Nanocage Synthesis Essay

Abstract

Iron is essential for all life, yet can be dangerous under certain conditions. Iron storage by the 24-subunit protein ferritin renders excess amounts of the metal non-reactive and, consequentially, ferritin is crucial for life. Although the mechanism detailing the storage of iron in ferritin has been well characterized, little is known about the fate of ferritin-stored iron and whether it can be released and reutilized for metabolic use within a single cell. Virtually nothing is known about the use of ferritin-derived iron in non-erythroid cells. We therefore attempted to answer the question of whether iron from ferritin can be used for haem synthesis in the murine macrophage cell line RAW 264.7 cells. Cells treated with ALA (5-aminolaevulinic acid; a precursor of haem synthesis) show increased haem production as determined by enhanced incorporation of transferrin-bound 59Fe into haem. However, the present study shows that, upon the addition of ALA, 59Fe from ferritin cannot be incorporated into haem. Additionally, little 59Fe is liberated from ferritin when haem synthesis is increased upon addition of ALA. In conclusion, ferritin in cultivated macrophages is not a significant source of iron for the cell's own metabolic functions.

Abbreviations: ACD, anaemia of chronic disease; ALA, 5-aminolaevulinic acid; ALA-S, ALA synthase; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; HO-1, haem oxygenase-1; IRE, iron response element; IRP, iron regulatory protein; PPIX, protoporphoryrin IX; RES, reticuloendothelial system; ROS, reactive oxygen species; SA, succinylacetone; SIH, salicylaldehyde isonicotinoyl hydrazone; TCA, trichloroacetic acid; Tf, transferrin; TfR, Tf receptor; UTR, untranslated region

  • © The Authors Journal compilation © 2010 Biochemical Society

Structural evolution from nanocube to nanocage

The etching strategy is summarized in Fig. 2a. In step (i), the seeds of Pd nanocubes are dispersed in aqueous compartment of reverse micelle. The Pd nanocube seeds are hydrophilic as their {200} facets are protected by a well-defined bilayer of cetyltrimethylammonium bromide (CTAB; Fig. 2ai)24,26,42,43,44. Next, in step (ii), heating slowly evaporates the water phase. In step (iii), the hydrophilic compartment becomes smaller and eventually transformed into one single layer of CTAB whose polar headgroups directly contact the well-defined bilayer of CTAB on Pd NPs, and then diffusion of the organic solvent destabilizes this capping structure. In step (iv), reduction in coverage and disorder in the packing structure of capping agents occur more dominantly at the centre of {200} facets. As a result, desorption of Pd atoms from the surface of seeds is triggered because of the surface energy gradient. In addition, the formation of stable Pd complexes between desorbed Pd atoms and CTAB in solution further promotes the release of Pd atoms and discourages the re-adsorption of Pd atoms on the seeds. This strategy enables the localized etching of Pd atoms on the nanocube seeds, and it creates concaved nanocubes and then the shape is further evolved into nanocages as shown in Fig. 2b.

Pd nanocubes for seeds

The Pd nanocubes were prepared in an aqueous solution at 95 °C. After sodium tetrachloropalladate(II) (Na2PdCl4), sodium hydroxide (NaOH) and CTAB were dissolved in water and stirred vigorously at 95 °C and freshly prepared ascorbic acid solution was immediately injected into the reaction system. The reaction was kept at 95 °C for 30 min. The product was collected using centrifugation and was washed with water. Transmission electron microscopy (TEM) image shows that as-prepared Pd nanocubes have an average size and the size distribution of 12 nm and <10%, respectively (Supplementary Figs 1a and 2). High-resolution TEM (HRTEM) image reveals the single crystal characteristics of the nanocubes with the lattice fringe spacing at 2.0 Å, corresponding to the {200} planes of face centred cubic (fcc) Pd (Supplementary Fig. 1b,c). The inset in Supplementary Fig. 1b is the indexed fast Fourier transform (FFT) pattern of Supplementary Fig. 1b, consistent with the single crystalline pattern of the Pd nanocubes.

Generation of Pd nanocages by etching Pd nanocube seeds

The etching treatment was conducted in a reverse micelle system made of CTAB/octanol/H2O. In 20 min after residual octanol and excess surfactants were removed by washing with ethanol and water, the edges of Pd NPs were excavated on six faces, while the overall size remained the same as Pd seeds (Fig. 3a). HRTEM image of the Pd concave nanocube in Fig. 3b reveals darker contrasts at the corners, indicating that the sample is thicker at the corners and is thinner in between after the etching treatment. The single crystal characteristics observed along the [100] axis is demonstrated by the corresponding FFT pattern in the inset of Fig. 3b. Figure 3c shows higher magnification of the corner region highlighted of a squared area in Fig. 3b. A large number of surface atoms are situated at ledge, ledge–kink and kink sites after etching, and it generates complex lattice fringe patterns on the surface. Figure 3d shows the two-dimensional (2D) lattice modelling corresponding to Fig. 3c, where a high-index {320} plane (marked in blue) composed of alternating subfacets of {110} (marked in red) and {210} (marked in black) is tentatively labelled. This result indicates that CTAB cannot completely protect these nanocrystals, and desorption of Pd atoms from the seed is predominant while the rate of re-adsorption on lower-energy surfaces is negligible at this early stage of shape evolution. This sparse growth may be attributed to the low concentration of and the low reactivity of Pd species in the hydrophobic solution.

After 1 h of further etching treatment, the concaved nanocubes were transformed into hollow nanocages (Fig. 4a). Figure 4b shows TEM images of individual nanocages at different angles. The average edge length of nanocage is 10 nm, 2 nm shorter than the one for the seed (Supplementary Fig. 2). It suggests that atoms on the corners of seeds started to dissolve into the solution at this aging time. The hollow structure of Pd nanocage was also confirmed from a series of TEM images obtained through tilting the sample stage at a variety of angles (Supplementary Fig. 3). The nearly pure composition of Pd of the nanocage was confirmed using the energy-dispersive X-ray spectrum (Supplementary Fig. 4). While the nanocages are single crystalline in general (Supplementary Fig. 5), some NPs show polycrystalline characteristics. As shown in the HRTEM image of Fig. 4c and its corresponding inverse FFT images (Supplementary Fig. 6), the edges of the nanocages are imaged along [100], where the (020) and (002) planes of fcc Pd are indexed. Meanwhile, the left upper corner area of the nanocages is imaged along [1–10], and lattice fringes corresponding to (020) and (111) planes of fcc Pd are resolved. A vacancy separating these two crystalline zones is marked with an arrow in Fig. 4d. The polycrystalline characteristics are also confirmed by the FFT pattern in the inset of Fig. 4c, where a superposition of [100] and [1–10] zones are indexed. The observation of shape transformation from nanocubes to concave nanocubes and finally hollow nanocages indicates that the shape evolution occurs thermodynamically so that the etching patterns are consistent with surface energy landscape created by the reorganization of capping agents in the diminishing reverse micelle compartment. The observation of atomic re-adsorption on the lowest-energy {111} facets45 with complicated lattice fringes with atoms on ledge, ledge–kink and kink sites (Fig. 4c) becomes more noticeable after 1 h of the shape evolution, probably because of the increased concentration of Pd in solution inducing their re-adsorption on these sites.

As illustrated in Fig. 2a, the shape evolution of seed nanocubes is driven by the structure change of capping layers on seed nanocrystals after water compartment is evaporated in the reverse micelle system. To probe the packing condition of CTAB capped on shaped NPs, Fourier transform infrared spectroscopy is applied to Pd nanocubes before and after etching, where peak positions and widths of C-CH2 asymmetric and symmetric stretching vibrations of the methylene chain of CTAB can be used to assess the nature of surfactant packing on solid surfaces40,46. In Supplementary Fig. 7, as compared with the crystalline CTAB, both stretching vibrations shift to the higher frequency for CTAB-capped Pd nanocubes (from 2,917 to 2,920 cm−1 for the asymmetric band and from 2,849 to 2,851 cm−1 for the symmetric band). The lower vibrational frequency and the narrower bandwidth for the crystalline surfactant correspond to more ordered structures of the methylene chains of CTAB40,46. Since the stretching bands for CTAB on Pd nanocubes are blue-shifted relative to crystalline CTAB and their frequencies are comparable to the ones on gold nanorods (2,921 and 2,851 cm−1, respectively), CTAB forms a well-defined bilayer capped on Pd nanocubes with less degree of packing order than the crystalline state38. As both symmetric and asymmetric bands for CTAB on Pd nanocages are further shifted to 2,922 cm−1 and 2,853 cm−1, respectively (Supplementary Fig. 7), it indicates that CTAB covers the surface of Pd nanocages with less packing order and surface coverage as compared with Pd nanocubes, yielding a large number of less-protected surface atoms exposed at ledge, ledge–kink and kink sites40,46, consistent with nanoscopic structures in Fig. 4c.

Effect of solvents on atomic desorption

In addition to the effect of the surface energy distribution, desorption of Pd atoms from the seed nanocrystals could be promoted by the formation of stable Pd complexes between desorbed Pd atoms and solute molecules. To confirm this hypothesis, we carried out UV–vis absorption measurements of solution during the shape evolution process. Under the experimental condition, Pd atoms dissolved from {200} facets in octanol can readily oxidized into CTA+[PdX4]2− complex species (X=Cl or Br)24,47, and a minute amount of the oxidized forms of ascorbic acid residue (for example, semidehydroascorbic acid and dehydroascorbic acid) in the Pd seed solution may serve as oxidants. After 1 h of etching and separation of Pd NPs with centrifugation, the remaining supernatant was analysed. While octanol solution containing only CTAB (20 mg ml−1) shows no absorption peaks, the spectrum of supernatant shows two peaks at 250 and 340 nm, respectively (Supplementary Fig. 8a). Both peaks are corresponded to the ones for CTA+[PdX42−] complex ions47. A series of UV–vis spectra of CTA+[PdX42−] with the concentration ranging from 0 to 180 μM in octanol also showed the similar absorption profile (Supplementary Fig. 8b), and they follow the Beer–Lambert law as shown in Supplementary Fig. 8c. This result suggests that the desorbed individual Pd atoms are highly reactive in solution and they can readily be oxidized and can form the stable complex during the etching treatment. Since the relatively high stability of complexes between Pd atoms and solute molecules promotes the desorption of Pd atoms from Pd nanocubes, types of solvents should also influence the etching process. As shown in Supplementary Fig. 9a, when the seeds were aged in ethylene glycol, a polar solvent, instead of octanol, NPs were grown in larger sizes while maintaining their cubic shape, indicating that the bilayer capping of CTAB on {200} facets was intact. As no obvious etching was observed on the seed nanocrystals, the reaction rates of dissolving of small NPs and the growth of large NPs are equally high and large NPs grow at the expense of smaller seeds as seen in Ostwald ripening. The faster crystal growth in EG is attributed to the higher reactivity of CTA+[PdX4]2− in the polar solvent48,49. When the reaction was conducted in a 1:1 EG/octanol solution, normal Ostwald ripening was still observed; however, NPs no longer maintained their cubic shape (Supplementary Fig. 9b). Adding octanol to EG solution increased the hydrophobicity of the solution, and it seems to destabilize the bilayer structure of CTAB on {200} facets of Pd nanocubes so that the shape of NPs became irregular after aging at elevated temperature.

Catalytic performance and mechanism

As the hollow Pd nanocages have the large surface area-to-volume ratio and they also exhibit the high density of catalytically active atoms on the ledge, ledge–kink and kink sites, these characteristics prompted us to investigate their potential catalytic performance. To this end, the catalytic activities of 10-nm Pd nanocages and 12-nm Pd nanocubes were compared in Suzuki coupling reactions. First, the coupling between phenylboronic acid (PhB(OH)2) and iodobenzene was investigated in 80% ethanol aqueous solution at room temperature. As shown in Fig. 5a, Pd nanocages show superior catalytic property that a nearly complete formation (>90%) of C–C bond is achieved within 30 min, whereas 12-nm Pd nanocubes can only promote a conversion of ~40% under the same condition and it required 75 min to reach a steady conversion of ~80%. In Fig. 5b, Pd nanocages also demonstrate excellent recycling performance while small loss of catalytic property is observed for Pd nanocubes after circles of the reuse. The cumulative turnover number (mol of product/mol Pd) over six runs for Pd nanocages (10 μg, 0.03 mol%) is 1.9 × 104, which is 1.3 times higher than the one for the Pd nanocubes. To compare them in more difficult catalytic environment, iodobenzene is replaced by iodotoluene, an electron-neutral aryl iodide, which is less reactive in the coupling reaction50,51. As shown in Fig. 5c, the reaction catalysed by Pd nanocages was nearly completed (>90%) after 90 min, while Pd nanocubes could convert only <30% of reactants at 50 °C. In this harder coupling reaction between PhB(OH)2 and iodotoluene, the turnover number for Pd nanocages (10 μg, 0.03 mol%) remains 1.9 × 104 over six runs; however, it is 4.5 times higher than the one for Pd nanocubes, and the superior recyclability for Pd nanocages over Pd nanocubes is also demonstrated (Fig. 5d). It should be noted that no homocoupled product was observed during the coupling between PhB(OH)2 and iodotoluene, as supported by nuclear magnetic resonance (NMR) spectra (Supplementary Fig. 10).

It is unlikely that leaching Pd atoms from Pd nanocages catalyse the Suzuki coupling reaction between aryl iodide and PhB(OH)2 in Fig. 5 because if Pd atoms are released from Pd nanocages as homogeneous catalysts, deformation of the shape of nanocages along with declined catalytic turnovers should be observed in the recycling process even though some of these Pd atoms are readsorbed on the nanocage. To firmly confirm that no leaching Pd species are involved in the Suzuki coupling reaction, the three-phase test was examined under the standard condition (0.6 mmol PhB(OH)2, 0.3 mmol iodobenzene or iodotoluene, 1 mmol K2CO3, 10 μg Pd nanocages (0.03 mol%) and 5 ml 80% ethanol aqueous solution) in the presence of NovaSyn TGR resin-supported aryl iodide (2 in Supplementary Fig. 11)52,53. While the quantitative recovery of biphenyl products was still achieved in the solution phase, no biphenylamide (6 in Supplementary Fig. 11) was detected after cleavage of the resin with trifluoroacetic acid (TFA) by gas chromatography–mass spectrometry (GC–MS) and NMR (Supplementary Figs 12 and 13). Thus, these results indicate that no free Pd atoms or Pd-organic complexes are involved in the Suzuki coupling reactions in the aqueous solution at relatively low reaction temperature, and the catalytic reactions only take place on the surface of Pd nanocages54. The heterogeneous catalytic nature of Pd nanocages indirectly supports the hypothesis above that the superior catalysis is related to the large number of active Pd atoms exposed on the surface.

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