Core-shell Nanoparticles Containing Peptide Den-drimers Anas Lataifeh,† Lyudmila V. Goncharova,§ Heinz–Bernhard Kraatz*,‡ †Department of Chemistry, Tafila Technical University, Tafila 66110, Jordan. Tel. +962(079) 730 123. E–mail: latafeh@ttu.edu.jo. ‡ Department of Physical and Environmental sciences, University of Toronto at Scarborough, 1265 Military trail, Toronto, Ontario, M1C 1A4, Canada. Tel: +1 (416) 287 7278; E–mail: hkraatz@utoronto.ca. § Department of Physics and Astronomy, University of Western Ontario, 1151 Richmond Street, London, Ontario, N6A 3K7, Canada. Tel: +1 (519) 661 2111 ext. 81558; E–mail: lgonchar@uwo.caABSTRACT: A gold core–silver shell nanoparticles (NPs) containing peptide dendrimers innerlayer were prepared using peptide dendrimers from generation 1 to generation 3. The resulting NPs exhibit optical properties characteristic of Au/Ag core–shell NPs. Transmission electron microscopy and x–ray photoelectron spectroscopy of the core–shell NPs exhibit a size and composition de-pendence on the dendrimer generation number. Rutherford backscattering spectrometric analyses of core–shell NPs containing dendrimer innerlayer indicates that the NPs are consisting of discrete domains of Ag–shell and Au–core.

Metal NPs have attracted much attention recently due to their unique optical,1 electronic,2,3 and cat-alytic properties.4 For the successful and wide ap-plication of these nanomaterials, it is critical to control the size, shape and composition of such NPs. Multicomponent core–shell NPs have the po-tential to find more diverse applications than sin-gle component NPs because it offer more handles in controlling material structure and properties, and allow both the core and shell components to be taken advantage of.5,6 Among gold (Au) and sil-ver (Ag) based core–shell NPs, the use of Au/Ag core–shell would be more beneficial than Ag/Au core–shell NPs, because the Ag component with potentially promissing optical properties (the ex-tinction coeffiecientcoefficient of AgNPs could be ~4 times larger than that AuNPs of same size and shape) is in the outer layer in Au/Ag core–shell NPs.6,9 Encapsulation of spherical,7 triangular,8

and rod9 shape Au core with Ag shell has been demonstrated so far. In these systems, the Au–Ag interface is characterized as an alloy mixture of both metals.8,10 Making discrete hybrid core–shell structures has been of limited success,5 and re-mains on the theoretical analysis level.11 Herein

we are testing the feasiblityfeasibility of separat-ing the core from the shell in core–shell NPs by a distance of < 2 nm using nano–sized peptide den-drimers, see Fig. 1. Peptide dendrimers based on glutamic acid (Glu) have exhibited intramolecular hydrogen bonding (H–bonding) interactions, which “crosslink” the dendrimer branches producing globular and rigid, nanometer–sized structures particularly at higher dendrimer generation number (G3 (~1 nm) and G4 (~1.5 nm)).12 In the context of core–shell NPs, the use of dendrimers to seperateseparate Au core from Ag shell is proposed based on the abil-ity of the dendrimer branches to reduce ion mi-gration.13,14 Therefore, a question may arise: Does H-bonding interactions between dendrimer branches in Glu dendrimers assist the reduction of silver ion diffusion in the synthesis of Au/Ag core–shell NPs?

Fig. 1 Synthesis of core–shell NPs separated by den-drimer inner layer: The NP core is prepared from cit-rate–stabilized AuNPs (20 nm) and peptide den-drimers Thc–[Gn]OH, n= 1–4, containing single disulfide ligand and carboxylate groups at their sur-faces (2, 4, 8 and 16 carboxylate groups in G1, G2, G3 and G4 respectively). The Ag+ ions bind to the

1

carboxylate groups at the outmost surface of the dendrimer shell. Reduction of the Ag+ ions was car-ried out using 100 µL of L-ascorbic acid (0.1 M), 10 or 20 µL of AgNO3, (0.01 M) and 150 µL of NaOH (0.1 M) at 24 °C. The reaction conditions were set to 1.3 nM Au[Gn]OH. The silver shell encapsulated peptide dendrimer conjugates of AuNPs is are prepared as follow. Briefly, the dendrimer coated AuNPs are obtained by stabilizing AuNP core with Glu dendrimers. The dendrimers Thc–[Gn]OH, n = 1–4, are functional-ized with a single disulfide (S–S) ligand and car-boxylate groups at the surface, Fig. 1 and Scheme S1. The disulfide group binds to the Au core, while the carboxylate groups are available for metal binding,15 then, the addition of Ag+ ions to the aqueous solution containing AuNP–dendrimer conjugates, followed by reduction of Ag+ ions af-ford the Au/Ag core–shell construct.The peptide dendrimers Thc–[Gn]OH are deriva-tives of poly L–Glu dendrons, [Gn]OMe,12 and thioctic acid (Thc), Scheme S1. The dendrimers are prepared sequentially from [Gn]OMe Thc–[Gn]OMe Thc–[Gn]OH, n = 1–4 in acceptable yields. The 1H NMR spectra of Thc–[Gn]OH show the characteristic signal of the carboxylate groups at ~ δ12.4 while the amide protons of the den-drimers exhibit their charactersticcharacteristic signals in the range δ8–10, see experimental and Fig. S1. The assembly of Thc–[Gn]OH dendrimers on the gold surface is achieved by displacement of cit-rate ligands from commercially available citrate–stabilized AuNPs (20 nm) yielding Au[Gn]OH con-jugates in quantitative yields, n = 1–4. The den-drimers were attached to AuNP surface using Au–S bonds. The x–ray photoelectron spectroscopy (XPS) of the Au[Gn]OH conjugates, Fig. S2, ex-hibit the binding energy of the Au 4f at ~84 and ~87.8 eV, which are in line with those reported in monolayer protected gold clusters functionalized with alkane thiolate capping agents (Au–MPCs).16,17 This result suggest that dendrimers binds to gold core preferentially from the disulfide terminal.The resulting conjugates were characterized by UV–vis spectroscopy, Fig. S3. The surface Plas-mon resonance (SPR) has shifted from 519 nm in citrate–stabilized AuNPs to ~524 nm in Au[Gn]OH conjugtesconjugates. The H–bonding interactions in [Gn]OH dendrimers before and after binding to AuNPs have been evaluated using IR spec-troscopy. , Fig. S4. The results suggest that H–bonds on the attached dendrimers are disrupted (except in Au[G4]OH) after binding to AuNPs, in-dicated by the shifts to a lower wavenumbers of the amide II bands in the IR spectra of Au[Gn]OH, n = 1–3. The disruption of H–bonds is known to be due to surface curvature of the Au NPs.18

Silver shell encapsulated Au[Gn]OH NPs are pre-pared as demonstrated in Fig. 1. The Ag+ ions co-ordinate to the carboxylate groups at the outer-most layer of the Au[Gn]OH conjugates.15 Reduc-tion of the Ag+ ions by L–ascorbic acid has re-sulted in a color change (redyellow) for the con-jugates Au[Gn]OH, n = 1–3. The L–ascorbic acid promoted Ag+ reduction chemistry was not suc-cessful to induce color change for the solution containing Au[G4]OH conjugate (remains red). Similar reaction conditions were used to encapsu-late Au–citrate core with a silver shell (control), see experimental. The resulting yellow solutions were centrifuged twice and the supernatant (yel-low) was discarded. To further stabilize the ob-tained NPs, The solid precipetateprecipitate was redispersed in 0.5 mL of 0.02 M sodium citrate,19

giving a brown–red color solution. The optical spectra of the synthesized NPs exhibit two absorption maxima at ~415 nm and ~520 nm and are assigned to the SPR of Ag and Au in bimetallic NPs respectively, Fig 2. Comparing the absorption spectra of the bimetallic NPs with ad-mixture of separately prepared monometallic AuNPs and AgNPs, Fig. S5, it is concluded that the Au and Ag interact with each other in the case of bimetallic NPs judging from the blue shift of the SPR band in Au–Ag bimetallic NPs. The optical spectra of the NPs suggest the forma-tion of two clearly absorption bands originated by the presence of unmixed Au and Ag domains dis-tributed in different regions of the NPs, Fig. 2.7

The structural order of the NPs suggest that the silver is at the outermost layer which is confirmed by the reduction of the Au SPR intensity in (Au[G3]OH)core–Agshell spectrum when the equiv-alents of Ag+ ions are doubled.7,20 Fig. 2. Such changes in the SPR band intensities are found to be proportional to amount of Ag+ added.20 Fur-thermore, The the optical spectra shows the SPR of gold core has blue shifted in all of the synthe-sized NPs, in which the magnitude of the shift in (Au–citrate)core–Agshell NPs is the largest among the prepared core–shell NPs, inset of Fig. 2.

Fig. 2: Partial UV–vis spectra of the as prepared core–shell NPs: (Au–citrate)core–Agshell (———, 20 µL

of 0.01 M AgNO3), (Au[G1]OH)core–Agshell (____, 20 µL of 0.01 M AgNO3), (Au[G2]OH)core–Agshell (····, 20 µL of 0.01 M AgNO3), (Au[G3]OH)core–Agshell (—·—, 10 µL of 0.01 M AgNO3) and (Au[G3]OH)core–Agshell (—··-—, 20 µL of 0.01 M AgNO3). Inset,. : A a blue shift of the gold SPR is noted in (Au–citrate)core–Agshell NPs (519500 nm), while the gold SPR in (Au[Gn]OH)core–Agshell NPs, n = 1–3 is only slightly blue shifted (525520 nm).Our earlier observations reports on the synthesis of (Au[Gn]OH)core–Agshell NPs, n = 1–3, showed that the reduction with L–ascorbic acid (Eº = –0.078 V vs. NHE) is only possible when Ag+ ions are accessible to Au core (Ag+/Ag0, Eº = +0.799 V vs. NHE).22 This suggest that Ag+ ions diffuse through the dendrimer branches and monolayer defects. Furthermore, the dendrimer monolayer of [G4]OH seems to isolate the Au core from the surrounding medium and thus reduction of iso-lated Ag+ ions (Ag+/Ag0, Eº = –1.800 V vs. NHE) becomes unattainable by L–ascorbic acid.18 Ag layer formation at the surface of Au[G4]OH by citrate reduction method or reduction by NaBH4 method yields AgNPs of different sizes and shapes.Both chemical reduction experiments and IR re-sults suggest an open structure for the branches of the dendrimers in Au[Gn]OH NPs, n = 1–3, while a rigid compact structure for the branches of dendrimers in Au[G4]OH NPs, this conclusion supports that extensive H–bonding interactions in peptide dendrimers of [G4]OH reduces/stops Ag+

ion migration in core–shell NPs synthesis.The transmission electron microscopy (TEM) mea-surements of the core–shell NPs containing den-drimer innerlayer exhibit a spherical particles having sizes from 24–27 nm, with a narrow size distribution (±4 nm), Fig. S6. The TEM image of Au[G3]OH, Fig. 3a, shows a monodisperse gold particles with average core diameter of 21 ± 4 nm, while the average diameter of core–shell NPs made from Au[G3]OH has increased to 27±3 nm, Figs 3d and S11. Similar results were ob-tained for (Au[Gn]OH)core–Agshell particles, n = 1, 2, , Figs 3b–c and S9–S10.

Fig. 3: TEM images of (a) Au[G3]OH, mean particle size = 21 ± 4 nm, scale bar = 100 nm. (b) (Au[G1]OH)core–Agshell, mean particle size = 24 ± 4 nm, scale bar = 100 nm. (c) (Au[G2]OH)core–Agshell, mean particle size = 27 ± 4 nm, scale bar = 20 nm and (d) (Au[G3]OH)core–Agshell, mean particle size = 27 ± 3 nm, scale bar = 20 nm.The TEM measurements of (Au–citrate)core–Agshell NPs shows a nearly spherical, poly–dis-perse particles with an average core diameter = 35±8 nm, Fig. S7. Such particles exhibit the largest size among the prepared core–shell NPs, this result explains the largest blue shift for the SPR of the Au core in the optical spectrum of (Au–citrate)core–Agshell.8,9

Although the synthesis of the core–shell NPs were carried out under similar reaction conditions, the size of the resulting NPs seem to depend on the Au surface functionalization, Fig. 4 (blue circles, right axis). For example, The the Ag–shell thick-ness increases with the increase in generation number of the dendrimer attached to the Au sur-face. The TEM images, however, does not show a transparent gap between the core and the shell phases which would be expected to be visualized in TEM for low electron density organic den-drimers,5 particularly in NPs having Au[G3]OH as a core, since G3 dendrimers has a size of ~ 1 nm.The elemental composition of the core–shell NPs, probed by XPS spectroscopy, confirms the pres-ence of Au, S and Ag elements, Fig. S12, this re-sult is in accord with the proposed structure of a gold core, dendrimer containing sulfur atoms and silver shell. The high resolution XPS of the Au core shows two doublets for Au 4f at ~84 and ~87.8 eV and two doublets for Ag 3d at ~369 and ~375 eV consistent with Au0, Ag0 atoms respectively, Fig. S13.8 The Ag/Au atomic ratio (obtained from XPS) of core–shell NPs increases with the increase in the average size of core–shell NPs containing den-drimers, Fig. 4 (black squares, left axis). The Ag/Au atomic ratio of core–shell NPs made from Au–citrate core is the smallest among the prepared NPs. This result is unexpected in light of the largest size of the Ag shell thickness (15 nm) compared with the Ag shell thickness of the other core–shell NPs (~5 nm).

Fig. 4: A plot of the average core diameter of core–shell NPs vs. the type of Au surface functionalization (blue circles, right axis). The plot shows the effect of dendrimer generation number on the size of the re-sulting NPs. The error bar corresponds to experimen-tal standard deviation. A correlation between the av-erage core diameter and the Ag/Au atomic ratio at the surface (obtained from XPS) of the NPs (black squares, left axis). The increase in Ag/Au atomic ra-tio in core–shell NPs containing dendrimers is due to increase in distance (dendrimer size + Ag shell thickness) for the Au core electrons to travel from the Au core to NP surface. This result suggests that the growth of the Ag shell in Au–citrate core either occurs preferen-tially in particular faces of the gold core leading to the formation of islands of silver layers Fig.5a, or the Ag shell uniformly covers the Au–citrate core in which some of the Au surface atoms mi-grate from the core with concurrent position re-placement by Ag atoms. Thus, forming a mixture of both Ag and Au atoms (alloy) interface, Fig.5b,21 While NPs having Au–dendrimer as a core produce a homogeneous, ordered Ag shell thickness, Fig.5c, as indicated by the gradual in-crease in Ag/Au atomic ratio, Fig. 4 (black squares, left axis).

Fig. 5: Pictorial representation of the three possible structures for the metal interface in core–shell NPs prepared in this study. (a) Formation of individual is-lands of silver atoms as a result of random growth above the Au core. (b) Formation of bimetallic (alloy) interface resulted from diffusion of Ag atoms form the shell into the Au surface atoms in the core. (c) Homogeneous Ag shell formation templated by the dendrimers carboxylate groups at the top of Au core without Ag, Au mixing.Elemental depth profiles for the core–shell NPs at various stages of the preparation were examined by Rutherford backscattering spectrometry (RBS), Figs 6 and S14–S16. In RBS the structure and composition of materials is determined by mea-suring the backscattering of high energy2MeV

He2+ (2 MeV) radationbeam. The energy loss of the backscattered ions depends on the sample nuclei, .x in which tTwo different elements scatter ions to different energies, thus producing two peaks in the energy spectrum. The second com-ponentThe width of RBS peak is related forto the energy loss of the scattered ions is due to the electron density of elements and the distance transverse in the sample, where small energy loss of the backscatterdbackscattered ions produces sharper(narrower peaks) can be correlated with a narrow depth distribution (smaller thicknesses or diameters).x, y For pure AuNPs, only gold peak observed at the energies arround 1.8 MeV, Fig. S14. The width of the peak is slightly wider compared to the calcu-lations based on a model for an average NP diam-eter of 24 nm. One can argue that small portion of nanoparticles (< 10%) cluster togather to-gether on the surface forming “two layered” structures.For the core–shell NPs having Au–citrate core small Ag peak is detected around 1.7 MeV, which corresponds to the Ag thickness not more than 5–625 nm, though while the Ag shell thickness ob-tained from TEM is 15 nm, Fig. 6 (blue solid cir-cles). In contrast, very different Au peak position and Ag peak area density is observed in (Au[G1]OH)core–Agshell system, Fig. 6 (black open circles). The Au peak is shifted by ~60 KeV in (Au[G1]OH)core–Agshell compared with its surface position in (Au–citrate)core–Agshell, which can only be the case if Au particles are completely covered with Ag, thus ruling out the formation of core–shell NPs of the type shown in fig. 5a. In addition, the Ag depth distribution (as based on the width of Ag peak, compared to the Au peak) is larger in (Au[G1]OH)core–Agshell NPs, though the silver shell thickness in such NPs (25 atomic layer) is thinner than the silver shell in (Au–citrate)core–Agshell NPs (59 atomic layer).23,24 This result indi-cates that the Au core in such NPs is completely and uniformly covered with Ag atoms resulting in a great significant energy loss of the RBS beam. Similar RBS results were obtained for the samples of (Au[Gn]OH)core-Agshell NPs, n = 2, 3, see Figs S15–S16.

Fig. 6: Rutherford backscattering spectra for (Au–citrate)core–Agshell (blue solid circles), and (Au[G1]OH)core–Agshell (black open circles). The number of silver atomic layers (obtained from TEM) in (Au–citrate)core–Agshell is 59, and in (Au[G1]OH)core–Agshell is 25.24 Considering models for the interface structure in core–shell NPs, one can assume a phase segre-gated core–shell NPs of the type shown in Fig 5c for the NPs containing Au-dendrimer inner core. This result is based on the unique RBS spectra ex-hibited by such NPs, supported by a weak elec-tronic coupling between the core and shell SPR in the UV–vis spectra. While the RBS spectrum of NPs containing Au–citrate core conclusively showed that the thickness of pure Ag shell is 5–6~25 nm and the rest of the growing shell is a mixture of gold and silver atoms (alloy interface) as shown in Fig 5b. This work is significant for the following reasons: First, it demonstrates that H–bonding interactions in peptide dendrimers preserve a compact rigid structure of the dendrimers at the NP surface, thus preventing silver ion migration in Au/Ag core–shell NP formation. Second. , The the thick-ness and the order of the Ag shell layer at the subatomic level are controllable through the num-ber of carboxylate groups at the surface of the dendrimer. For the first time in NP characteriza-tion, the RBS technique has been used to distin-guish between complex NP layers structure. The RBS data has unequivocally showed the differ-ence between alloy structure interface in core–shell NPs and separate domains of the core and shell materials. Taken together, the results sug-gest that the use of ligands terminated with carboxylate groups is a facile method to control the size and composition of core–shell NPs. This method could be potentially extended to prepare core–shell particles with using QD, nano–rod cores.

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