carrier for the sustained release of a proteinaceous pharmaceutical. This method makes use of the silicification activity of the protein itself, which autonomously formed a protein-silica composite upon simple incubation with a silica precursor. The composite was dissolved, and the encapsulated protein was released into a culture medium, thereby sustaining the protein’s activity for a long period of time.
Introduction Targeted delivery of anticancer agents to tumors and their controlled release are prerequisites for the development of the next generation of anticancer drug delivery systems (DDSs).1 Such delivery systems must be compatible not only with low molecular weight anticancer drugs but also with proteinaceous biologics, including antibodies, cytokines, and rationally engineered proteins. Because of their excellent biocompatibility, regular sol-gel silica xerogel nanoparticles have been attracting attention as potential carriers for anticancer agents.2,3 Because these nanoparticles are prepared using the method that employs harsh reaction conditions,4 proteins should be loaded onto the particles after the synthesis, requiring a two-step preparation. Therefore, a facile one-step synthesis method for preparing the protein loaded silica particles under milder conditions must be developed. Number 284 is a 24.2 kDa artificial protein with the ability to penetrate cell membranes and then induce apoptosis.5 This protein was synthesized using the MolCraft system by combinatorially assembling PTD and BH3 motifs (Figure 1a), which are, respectively, associated with protein transduction and induction of apoptosis.6 Subsequent characterizations using 39 cancer cell lines showed that #284 preferentially inhibits the growth of several cancer cell types with a GI50 of approximately 5 μM (which is within the range of conventional anticancer drugs), indicating its potential utility as a novel agent for cancer treatment.7 Here, we present a novel method in which the mineralization activity of a protein is utilized to encapsulate it within silica *To whom correspondence should be addressed. Address: Department of Protein Engineering, Cancer Institute, Japanese Foundation for Cancer Research, 3-10-6 Ariake, Koto, Tokyo 135-8550, Japan. Telephone þ81-33570-0489. Fax: þ81-3-3570-0461. E-mail:
[email protected].
(1) Brigger, I.; Dubernet, C.; Couvreur, P. Adv. Drug. Delivery Rev. 2002, 54, 631–651. (2) Rosenholm, J. M.; Meinander, A.; Peuhu, E.; Niemi, R.; Eriksson, J. E.; Sahlgren, C.; Linden, M. ACS Nano 2009, 3, 197–206. (3) Avnir, D.; Coradin, T.; Lev, O.; Livage, J. J. Mater. Chem. 2006, 16, 1013– 1030. (4) Gill, I.; Ballesteros, A. Trends Biotechnol. 2000, 18, 282–296. (5) Saito, H.; Honma, T.; Minamisawa, T.; Yamazaki, K.; Noda, T.; Yamori, T.; Shiba, K. Chem. Biol. 2004, 11, 765–773. (6) Shiba, K. J. Mol. Catal. B: Enzym. 2004, 28, 145–153. (7) Saito, H.; Minamisawa, T.; Yamori, T.; Shiba, K. Cancer Sci. 2008, 99, 398– 406.
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particles. In living systems, it has been revealed that mineralization reactions proceed by the help of biomacromolecules including proteins.8 Natural proteins with the capacity for silicification are often enriched with cationic residues, and it has been shown that synthetic cationic peptides can increase the rate of silicification.8 Because both of the embedded motifs (PTD = YGRKKRRQRRR and BH3 = LRRFGDKLN) contain a number of cationic amino acids, #284 was also enriched with these cationic residues, and its pI was calculated to be 12.16.5 We therefore predicted that #284 would have a capacity for silicification that could enable it to autonomously form silica particles, within which a protein could be encapsulated. The encapsulated proteinous pharmaceutics would be expected to show sustained release. Furthermore, the encapsulation of protein can be expected to protect the protein from denaturation and degradation in physical environment.
Experimental Section Preparation of #284@silica. Expression and purification of protein #284 was carried out as described previously.9 The concentration of the purified #284 was determined by absorbance at 280 nm using an extinction coefficient calculated from the protein sequence. For silica encapsulation, 2 mg/mL #284 was incubated with 10% (volume) 1 M prehydrolyzed tetramethoxysilane (TMOS) in phosphate-buffered saline (PBS) for 20 min at room temperature, after which the #284-silica composite (#284@silica) was collected by centrifugation (1200g, 10 min). The prehydrolysis was done by incubating 1 M TMOS in 1 mM HCl for 5 min at 25 °C. The precipitate was washed twice in deionized water and lyophilized. Analysis of #284@silica. Scanning electron micrographs of #284@silica were obtained using a Keyence VE-8800 instrument. Thermogravimetric analysis was carried out using a Shimadzu TGA50 thermogravimetric analyzer. The temperature was increased from room temperature to 1000 °C at a rate of 2 °C/min under a flow of air (30 cm3/min). Cell Proliferation Assay. MCF7 human breast cancer cells were maintained in RPMI 1640 medium (Gibco) supplemented with 10% fetal bovine serum and an antibiotic/antimycotic solution (Sigma) at 37 °C under a humidified atmosphere of 5% CO2 and 95% air. (8) Kr€oger, N.; Deutzmann, R.; Sumper, M. Science 1999, 286, 1129–1132. (9) Shiba, K.; Takahashi, Y.; Noda, T. Proc. Natl. Acad. Sci. U.S.A. 1997, 94, 3805–3810.
Published on Web 01/19/2010
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Figure 2. Delayed release of #284 from the #284@silica composite in PBS. The arrow indicates the released #284 analyzed by SDS polyacrylamide gel electrophoresis.
Figure 1. (a) Primary structure of #284. PTD and BH3 motifs are shown in blue and red, respectively. (b) Scanning electron micrograph of the silica precipitate (#284@silica) formed. (c) Thermogravimetric analysis of #284@silica. For the WST-1 (4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H5-tetra-zolio]-1,3-benzene disulfonate) (Roche) assay, MCF-7 cells were plated to a density of 5 104 cells/well in a 24-well plate. After incubation for 22 h, the medium was replaced with 600 μL of RPMI 1640 supplemented with insulin-transferrinsodium selenite (ITS) medium (Sigma). In addition, #284, #284@silica, or hydroxylated silica dispersed in PBS were added to the MCF-7 cells via ThinCert cell culture inserts (pore size, 1 μm; Greiner Bio-one). Number 284 and #284@silica were added so that the final concentration of #284 would be 5 μM, and hydroxylated silica was added to the same weight as #284@silica. The cells were then incubated for 0, 2, 4, 6, 8, 10, or 12 h and assayed using a WST-1 kit. In addition, we used propidium iodide staining to confirm the identity of dead cells. To localize #284 within cells (Figure 3) by fluorescence microscope, we first synthesized fluorescein-conjugated #284 (F-#284) as previously described.5 Next, F-#284@silica was prepared under the same conditions as #284@silica. After incubation for 3 or 8 h, cells were washed with PBS containing 1 mM Mg2þ and 1 mM Ca2þ and then stained with propidium iodide for 15 min at 37 °C.
Results and Discussion To investigate the silicification activity of #284, we incubated the protein (2 mg/mL) with 100 mM prehydrolyzed TMOS in PBS at ambient temperature. Under these conditions without the protein, no precipitation of silica was seen, even after incubation for several hours (data not shown). By contrast, in the presence of #284, a silica precipitate formed within 1 min. Moreover, scanning electron microscopy revealed that the precipitate consisted of spherical particles with diameters of several hundred nanometers (Figure 1b). To estimate the #284 content of the precipitate, we first freeze-dried the sample to remove unbound water and then performed thermogravimetric analysis, during which the weight loss that accompanied increases in temperature was monitored as shown in Figure 1c. By the time the temperature reached 200 °C, approximately 9% of the original weight was lost. This initial loss at this relatively low temperature should represent vaporization of bound water. With further increases in temperature, the total loss of weight reached to 46%. We also determined the weight loss of 2232 DOI: 10.1021/la9045226
precipitated silica without additives to be approximately 15% (data not shown). From these data, we concluded that the protein content of the composite (which we termed #284@silica) was approximately 30% by weight. Because silica is slowly hydrolyzed in aqueous solution (the dissociation constant of biosilica is reportedly 2-4 mM),10 we expected sustained release of the encapsulated #284 from #284@ silica. To confirm this, we placed #284@silica containing 46.3 μg of #284 in 100 μL of PBS and tested for the presence of #284 in the supernatant (Figure 2). Under these conditions, no #284 was observed in the supernatant after incubation for 2 h. After 3 h, however, the protein was observed, indicating its delayed release. We then extended this release assay by assessing the activity of #284 released from silica. Because #284 is strongly apoptotic,4 its release could be monitored based on the loss of viability of cells in culture. For this purpose, we grew MCF7 cells (a human breast cancer line) in a culture plate, after which #284@silica was added so that the final #284 concentration would be 5 μM. To avoid direct contact between the #284@silica and the cells (which could cause unexpected physiological effects), we placed the #284@ silica in an insert having a porous (1 μm pore size) membrane bottom. For comparison, we also cultured cells with free #284 or with silica precipitate devoid of the protein (Supporting Information). As shown in Figure 3a, when incubated with free #284, the viability of MCF7 cells rapidly declined, and more than 80% of cells were killed within 4 h. By contrast, when #284 was entrapped within silica, there was no decline in cell viability during the first 6 h of incubation. Subsequently, however, cell viability gradually declined until 40% of the cells were killed after 12 h of incubation. Under the conditions used, silica itself had no harmful effect on cell viability. In the experiments summarized above, we assessed cell viability indirectly by measuring mitochondrial dehydrogenase activity using a WST-1 assay system (Figure 3a). To confirm that the cells were in fact dead, we also identified dead cells using propidium iodide (PI) staining. As shown in Figure S1 in the Supporting Information, most cells treated with free #284 for 2 h were stained by PI. However, after incubation for 2 h with #284@silica, the cells were not stained with PI, and only a portion of the cells were stained after 8 h, which confirmed the results of the WST-1 assay (Supporting Information). To confirm that this killing was caused by #284, we labeled #284 with fluorescein (F-#284) and then prepared F-#284-silica composite particles (F-#284@silica). F-#284 retained the killing activity of free #284 (Figure 3b). After incubation with F-#284@silica for 3 h, most cells remained viable, and no fluorescein signal was observed (note that F#284@silica was placed in an insert and was separated from the cells by a porous membrane) (Figure 3c). After incubation for 8 h, however, cells started to die, and the dead cells were found to have incorporated #284, as indicated by the colocalization of PI and fluorescein. From these data, we conclude that the killing of MCF-7 cells was induced by F-#284 released into the culture medium from the F-#284@silica composite. (10) Perry, C. C. Rev. Mineral. Geochem. 2003, 54, 291–327.
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Figure 3. (a) Viability of MCF7 cells incubated in the presence of #284@silica (red), free #284 (green), or silica (blue). Bars represent standard deviations (n = 3). (b) Cells were incubated in the presence of F-#284 for 3 h and then stained with propidium iodide. (c) Colocalization of F-#284 (green) and dead cells (red) in F-#284@silica treated cells.
The encapsulated protein is expected to be resistant to digestion by proteases and the composite is also expected to be robust against the gastric acidic environment, suggesting that this system may be useful for oral administration of medication. In this report, we used apoptosis-inducing #284 proteins as an illustrative example. Because many natural proteins contain cationic amino acids,11 we believe that this system could be applied to biologics other than #284. And (11) Luckarift, H. R.; Dickerson, M. B.; Sandhage, K. H.; Spain, J. C. Small 2006, 2, 640–643. (12) Sano, K.; Sasaki, H.; Shiba, K. Langmuir 2005, 21, 3090–3095. (13) Luckarift, H. R.; Spain, J. C.; Naik, R. R.; Stone, M. O. Nat. Biotechnol. 2004, 22, 211–213.
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if a directed protein does not produce sufficient silicification, addition of a peptide known to strongly mediate mineralization12-14 to the protein would facilitate formation of the composite.
Conclusions In summary, we have presented a novel method for preparing a silica carrier for the sustained release of a proteinaceous pharmaceutical. This method makes use of the silicification activity of the protein itself, which autonomously formed a protein-silica (14) Naik, R. R.; Tomczak, M. M.; Luckarift, H. R.; Spain, J. C.; Stone, M. O. Chem. Commun. (Cambridge, U.K.) 2004, 1684–1685.
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composite upon simple incubation with a silica precursor. Because this mineralization reaction proceeds under very mild conditions, this method would be expected to avoid unexpected deactivation of the protein, which has often been problematic when attempting to immobilize a protein through physical adsorption onto an inorganic carrier.15 The encapsulated protein was slowly released from the composite, (15) Vallet-Regi, M.; Balas, F.; Arcos, D. Angew. Chem., Int. Ed. 2007, 46, 7548– 7558.
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thereby sustaining the protein’s activity for a long period of time. Acknowledgment. We thank Drs. S. V. Patwardhan, G. E. Tilburey, and C. C. Perry for invaluable discussions. Supporting Information Available: Cell killing assay (Figure S1). This material is available free of charge via the Internet at http://pubs.acs.org.
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