pubs.acs.org/Langmuir © 2009 American Chemical Society
Complexity in Nanoparticle Assembly and Function Obtained by Direct-Grafted Peptides Wade K. J. Mosse,† Merran L. Koppens,† Sally L. Gras,†,‡ and William A. Ducker*,†,§ †
The Department of Chemical and Biomolecular Engineering, The University of Melbourne, Victoria, 3010, Australia, ‡The Bio21 Molecular Health and Biotechnology Institute, The University of Melbourne, Victoria, 3010, Australia, and §Department of Chemical Engineering, Virginia Tech, Blacksburg, Virginia 24061 Received July 9, 2009. Revised Manuscript Received September 22, 2009 We synthesize peptide-functionalized nanoparticles by growing the peptide directly from the nanoparticles in a grafting-from process. We demonstrate the procedure by grafting a short, pH and oxidation responsive peptide sequence from 300 nm silica nanoparticles. The peptide allows destabilization of the particles in response to pH by neutralization of electrostatic charge, while manipulation of oxidizing conditions in the system offers the ability to select for irreversible, covalent bonding between the particles. In one system, we show the assembly of an asymmetrically functionalized set of particles, which may have applications in the formation of binary particle networks. The method of preparing peptide-coated particles should greatly simplify existing processes used to create peptide-functionalized nanoparticles.
Introduction Control over the aggregation and self-assembly of nanoparticles is an area of broad interest, with many potential applications in nanotechnology and the development of new materials.1-5 Previous work has used many strategies to allow the controlled assembly of nanoparticles, usually relying on noncovalent interactions to drive assembly.1,6 More recent advances have involved the applications of biomolecules such as peptides and DNA to allow more precise control of particle ordering.2,7-10 Here we demonstrate a new method of modifying nanoparticles by growing peptide molecules directly from the nanoparticle surface.11 The same method has previously been widely applied to the preparation of peptide libraries on microscopic beads,12 but this has not been extended to inorganic nanoparticles, nor have the products been used for modification of colloidal stability. In addition to stabilization, peptide films can be used to introduce diverse and complex functionality to a particle surface. The diversity arises from the fact that there are 20 naturally occurring *Corresponding author. (1) Galow, T. H.; Boal, A. K.; Rotello, V. M. Adv. Mater. 2000, 12, 576–579. (2) Mirkin, C. A.; Letsinger, R. L.; Mucic, R. C.; Storhoff, J. J. Nature 1996, 382, 607–609. (3) Shevchenko, E. V.; Talapin, D. V.; HKotov, N. A.; O’Brien, S.; Murray, C. B. Nature 2006, 439, 55–59. (4) von Maltzahn, G.; Harris, T. J.; Park, J.-H.; Min, D.-H.; Schmidt, A. J.; Sailor, M. J.; Bhatia, S. N. J. Am. Chem. Soc. 2007, 129, 6064–6065. (5) Levy, R.; Thanh, N. T. K.; Doty, R. C.; Hussain, I.; Nichols, R. J.; Schiffrin, D. J.; Brust, M.; Fernig, D. G. J. Am. Chem. Soc. 2004, 126, 10076–10084. (6) Cusack, L.; Rizza, R.; Gorelov, A.; Fitzmaurice, D. Angew. Chem., Int. Ed. 1997, 36, 848–851. (7) Nykypanchuk, D.; Maye, M. M.; van der Lelie, D.; Gang, O. Nature 2008, 451, 549–552. (8) Shim, J.-Y.; Gupta, V. K. J. Colloid Interface Sci. 2007, 316, 977–983. (9) Stevens, M. M.; Flynn, N. T.; Wang, C.; Tirrell, D. A.; Langer, R. Adv. Mater. 2004, 16, 915–918. (10) Ryadnov, M. G.; Ceyhan, B.; Niemeyer, C. M.; Woolfson, D. N. J. Am. Chem. Soc. 2003, 125, 9388–9394. (11) Mosse, W. K. J.; Koppens, M. L.; Gengenbach, T. R.; Scanlon, D. B.; Gras, S. L.; Ducker, W. A. Langmuir 2009, 25, 1488–1494. (12) Lam, K. S.; Salmon, S. E.; Hersh, E. M.; Hruby, V. J.; Kazmierski, W. M.; Knapp, R. J. Nature 1991, 354, 82–84. (13) Nelson, D. L.; Cox, M. M. Lehninger Principles of Biochemistry, 3rd ed.; Worth Publishers: New York, 2000.
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amino acids (as well as many other synthetic amino acids), any one of which can occupy each position in the peptide sequence.13 The biological or catalytic activity of many of these sequences is known and can be introduced to nanoparticle or colloidal suspensions through adsorption of the peptides. Applications of to functionalized nanoparticles include gene delivery,14 chemical and radiation therapy,15,16 and contrast agents.17,18 We anticipate that new methods of functionalizing nanoparticles could find applications in all these areas. For short peptides (n < 30), the standard method of adsorbing peptides (e.g., refs 10, 19, and 20) is to synthesize the peptide on a resin, obtain the free peptide by cleavage, and then to attach the peptide to the nanoparticle, a procedure known as “grafting to” (Scheme 1). Clearly, it is much simpler to grow the peptide directly from the desired nanoparticle, “grafting from”, as described here. Direct grafting simplifies the process, as well as reducing losses of peptide that are incurred on cleavage and grafting of the peptide. In addition, “grafting from” allows complete freedom of the desired sequence whereas the sequence used in the “grafting to” procedure is constrained both by the need to incorporate groups that will bind to the surface and by the need for other groups not to bind the surface.5 In the grafting from procedure, the binding to the nanoparticle is covalent and controlled by the order of synthesis. The principal disadvantage of “grafting from” is the inability to separate unwanted peptide products, for example peptides where one amino acid has failed to attach in the (14) Bharali, D. J.; Klejbor, I.; Stachowiak, E. K.; Dutta, P.; Roy, I.; Kaur, N.; Bergey, E. J.; Prasad, P. N.; Stachowiak, M. K. Proc. Natl. Acad. Sci. U. S. A. 2005, 102, 11539–11544. (15) Zhang, Y.; Kohler, N.; Zhang, M. Q. Biomaterials 2002, 23, 1553–1561. (16) Oldenburg, S. J.; Averitt, R. D.; Westcott, S. L.; Halas, N. J. Chem. Phys. Lett. 1998, 288, 243–247. (17) Loo, C.; Lowery, A.; Halas, N.; West, J.; Drezek, R. Nano Lett. 2005, 5, 709–711. (18) Loo, C.; Lin, A.; Hirsch, L.; Lee, M. H.; Barton, J.; Halas, N.; West, J.; Drezek, R. Technol. Cancer Res. Treat. 2004, 3, 33–40. (19) Scarberry, K. E.; Dickerson, E. B.; McDonald, J. F.; Zhang, Z. J. J. Am. Chem. Soc. 2008, 130, 10258–10262. (20) Wang, Z. Y.; Zhao, Y.; Ren, L.; Jin, L. H.; Sun, L. P.; Yin, P.; Zhang, Y. F.; Zhang, Q. Q. Nanotechnology 2008, 19, 14.
Published on Web 10/21/2009
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Figure 1. Schematic of peptide structure, with approximate charging at pH 7. Scheme 1. Existing Method for Peptide Functionalization of Nanoparticles, Contrasted with the Direct Method Employed in This Papera
enabling particle cross-linking via the formation of disulfide bonds under oxidizing conditions at pH > 8.22
Materials and Methods
growing sequence. This effect is more serious for biological recognition where the forces are often more sequence dependent than in particle processing. We demonstrate the method of peptide grafting from nanoparticles through the development of a complex film on silica nanoparticles. Silica particles serve as a useful demonstration platform, but also find application in gene delivery.14 The peptide was synthesized by the same standard Fmoc procedures as used in conventional solid phase peptide synthesis,21 with aminosilanized silica nanoparticles as a substrate. The film has the sequence particle-aminosilane-aminohexanoic acid-(lysine)4-cysteine-NH2, and is shown in Figure 1. This sequence was chosen to allow changes in colloidal stability that can be addressed though pH and oxidation state; more complex film properties could be produced by the same method simply by preparing a more complex sequence. The aminosilane is used to create a free amine (the starting point for peptide synthesis) and the aminohexanoic acid is simply a spacer. The four lysine residues (as well as the N-terminus) are used to introduce pH-sensitivity to the colloidal stability: with pKa = 10.5,13 the lysine residues introduce stabilizing positive charge at acidic and neutral pH. This charge is gradually lost as the pH rises to 10.5. The cysteine residue at the N-terminus of the peptide chain has a thiol group with pKa = 8.5,
Reagents. Silica nanoparticles (300 nm diameter) were obtained from Fuso Chemical Co (Osaka, Japan). Fluorenylmethoxycarbonyl-L-amino acids (Fmoc amino acids), 2-(1H-benzotriazol-1-yl)1,1,3,3-tetramethylaminium hexafluorophosphate (HBTU) and N-hydroxybenzotriazole (HOBt) were obtained from MerckNovabiochem (Melbourne, Australia). Trifluoroacetic acid (TFA) was purchased from Scharlau S.A. (Sentmenat, Barcelona, Spain). Dimethylformamide (DMF), aqueous ammonia and piperidine were purchased from Merck (Darmstadt, Germany). All other chemicals were purchased from Sigma-Aldrich (Milwaukee, WI). Toluene was dried over 4 A˚ molecular sieves prior to use. All other chemicals were analytical reagent grade or higher and used as received. Water was purified with a Millipore Simplicity purification system (Millipore, Watertown, MA). Amine Functionalization. A 1.0% (w/v) aminopropyltriethoxysilane (APTES) sample was added to a stirred solution of 1.5% (w/v) silica nanoparticles in dried toluene. The stirred mixture was allowed to react overnight; once stirring stopped, the mixture gradually settled. The supernatant was decanted, and the particles rinsed in more toluene, followed by three centrifuge/decant cycles in methanol. Finally, the aminated particles were dried at 80 °C. Peptide Synthesis. Peptides were synthesized directly onto aminated particles. Standard Fmoc chemistry21 was used employing N-R-Fmoc-L-amino acids with HBTU, HOBt, and N,N-diisopropylethylamine as a 0.2 M solution in DMF for 18 h. Fmoc deprotection after each amino acid coupling was achieved using 20% (v/v) piperidine/ DMF for 4 h. The particles were washed in between each step with DMF (3 2.5 mL) and isolated after each wash by centrifugation. After the final amino acid had been added, the side-chain protecting groups were removed by treatment with 2.5% (v/v) triethylsilane/2.5% (v/v) water/TFA for 3 h, followed by washing with DCM and air-drying. Cysteine Activation. Peptide modified particles (60 mg) were solvated with water (0.8 mL). Separately, 2,20 -dithiodipyridine (7.7 mg) was dissolved in methanol (0.6 mL) then added to the solvated particles and allowed to react for 1 h. The particles were then washed with ethanol (3 2 mL) and air-dried. Zeta Potential Measurement. The zeta potential was measured using a Malvern Zetasizer 2000, with results calculated from five measurements. Particles were initially dispersed at 2% (w/v) in 10 mM phosphate buffer (pH = 6.5) by ultrasonication, then diluted with water to a final concentration of 0.2% (w/v) for measurement. Mass Spectrometry. To allow cleavage of the peptide for analysis, the base-labile 4-(hydroxymethyl)benzoic acid (HMBA) linker was inserted between the amine-derivatized silicon particle and the aminohexanoic acid. The particle was exposed to a 0.2 M solution of linker in DMF with equimolar amounts of HOBt and diisopropylcarbodiimide. The peptides were then synthesized as
(21) Atherton, E.; Fox, H.; Harkiss, D.; Logan, C. J.; Sheppard, R. C.; Williams, B. J. J. Chem. Soc., Chem. Commun. 1978, 537–539.
(22) Stryer, L. Biochemistry, 4th ed.; W. H. Freeman and Company: New York, 1995.
a In the standard scheme, the peptide is synthesized on a resin, R, cleaved and purified, and then ligated to the nanoparticles, P. In our method, the peptide is grown directly from the particles.
1014 DOI: 10.1021/la903466b
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Figure 2. Mass spectrograph of the solution obtained when the peptide was cleaved from the silicon nanoparticle. The peak at 746.5 g mole-1 corresponds to the full peptide, while 634.3 g mole-1 is the weight expected for the peptide without aminohexanoic acid incorporation. described above and cleavage with concentrated aqueous ammonia for 2 h left a free peptide. The particles were washed with concentrated aqueous ammonia then acetonitrile (twice) and the combined deprotection solution and washings were evaporated to dryness. The peptide was redissolved in a solution of 2% (v/v) TFA and 50% (v/v) acetonitrile in water and electrospray mass spectrometry was used to confirm the molecular weight of the peptide. TNBSA Assay. Small aliquots of particles were treated with a mixture of 0.1 M N,N’-diisopropylethylamine and 10% (w/v) trinitrobenzenesulfonic acid (TNBSA) in DMF. A positive result was indicated by a red color of the particles, judged by visual inspection.23 Ellman’s Reagent. Aliquots of particles were treated with 5,50 -dithiobis(2-nitrobenzoic acid) (0.13 mg/mL) in 0.1 M phosphate buffer for 15 min. A positive test for thiols was indicated by a yellow solution and could be quantified by measuring the absorption at 410 nm (ε = 13650 M-1 cm-1).24 Nanoparticle Assembly. Initially, particle dispersions were made with 0.5% (w/v) peptide-particles in 5 mM sodium chloride solution, followed by ultrasonication in a bath to disperse the nanoparticles. Details of compositions and treatments for the three routes are described below: Route 1. The initial solution of 0.1% (w/v) peptide particles (with untreated cysteine residues) in 5 mM NaCl was made in a cuvette, where the total volume was 2.5 mL. Particles were precipitated by the addition of 50 μL of 100 mM NaOH solution to a final concentration of 2 mM, then left for at least 48 h to ensure disulfide bond formation. After this time, the solution was neutralized by the addition of 50 μL of 100 mM HCl. Route 2. The initial solution of 0.1% (w/v) peptide particles (with untreated cysteine residues) in 5 mM NaCl with 1 mM tris(2-carboxyethyl)phosphine (TCEP) was made in a cuvette, where the total volume was 2.5 mL. Particles were precipitated by the addition of 128 μL 100 mM NaOH solution to a final concentration of 5 mM (extra base was required relative to route 1 to overcome acid properties of TCEP), then left overnight to settle. After this, the supernatant was decanted and exchanged for a 2 mM NaOH TCEP-free solution as described for route 1, and the sample was left 48 h for disulfide formation. After this time, the solution was neutralized by the addition of 50 μL of 100 mM HCl. (23) Goodwin, J. F.; Choi, S.-Y. Clin. Chem. 1970, 16, 24–31. (24) Ellman, G. L. Arch. Biochem. Biophys. 1959, 82, 70–77.
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Route 3. A set of activated particles and a set of unactivated particles were mixed in equal particle mass to yield 2.5 mL of 0.1% (w/v) peptide-particles suspension in 5 mM NaCl. Particles were precipitated by the addition of 50 μL of 100 mM NaOH solution to a final concentration of 2 mM, then left for at least 48 h to ensure disulfide bonds were able to form. After this time, the solution was neutralized by the addition of 50 μL of 100 mM HCl. Sedimentation Measurements. Neutralized particle samples were dispersed by gentle shaking and inversion. Sedimentation of the particle suspensions over time was then monitored via light transmission (λ = 500 nm) using a Cary 3E UV-visible spectrophotometer. Electron Microscopy. Samples were prepared for electron microscopy by dispensing 10 μL of a 1:100 dilution of the sample solution onto a carbon disk. After drying, samples were sputter coated with 4-5 nm of gold using a Dynavac Mini sputter coater (Massachusetts) and imaged on a Quanta 200F scanning electron microscope (FEI, Oregon) equipped with an Everhart Thornley Detector at a voltage of 2 or 5 kV and under high vacuum.
Results and Discussion Successful preparation of the target sequence was verified in two ways. First, we verified the molecular mass of the peptide. To verify the mass, a parallel synthesis was performed with the addition of a cleavable linker, 4-(hydroxymethyl)benzoic acid, between the silane and the aminohexanoic acid so the peptide could be freed from the particle for mass spectrometry. Mass spectrometry (Figure 2) of the cleaved peptide showed a peak at m/z = 746.5, consistent with the full peptide sequence. A smaller peak at m/z = 634.2 indicated the presence of a small number of peptides in which aminohexanoic acid was absent. The absence of other significant peaks confirmed successful coupling of all lysines and the cysteine. Zeta potential measurements of the peptide nanoparticles at pH 7 confirmed that silanization introduced a positive charge to the silica particles because the zeta potential increased from - 40 ( 1 to þ26 ( 3 mV. After synthesis of the peptide on the particles, the zeta potential had risen further, to þ33 ( 5 mV, as expected for the predominantly positively charged peptide. A strong positive result to a TNBSA assay23 showed the presence of amines on the particles, and testing with Ellman’s reagent showed a thiol concentration of 2.9 μmol g-1 particles. This is equivalent DOI: 10.1021/la903466b
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Mosse et al. Scheme 2. Illustration of the Assembly Routes Available to the Peptide-Functionalized Particlesa
a In route 1, mainly intraparticle disulfide bonds are formed, so free thiols are not available for particle cross-linking. The stability of the particle then depends on pH. In route 2, the interparticle disulfide bonds formed cause coagulation, which persists independent of the pH. In Route 3, activated cysteine residues are unable to form intraparticle bonds, and instead bond with any free cysteine residues remaining on the normal particles, again resulting in durable aggregates.
to approximately 54 000 peptide molecules per particle, or 0.2 molecules per nm2 of surface area. From the volumes of the amino acids,25 and the extended length, we estimate that a fully packed surface of fully extended peptide would have a density of about 2 molecules per nm2. Thus, our peptide layer is about 10% of a fully packed layer. Note that the incorporation of protecting groups during the synthesis places a limit on the maximum density of about 50% of a fully packed layer after deprotection. The peptide-particles showed the expected pH and oxidation response. The particles were easily dispersed in water at pH 7 by ultrasonication, and remained stable over a period of hours. Although settling occurred overnight, the particles could be immediately resuspended by inverting the mixture. When the solution pH was increased to ∼11 by the addition of NaOH, the peptide-particles flocculated rapidly because the stabilizing positive charge was neutralized (Scheme 2, route 1). When the mixture was returned to pH 7 the particles were once again easily resuspended by simple inversion and showed little settling, in line with that of newly suspended particles, as shown in Figures 3 and 4. After this procedure a test for free thiols (Ellman’s reagent) was negative, a change from the positive result before exposure to basic conditions. This indicates that the thiols initially present have become oxidized, and are now participating in disulfide bonds. The good stability of the particles at pH 7 shows that the disulfide bonds are between thiols on the same particle (intraparticle) as interparticle disulfide bonds would prevent dispersion of the particles. Electron microscopy confirmed that little cross-linking had occurred, with most of the particles existing as either monomers or very small aggregates (Figure 5). Changing the oxidation conditions of the solution gave us additional control of the stability of the suspension. Starting with (25) Tsai, J.; Taylor, R.; Chothia, C.; Gerstein, M. J. Mol. Biol. 1999, 290, 253– 266.
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Figure 3. Settling behavior of the particles after resuspension at pH 7 based on transmittance of light (λ = 500 nm). The transmittance was normalized to correct for small variations in turbidity between samples. Freshly suspended particles (red circles) and those treated by route 1 (blue circles), are mostly single particle and show little settling over 30 min. Particles assembled by route 2 (green triangles) and route 3 (black squares) form large durable aggregates that settle quickly, even though the particles are highly charged at pH 7.
an identical suspension to that used to begin route 1, we used the reducing agent tris(2-carboxyethyl)phosphine (TCEP) to ensure thiol groups were unable to form disulfide bonds when the solution was precipitated at pH ∼ 11 (Scheme 2, route 2). TCEP was then removed, and the solution left at pH 11 for 48 h to allow disulfide formation while peptide chains from neighboring particles remained in close contact. Upon returning to pH 7, the settled particles showed rapid settling behavior after resuspension (Figures 3 and 4); this is a result of the particles remaining in Langmuir 2010, 26(2), 1013–1018
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Figure 4. Photographs of particle suspensions: Immediately after resuspension by inversion (top) and after 30 min settling (bottom). Cuvettes are, from left to right, particles treated by routes 1-3. Routes 2 and 3 result in obvious settling after 30 min, while route 1 only shows a slight settling near the top of the cuvette.
large aggregates, as observed under electron microscopy (Figure 5). These aggregates are cross-linked by interparticle disulfide bonds that bind clusters of particles that are large enough to sediment, independent of whether the particles are highly charged (pH 7) or uncharged (pH 11). Interparticle disulfides could also be formed by mixing two sets of particles under flocculating conditions (route 3); one set of particles were identical to those used in route 1, while in the other set the thiols were activated with 2,20 -dithiodipyridine. These mixed particles were then treated via the same path as was trialled in route 1. In this case, the activated thiols are only able to react with other free thiols, eliminating intraparticle bond formation on half of the particles present; this greatly enhances the probability of interparticle bond formation in concert with any remaining free thiols on the unactivated particles. Aggregates formed under these Langmuir 2010, 26(2), 1013–1018
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Figure 5. SEM images of treated peptide-particles. Particles treated by route 1 (top) were present mostly as isolated particles and small aggregates, while many larger aggregates were observed in samples treated by route 2 (middle) and route 3 (bottom).
conditions remained flocculated, even after a return to pH 7 where all particles are highly charged; this was again demonstrated by particle settling (Figures 3 and 4), as well as observation of aggregates by SEM (Figure 5). The difference in settling of the final product in Routes 2 and 3 are insignificant, as expected for two sets of particles that are each cross-linked by disulfide bonds. Route 3 also offers a method by which a particle of one type can be forced to bind to another type in a mixture, which could in future be used to create composite materials. Recently, the stability of siliver nanoparticles with a similar peptide film was examined.26 The peptide was a dimer of (26) Graf, P.; Mantion, A.; Foelske, A.; Shkilnyy, A.; Masic, A.; Thunemann, A. E.; Taubert, A. Chem.;Eur. J. 2009, 15, 5831–5844.
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particle is forced by the experimenter and is a free choice of the experimenter. We are thus able to add a cysteine (or any other group) to the free end. In contrast, when the complete peptide is added to the particle, the peptide will bind by whatever groups are thermodynamically or kinetically favored. This concept is illustrated schematically in Figure 6.
Figure 6. Depiction of the “grafting-from” and “grafting-to” methods. One advantage of the “grafting from” method is that each amino acid is added in order, so the binding configuration is known. The solid can even be protected or reaction conditions changed so that subsequent binding units can be added without forced binding, as in the example shown. In the example here, B is a binding unit and W, X, Y, and Z are other units. In the “grafting to” technique, all the monomer units are added together (as one unit), so the polymer can adopt a variety of binding configurations. In this example, the added peptide can bind via either or both ends, giving a chemically inhomogenous layer. There may also be some binding via W, X, Y and Z. The inhomogeneity in binding becomes increasingly problematic with more complex peptides. Also, the density in “grafting from” is controlled via the density of grafting sites, whereas in ‘grafting to” it is also determined by the interactions between neighboring groups.
Lys-Lys-Cys (KKC) covalently linked with a disulfide bond. The peptide was synthesized first, and then introduced during the growth of the silver nanoparticles to offer the opportunity of templating the formation and growth of nanoparticles. In common with the peptide particles described here, the KKC particles also showed evidence of destabilization at pH 11, as expected for a peptide containing positive residues with pK ∼ 10.5. The KKC dimer or monomer was bound to the silver surface via the cysteine, so the cysteine was not available for cross-linking the particles. That procedure also illustrates one advantage of our technique: the sequence is not subject to constraints of binding. In our technique, the connection of the amino acid proximal to the
1018 DOI: 10.1021/la903466b
Conclusions In summary, we have demonstrated a facile method for preparing nanoparticles with a covalently attached peptide layer: the peptide is grown directly from the particle. The method allows the preparation of complex and functional coatings on nanoparticles. In particular, it allows for the forced binding of the peptide via a particular group, and liberates the experimenter from the severe constraint of designing a peptide sequence that spontaneously binds in the desired configuration. As with other graftingfrom procedures, the density of peptide can be controlled by the density of grafting sites on the particle, rather than by interactions between adsorbed particles. This should allow greater control of the density of peptide on the particle. Finally, the grafting-from procedure involves fewer steps than the traditional method in which the peptide is first grafted from a resin, then cleaved, then grafted back onto another particle. We have demonstrated this capability through the preparation of nanoparticles where the colloidal stability is addressable by both solution pH and oxidation conditions, and introduce the use of cysteine-based disulfide bonds as a “locking mechanism” for the self-assembled particles, allowing control of whether a nanoparticle suspension forms reversible or irreversible aggregates. By activation of one set of particles in a particle mixture we also describe a method whereby binding between dissimilar particles is favored over binding between similar particles. The same synthetic method with different peptide sequences may be used in future to produce nanoparticles with peptide sequences that include biological recognition sites or other functional elements with applications such as gene delivery, chemical and radiation therapy and contrast agents Acknowledgment. The authors wish to acknowledge the Electron Microscopy Unit of Bio21 Institute, the University of Melbourne, for assistance with electron microscopy performed in the course of this research. This research was supported by the Australian Research Council (DP0664051 and FF0348620).
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