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Amine-Terminated Dendrimers as Biomimetic Templates for Silica Nanosphere Formation Marc R. Knecht and David W. Wright* Department of Chemistry, Station B 351822, Vanderbilt University, Nashville, Tennessee 37235 Received March 8, 2004 In diatoms, silica synthesis occurs by use of complex posttranslationally modified peptides, termed silaffins, and highly complex biological polyamine structures. Silaffin peptides have lysine residues that are modified to long-chain polyamine moieties of N-methyl derivatives of polypropylenimine to drive silica synthesis at slightly acidic pH conditions. Using polypropylenimine (PPI) and PAMAM amine-terminated dendrimers as a biomimetic analogue of the polyamine modifications of silaffins, we have demonstrated the condensation of silica nanospheres. We have shown that the dendrimers react in an amine concentrationdependent fashion yielding silica nanospheres with a distinct size distribution reminiscent of the structures produced from both the modified and nonmodified peptides extracted from diatoms. Additionally, the templates were encapsulated by the growing nanospheres and precipitated from solution in a manner similar to that previously described for the bioactive peptides and polyamines.
Introduction In contrast to many current materials engineering approaches to the synthesis of patterned silica,1 biogenic silica is formed rapidly under mild conditions. The diverse array of nanopatterned silica in many diatoms2 or the protective glass spicules of some sponges3 are exemplars of such biosilicification. The exquisite species-specific control of silica architectures over a wide length scale has focused attention on the involvement of biopolymers in the condensation of silica. In diatoms, cationic polypeptides named silaffins are highly posttranslationally modified peptides that have been shown to precipitate silica from monosilicic acid at pH 5.5.4 Within the native peptide, many of the lysines have been modified to either -Ndimethyllysine, phosphorylated -N-trimethyl-δ-hydroxylysine, or long-chain polyamine moieties of N-methyl derivatives of polypropylenimine,5,6 while the serines have been posttranslationally phosphorylated. Functional analysis of native silaffins and isolated polyamines has shown that the peptides self-assemble into supramolecular structures, providing a template for silicic acid polycondensation by long-chain polyamine moieties.6,7 The nonmodified peptide R5 (H2N-SSKKSGSYSGSKGSKRRIL-CO2H) has also been shown to produce silica.4 Studies have shown that the R5 peptide is able to selfassemble through the RRIL motif to form larger structures capable of condensing silica. Furthermore, this study demonstrated that multiple truncates and mutant pep* To whom correspondence should be addressed: e-mail
[email protected]; phone (615) 322-2636; fax (615) 3431234. (1) Zaremba, C. M.; Stucky, G. D. Curr. Opin. Solid State Mater. Sci. 1996, 1, 425. (2) Round, F. E.; Crawford, R. M.; Mann, D. G. The Diatoms: Biology & Morphology of the Genera; Cambridge University Press: Cambridge, U.K., 1990. (3) Sundar, V. C.; Yablon, A. D.; Grazul, J. L.; Ilan, M.; Aizenberg, J. Nature 2003, 424, 899. (4) Kro¨ger, N.; Deutzmann, R.; Sumper, M. Science 1999, 286, 1129. (5) Kro¨ger, N.; Deutzmann, R.; Sumper, M. J. Biol. Chem. 2001, 276, 26066. (6) Kro¨ger, N.; Lorenz, S.; Brunner, E.; Sumper, M. Science 2002, 298, 584. (7) Sumper, M.; Lorenz, S.; Brunner, E. Angew. Chem., Int. Ed. 2003, 42, 5192.
tides of the R5 displayed similar activity, despite being drastically shorter in peptidyl length, as long as the RRIL motif was present.8 The R5 and associated peptides produce nanospherical particles of silica, but added shear stress can induce formation of different columnar structures of silica.9 Recent work with a variety of polyamines such as polyL-lysine,10 pentapropylenehexamine,11 or poly(allylamine hydrochloride)12,13 have shown that they too can act to form silica nanospheres, most likely in an aggregationdependent manner. Dendrimers represent unique unimolecular polymer templates whose functionality can be tuned through the judicious choice of branching elements and terminal groups. Previously, dendrimers have been used as templates for the stabilization of a wide variety of nanoparticles, including sol-gel composites.14-18 In this study, dendrimers represent well-defined templates capable of presenting highly localized concentrations of biomimetic moieties. Polypropylenimine dendrimers (PPI) are composed of repeating propylenimine units analogous to one of the major posttranslational modifications found in the lysines of the siliaffins, while the terminal amines of the PAMAM dendrimers are reminiscent of the unmodified lysines within the R5 peptide (Figure 1). Herein, we report the use of amine-terminated PPI and PAMAM dendrimers as templates for biomimetic silica nanosphere production. (8) Knecht, M. R.; Wright, D. W. Chem. Commun. 2003, 3038. (9) Naik, R. R.; Whitlock, P. W.; Rodriguez, F.; Brott, L. L.; Glawe, D. D.; Clarson, S. J.; Stone, M. O. Chem Commun. 2003, 238. (10) Patwardhan, S. V.; Mukherjee, N.; Clarson, S. J. J. Inorg. Organomet. Polymers. 2001, 11, 193. (11) Noll, F.; Sumper, M.; Hampp, N. Nano Lett. 2002, 2, 91. (12) Patwardhan, S. V.; Clarson, S. J. Mater. Sci Eng. 2003, 23, 495. (13) Brunner, E.; Lutz, K.; Sumper, M. Phys. Chem. Chem. Phys. 2004, 6, 854. (14) Crooks, R. M.; Zhao, M.; Sun, L.; Chechick, V.; Yeung, L. K. Acc. Chem. Res. 2001, 181. (15) Grohn, F.; Bauer, B. J.; Akpalu, Y. A.; Jackson, C. L.; Amis, E. J. Macromolecules 2000, 22, 6042. (16) Balogh, L.; Tomalia, D. A. J. Am. Chem. Soc. 1998, 120, 7355. (17) Larsen, G.; Lotero, E.; Marquez, M. J. Mater. Res. 2000, 15, 1842. (18) Larsen, G.; Lotero, E.; Marquez, M. J. Phys. Chem. B 2000, 104, 4840.
10.1021/la0494019 CCC: $27.50 © 2004 American Chemical Society Published on Web 04/30/2004
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Figure 1. Chemical structures of biological and biomimetic silica precipitating amines. (a) Structure of silaffin peptide obtained from C. fusiformis. Underlined residues are posttranslationally modified in vivo; note the repeating N-methylpropylenimine units. (b) Structure of poly(N-methylpropylenimine) extracted from the silica produced by the diatom S. turris, where R represents a repeating N-methylpropylenimine unit of n ) 15-21. (c) Branched structure of G-3 PPI dendrimer. (d) Branched structures of G-1 PAMAM.
Experimental Section Materials. DAB-Am-4 poly(propylenimine tetraamine) dendrimer, DAB-Am-8 poly(propylenimine octaamine) dendrimer, DAB-Am-16 poly(propylenimine hexadecaamine) dendrimer, DAB-Am-32 poly(propylenimine dotriacontaamine) dendrimer, and DAB-Am-64 poly(propylenimine tetrahexacontaamine) dendrimer (G1-G5), PAMAM dendrimers (G0-G3 and G5), and tetramethyl orthosilicate were purchased from Aldrich Chemical Co. PAMAM dendrimers (G4 and G6) were purchased from Dendritech Inc. Silica Precipitation Assays. The silica precipitation assay was modified from literature methods.4 Tetramethyl orthosilicate was diluted in 1 mM aqueous HCl to a final concentration of 1 M. The solution was allowed to stir at low speeds for approximately 15 min to ensure complete hydrolysis to form free monosilicic acid. Silica-precipitating templates were dissolved in 100 mM aqueous phosphate buffer to the desired concentration of amine in a final volume of 200 µL. To this solution was added 20 µL of the preformed silicic acid, and the assays were shaken on an orbital shaker plate for 5 min at room temperature. The assays developed silica within seconds. Each assay was then centrifuged for 5 min at 10 000 rpm, the supernatant was decanted, and the pellet was isolated. The pellet was resuspended in water and washed multiple times with water to remove excess unreacted reagents. Silica Quantitation. Synthesized silica was quantitated by the β-silicomolybdate method developed by Iler.19 Individual samples were dissolved in 0.5 M NaOH and incubated at 95 °C for 30 min to ensure complete dissolution. After incubation, the liberated dendrimers in solution were removed from solution, as they were interferants with the molybdate assay, by Centricon filtration (Amicon Centricon filtration devices, Millipore Inc.). (19) Iler, R. K. The Chemistry of Silica; Wiley: New York, 1979; pp 96-98.
Due to available filtration cutoffs, only samples with templates of molecular weights greater than 3000 were quantitated. To the filtered solution was added the molybdate reagent, to initiate the formation of the bright yellow product that was monitored by UV-vis spectrophotometry (Agilent Technologies model 8453 UV-Vis) at 410 nm and quantitated from a standard curve of silicate standards. Scanning Electron Microscopy. Silica samples were suspended in ethanol and pipetted onto the surface of an aluminum SEM sample stage (Ted Pella Inc.) to dry. Each sample was sputter-coated with a thin layer of gold by use of a Pelco Model 3 Sputter Coater (Ted Pella Inc.). Once sputtered, each sample was imaged on a Hitachi S4200 scanning electron microscope operating at variable voltage. Infrared and Etching Analysis. Silica samples for IR analysis were prepared as above. After the final washing, each sample was dried under vacuum. Dried samples were pressed into KBr pellets and examined on a Mattson Genesis series FTIR. Silica nanospheres were synthesized and purified as above by use of the PAMAM (G5) and PPI (G5) dendrimers at an amine concentration of 20 mM. The nanospheres were suspended in 500 µL of a 10 mM aqueous NaOH solution and shaken for 10 s. The etched spheres were centrifuged for 5 min at 10 000 rpm and the supernatant was removed. The pellet was again washed repeatedly with water and analyzed by IR spectroscopy and SEM.
Results and Discussion A series of dendrimers (PPI G1-G5 and PAMAM G0G6) were assayed for their ability to rapidly precipitate silica nanospheres from a solution of metastable silicic acid (Table 1). All templates assayed were active and yielded silica nanospheres of an appropriate morphology (see Supporting Information). Quantitation of silica was based upon modifications of the β-silicomolybdate assay.19
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Table 1. Silica Condensation Activity for Amine-Containing Templates template
template surface diameter20 (Å) amines
G1 PPI G2 PPI G3 PPI G4 PPI G5 PPI G0 PAMAM
8.8 13.8 18.6 23.2 27.8 15
4 8 16 32 64 4
G1 PAMAM
22
8
G2 PAMAM G3 PAMAM G4 PAMAM G5 PAMAM G6 PAMAM
29 36 45 54 67
16 32 64 128 256
specific activitya ND ND ND 7.47 ( 1.2 23.7 ( 2.6 ND ND 8.1 + 0.7 17.2 ( 2.2 46.1 ( 4.4 63.0 ( 8.7 108.3 ( 19.3
nanosphere diameter (nm) 180 ( 27 169 ( 45 242 ( 76 219 ( 91 258 ( 76 95 + 25 350 ( 50 130 ( 20 400 ( 40 380 ( 40 360 ( 75 292 ( 73 275 ( 105 334 ( 80
a Nanomoles of SiO per minute per nanomole of dendrimer. 2 ND, not determined due to template interference.
As the dendrimers were interferants in the assay system, quantitation was possible only after removal of the template from the dissolved silica solution. As the monosilicic acid precursor became limiting, silica condensation activity was seen to plateau. For the PPI dendrimers, analysis of the reaction product revealed a sigmoidal correlation between amine concentration and silica production activity. A linear correlation between primary amine concentration and silica production activity was observed for the PAMAM dendrimers (Figure 2) under nonlimiting conditions. The sigmoidal reaction profile of PPI dendrimers may result from differences in dendrimer size and surface charge density when compared to the respective PAMAM dendrimers [i.e., PPI Gx vs PAMAM G(x-1)]. As shown in Table 1, the diameter of PPI dendrimers are essentially 60% the diameter of the corresponding PAMAM with the same number of surface primary amines.20 Consequently, PPI dendrimers have a higher amine and surface charge density. In the polycondensation reaction, the silica particles with PPI templates more rapidly precipitate from solution, resulting in the observed profile and decreased specific activity. In contrast, an analogous hydroxyl-terminated series of PAMAM dendrimers did not yield silica. A variety of alternate reaction conditions were also examined. Recently, Sumper et al.7 demonstrated an apparent requirement for multivalent anions in the biomimetic condensation of silica using polyamines isolated from the diatom Stephanopyxis turris. The effect of anions in the dendrimer-mediated condensation of silica nanospheres was examined. At pH 7.5 there was no observed difference in the amount of silica formed in buffer systems composed of phosphate, sulfate, or acetate anions (see Supporting Information). This difference may be attributed to the fact that, with the biologically derived polyamines, the multivalent anions purportedly play an important role in the supramolecular assembly of the silica-precipitating peptides. With unimolecular dendrimers, the anions assume the traditional role of charge balance of the template and the surface of the growing silica nanosphere. The precipitates were examined by scanning electron microscopy (Figure 3). The average nanosphere diameter for the isolated silica is shown in Table 1. Silica precipitated by the PPI dendrimers G1 and G2 yielded particles sizes ranging from 170 to 180 nm, while G3-G5 produce particles with diameters from 220 to 260 nm. Silica formed (20) Tande, B. M.; Wagner, N. J.; Mackay, M. E.; Hawker, C. J.; Jeong, M. Macromolecules 2001, 24, 8540.
Figure 2. Biomimetic silica condensation as a function of primary amine concentration for (a) PPI dendrimers G4 and G5 and (b) PAMAM dendrimers G2-G6.
by PAMAM G0 and G1 dendrimers present a bimodal population with smaller particle diameters averaging 95 and 130 nm and larger diameter particles of 350 and 400 nm, respectively, that is not observed for higher generations. PAMAM dendrimers G2-G6 show silica particles ranging from 275 to 390 nm. A comparison between PPI and PAMAM dendrimers with the same number of amines reveals that the ratio of the template diameter approximates the ratio of silica nanosphere diameters (Table 1), suggesting that the silica-encapsulated dendrimer is the aggregating unit of silica growth. These silica nanosphere sizes were consistent with those produced from native silaffins,4 the R5 peptide as reported by Naik et al.,9 and a series of R5 truncates reported by Knecht and Wright.8 Infrared analysis of isolated and exhaustively washed silica nanospheres clearly indicated that the dendrimer is associated with the silica (see Supporting Information). Vibrations associated with the encapsulated amine moieties of the PPI dendrimers and the amine and amide moieties of the PAMAM dendrimers were observed, while alkane hydrogen-bond vibrations for both PPI- and PAMAM-encapsulated dendrimers were seen in both samples. Within the precipitate, the molar ratio of silica to PPI dendrimer under nonlimiting monosilicic acid conditions was 37:1 for G4 and 137:1 for G5. In the PAMAM
Biomimetic Silica from Dendrimers
Figure 3. SEM micrographs of nanospherical silica produced by (a) G4 PPI and (b) G4 PAMAM.
dendrimers under similar conditions, the same ratio ranged from 40:1 for G2 to over 500:1 for G6. In comparison, the molar ratio of silica to silaffin-1A with its posttranslationally modified lysines (but not serines) was reported as 12.4 In previously reported dendrimer nanocomposites, the dendrimers have encapsulated the colloids, imparting stability to the aqueous colloidal solution.21,22 The preponderance of the biological evidence suggests that the silaffin peptides are encapsulated within the diatom’s siliceous skeleton.4-6 Similarly, in vitro studies with silicaprecipitating peptides NatSil 1-A,4 R5,8,9 and a number of truncates9 have suggested that the peptides are captured within the precipitated silica nanospheres. Several lines of evidence suggest that the dendrimers, like their biological analogues, are encapsulated within the isolated biomimetic silica particles. Purified silica nanospheres synthesized from either PPI or PAMAM templates were etched with 10 mM NaOH. The etched particles were extensively washed to remove the liberated silicic acid and any other released substances. SEM revealed that etching led to the removal of ∼60-100 nm of silica from the nanosphere surface. IR analysis of the etched nanospheres gave rise to strong vibrations resulting from template moieties still intimately associated with the particles. If the dendrimers were merely acting as a simple surface passivant, the etching process should have removed them from the silica. As they remain associated with the silica after etching, it is likely that the templates are encapsulated in the growing nanospheres during the condensation reaction (Figure 4). Similar results were obtained in the etching of PPIencapsulated silica nanospheres. (21) Sooklal, K.; Hanus, L. H.; Ploehn, H. J.; Murphy, C. J. Adv. Mater. 1998, 10, 1083. (22) Garcia, M. E.; Baker, L. A.; Crooks, R. M. Anal. Chem. 1999, 71, 256.
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Additional evidence of template encapsulation is seen in reseeding experiments. Nanospheres of silica produced from PPI or PAMAM dendrimers were isolated, washed copiously, and reseeded into solutions of monosilicic acid. If the templates were acting as a surface passivant, then further silica condensation should continue from the freely exposed amines, leading to significant silica production. In fact, silica formation was significantly reduced from the reseeded nanospheres (Figure 5). This is further suggestive of the template being buried within the silica. Previous work examining the polycondensation reactions of silica has highlighted a complex reaction broadly categorized into three stages: polymerization of monomer units to form stable nuclei, growth of nuclei to form spherical particles, and aggregation of particles to form structural motifs.23 The presence of additives, ranging from simple inorganic or organic molecules to complex polymers, are capable of dramatically influencing each of these stages. Consistent with such a model, light scattering profiles of the dendrimer-driven silica reaction (both PPI and PAMAM) suggest rapid nucleation followed by ripening and ultimately precipitation/flocculation. The reaction displays a significantly shorter lag phase (see Supporting Information) than recently reported in the reaction of polyamines from the diatom S. turris.7 This is likely due to the significant number of primary amines associated with the templates relative to the tertiary amines of S. turris. In the formation of dendrimer/silica nanocomposites, the dendrimers likely serve several roles in the silica condensation reaction. As has been suggested by several groups,24,25,8 the dendrimer’s primary amine moieties could act as a generalized acid-base catalyst in which deprotonated residues (base) accept a proton from silicic acid, forming a reactive silanolate group and protonated residues (acid) that drive the release of water by protonation of silicic acid substrates. Further, the positively charged patches on the surface of the dendrimers likely interact electrostatically with silanolate or growing negatively charged silica species to concentrate these species at the dendrimer surface. This interaction undoubtedly accounts for the observed encapsulation of the dendrimers within the silica nanospheres. Further, differences between the PPI and PAMAM dendrimer/silica composites are likely attributable to these differences in reactive amine and/or charge density on the surface of the template. Conclusions In a recent review, Coradin and Lopez26 asked whether biogenic silica patterning was “simple chemistry or subtle biology”. In vitro, it seems as if the simple chemistry dominates. To achieve the level of sophisticated silica processing seen in biological systems, more sophisticated organic templates with highly tunable architectures must be developed. Dendrimers represent an important class of such tunable templates. We have shown that PPI and PAMAM dendrimers react in an amine concentrationdependent fashion to condense monosilicic acid, yielding silica nanospheres. These nanospheres are analogous in size and shape to silica produced from both modified and nonmodified peptides extracted from diatoms. Additionally, these dendrimeric templates are encapsulated within (23) Perry, C. C.; Keeling-Tucker, T. J. Biol. Inorg. Chem. 2000, 5, 537. (24) Mizutani, T.; Nagase, H.; Fujiwara, N.; Ogoshi, H. Bull. Chem. Soc. Jpn. 1998, 71, 2017. (25) Kro¨ger, N.; Deutzmann, R.; Bergsdorf, C.; Sumper, M. Proc. Natl. Acad. Sci. U.S.A.. 2000, 97, 14133. (26) Coradin, T.; Lopez, P. J. ChemBioChem 2003, 4, 251.
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Figure 4. Etching analysis of silica nanospheres. a) unetched silica spheres produced from G5 PAMAM dendrimers b) etched silica nanospheres c) particle size distributions for unetched and etched silica nanospheres and d) qualitative IR analysis of PAMAM template and silica samples.
were subsequently removed by thermal treatment. In the formation of biomimetic silica nanospheres, PPI and PAMAM dendrimers act as templates that concentrate and drive the condensation of monosilicic acid through the dendrimer’s surface primary amine moieties. This dendrimer-mediated formation of silica may provide further routes to more complex composites. For example, dendrimers have been preciously used to stabilize colloidal suspension of gold19 and semiconductor quantum dots.18 These systems could be subsequently stabilized by precipitation within silica nanospheres. Such approaches to new composites are currently under investigation. Figure 5. Comparison of the silica condensation activity of representative PPI and PAMAM dendrimers (G5) and reseeded dendrimer precipitated silica nanospheres.
the growing nanospheres to form a hybrid silica/polymer composite structure. Increasingly, there is recognition that dendrimers can serve as important components in supramolecular aggregates.27 In previous studies, dendrimers have been used as porogens for silica gels formed in acidic alcohol solutions to imprint a cavity within the gel.17,18 These dendrimers
Acknowledgment. We thank the NSF for financial support through an NSF career award (CHE-0304124) and the NSF NER program (NSF 0196540). Additionally, we thank the Vanderbilt Institute of Nanoscale Science and Engineering for student financial support. Supporting Information Available: SEM micrographs, anion analysis, IR spectra, etching analysis, reseeding analysis, and light scattering analysis. This information is available free of charge via the Internet at http://pubs.acs.org. LA0494019 (27) Haag, R.; Vo¨gtle, F. Angew. Chem., Int. Ed. 2003, 204, 43, 272.
