Sustainable Synthesis and Improved Colloidal Stability of Popcorn

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Sustainable Synthesis and Improved Colloidal Stability of Popcorn-Shaped Gold Nanoparticles Mustafa Gharib, Mahmoud Khalaf, Sharmin Afroz, Neus Feliu, Wolfgang J Parak, and Indranath Chakraborty ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.9b00295 • Publication Date (Web): 06 May 2019 Downloaded from http://pubs.acs.org on May 7, 2019

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ACS Sustainable Chemistry & Engineering

Sustainable Synthesis and Improved Colloidal Stability of Popcorn-Shaped Gold Nanoparticles Mustafa Gharib,1,2,# Mahmoud Khalaf,3,# Sharmin Afroz,3 Neus Feliu,1 Wolfgang J. Parak,1 Indranath Chakraborty1,* 1Faculty

of Physics, Center for Hybrid Nanostructures (CHyN), Universität Hamburg, Hamburg,

Germany 2Radiation

Biology Department, Egyptian Atomic Energy Authority (EAEA), Cairo, Egypt Physik, Philipps Universität Marburg, Marburg, Germany #both authors contributed equally *corresponding author: [email protected] 3Fachbereich

Abstract Anisotropic popcorn-shaped gold nanoparticles (GNPCs) were synthesized through a sustainable route. Proteins were used as ligands, and among several proteins, bovine serum albumin (BSA) produced high-quality GNPCs. The method is scalable to produce liters of NPs (with almost mM elemental Au concentration) in a single batch. Structural changes of proteins due to GNPC formation were studied in detail using circular dichroism and fluorescence spectroscopy. The secondary structure of proteins was lost in the growth step, which facilitates the formation of GNPCs. Among other similar sized gold nanoparticles, GNPC@BSA was found to have high colloidal stability and biocompatibility. KEYWORDS

gold nanoparticles, nanopopcorn, surfactant free, protein, colloidal stability

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INTRODUCTION Gold nanoparticles (GNPs) are interesting materials due to their potential uses in emerging areas of nanoscience and nanotechnology.1-3 Their properties can easily be tuned by changing their size, shape, and surface chemistry.2, 4 There is a wide morphological variety of GNPs such as spheres, rods, stars, prisms, cubes, etc.4 that have been developed in the last few decades. In particular, anisotropic GNPs have drawn attention since they may have different physicochemical, optical, magnetic, catalytic, and electronic properties that are not quite the same as, and frequently surpassing, those of their isotropic counterparts, i.e., spherical GNPs, and have various potential applications probably due to their unique curvature structure.5 In contrast to isotropic spherical (non-hollow) GNPs, the most important stunning attribute of most anisotropic and hollow GNPs is apparently the presence of a plasmon peak in the NIR region (therapeutic window), where the absorption by biological tissues is very low.5 Therefore, these GNPs can be utilized for biodiagnostics and photothermal bioapplications (i.e., "theranostics").6 Another significant feature of anisotropic GNPs is the enhancement of the SERS activity.7 However, cytotoxicity is a major concern in biological/theranostic applications of anisotropic GNPs.8-9 Apart from the catalytic effects of the Au surface, the toxicity exhibited by typical anisotropic GNPs is mainly derived from the capping agents or ligands used in their synthesis. For instance, a number of in vitro studies have shown that the cationic surfactant cetyltrimethylammonium bromide (CTAB) which is used for the synthesis of rod-shaped GNPs renders them cytotoxic for many cell lines.9 Approaches exist to overcoat or exchange CTAB in post-synthesis procedures.10 Polymer coating is a common approach in this direction which is used to modify the surface chemistry by wrapping undesired ligands/surfactants such as CTAB, which in turn may reduce the exposure of biological compartments to the cytotoxic ligands.9, 11 Ligand exchange is another alternative strategy to remove bound surfactants.12-15 Nevertheless, 100% covering or removal by overcoating or ligand exchange, respectively, typically is not possible and the residual surfactant can induce so far some toxicity.9, 14 To solve this issue, a better sustainable approach would be needed to synthesize such anisotropic GNPs. Even though there are many green routes reported to create anisotropic GNPs using different bio-molecules, precise control over the nanoparticle size, stability, as well as large scale preparation remain major challenges.16-19 Apart from their cytotoxic surfactant coating (CTAB for instance), the surface chemistry of GNPs is strongly influenced in biological fluids by the formation of a protein corona.20 The protein corona increases the effective hydrodynamic diameter of GNPs.21 In case of GNPs with limited colloidal stability, the protein corona can result in aggregation of the GNPs and drastic modification of the physicochemical properties of the NPs.22 There are some cases where the formation of a protein corona has been reported to reduce toxic effects of nanoparticles.23 However, there is also a discussion that the presence of a protein corona may reduce potential applicability of nanoparticles in some drug delivery-based applications.24


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Summing up the above arguments, proteins should be good candidates for forming biocompatible surfaces around nanoparticles. In fact, proteins can be used as reducing, shapedirecting, and stabilizing agents25-26 to create biocompatible anisotropic GNPs. This avoids the use of potentially toxic surfactants. Besides, there is already a natural intrinsic pre-formed protein corona, which is, in fact, the surface capping of the GNPs directly after the synthesis, and thus later agglomeration due to the formation of a protein corona is not expected. Still, the preformed protein corona will be subject to dynamic exchange with proteins from the environment.27 Dynamic variability of the protein corona may affect even the interaction of protein-based nanoparticles with cells.28 Herein, we report an efficient, facile, benign and sustainable route for the synthesis of anisotropic popcorn-shaped gold nanoparticles ("gold nanopopcorn," GNPC). We use "popcorn" as a name in order to point out a morphology which resembles a structure of attached irregular spheres with variable diameter. In our study, different proteins were evaluated as shape directing and stabilizing agents. Among the various proteins, bovine serum albumin (BSA) was found to be the most effective ligand to synthesize GNPCs with narrow size distribution. The route is scalable, and liters of GNPCs (in the here presented large scale batch: CAu = 5.6·10-4 M) could be prepared in one single batch without compromising the quality and/or stability of the GNPCs. This study also provides structural insights into the proteins after GNPC formation using spectroscopic investigations. The tunability, stability in different biological media, as well as cytotoxicity of BSA-protected GNPCs have also been investigated. RESULTS AND DISCUSSION The synthetic route is straightforward and yields high quality branched GNPs (Figure 1A and Figure S1). The initial effort was devoted to optimizing the synthetic parameters to create such GNPs. BSA was used here as a shape-directing agent, since its potential role as a shape controlling agent is known for silver NPs.25-26 This sustainable route has the advantage of scalability without compromising the quality of the GNPs. Liters of materials can be created from a single batch. The corresponding TEM images show uniform popcorn-shaped NPs (hereafter will be termed as GNPC@BSA), similar to the structure reported by Lu et al.29 where CTAB was used as a shape-controlling agent. The TEM images also reveal good size distribution of GNPC@BSA with an average core diameter (dc) and arm’s length of 70 ± 5 nm and 12 ± 5 nm, respectively (Figure 1B). Dynamic light scattering (DLS) data further support the GNP size where the hydrodynamic diameter (dh) of GNPC@BSA was found to be 80 ± 8 nm (Figure S1B). High-resolution transmission electron microscopy (HRTEM) images of individual GNPC@BSA show the crystalline nature (Figure 1C-H) as revealed from the fast fourier transformation (FFT) pattern of different regions. Two major FFT patterns in GNPC@BSA were identified as the (111) and (200) planes of the face-centered cubic (fcc) structure of gold, which matches well with the reported branched GNPs (Figure1F, H).17, 30 Branched GNPs mostly formed due to the preferential growth of Au0 on the (111) plane, which suggests that this plane was the high-energy



facet for deposition.31 The tips of the arms of GNPC@BSA are found to be rounded compared to the analogous urchin-like structures reported by Bakr et al.32 and Li et al.33 which are prepared using thiols and hydroquinone, respectively.

Figure 1. Synthesis and characterization of GNPC@BSA: (A) Scheme of the synthesis using a sustainable two-step approach along with photographs of the corresponding GNPC@BSA in small scale (left) and large scale (right). (B) Transmission electron microscopy (TEM) images of GNPC@BSA showing the popcorn-shaped GNPs. The inset shows the core size (dc) distribution histogram of GNPC@BSA as analyzed by measuring the length from arm tip to tip from TEM images. (C) and (D) represent the TEM and scanning transmission electron microscopy (STEM) images of a single GNPC@BSA, respectively. (E, G) and (F, H) represent the HRTEM and corresponding FFT pattern of selected regions, respectively. The UV-vis absorption spectra of GNPC@BSA show a broad plasmonic peak (Figure 2A) centered around 588 ± 5 nm with full width at half maximum (FWHM) of 100 ± 10 nm, which is very similar to the reported branched GNPs.33 Such broadness in absorption spectra is mainly due to the difference in arm length of individual GNPCs. Along with BSA proteins, several other proteins (Figure 2A), amino acids (Figure S4) and ligands such as CTAB, PVP, and citrate (Figure S2D) were checked to explore the effective role of BSA in the formation of GNPCs. Proteins such as Cat, Lys, Blg, Hem, and Pep can effectively produce GNPCs (Figure S3), but they showed broader size distributions than the GNPCs@BSA (Figure S3). On the other hand, amino acids (Figure S4) are not capable of producing GNPCs, but instead, they form smaller


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GNPs as revealed from the UV-vis absorption spectra (Figure S4). Many of the GNPs prepared using amino acids are not stable for a longer time and decompose within few hours similar to the case of silver NPs.25 To further ensure the role of BSA in directing the shape of GNPs towards the nanopopcorn shape, a control experiment without the addition of BSA was carried out. The control experiment resulted in aggregated purple-colored GNPs that showed a plasmonic peak centered at 535 nm, along with a broad absorption band near 750 nm (due to aggregation10) (Figure S1C). UV-vis absorption spectra of GNPs synthesized using citrate and PVP showed the characteristics of spherical NPs (peak centered at ~530 nm)10 (Figure S2D), which suggests that these ligands facilitate the isotropic growth instead. In contrary, CTAB, which is known to form anisotropic GNPs (i.e., facilitates anisotropic growth),34 produced sponge-like GNPs of different sizes (Figure S3K) instead of popcorn shapes, although the absorption spectrum is similar to that of GNPC@BSA (~590 nm, Figure S2D). The tunability of GNPCs was evaluated by monitoring Au@Hem Au@HSA Au@Mb Au@Pep

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Figure 2. UV-vis absorption spectra and tunability of GNPC@BSA: (A) Normalized UV-vis absorption spectra A(λ) of GNPs using different ligands. (B) Representation of the tunability of the absorption peak maxima (λmax) using different synthetic parameters such as the volume of seed solution Vs, volume of Au in growth VAu(g), concentration of Au in seed solutions cAu(s), concentration of additional silver nitrate in growth cAg(g), different temperatures (T), pH, and different ligands (L). All the corresponding absorption spectra are presented in the SI (Figure S2). the position of the absorption maxima (λmax) of GNPC@BSA as a function of different reaction parameters (Figure 2B) such as the volume of seed NP solution (Vs), volume of gold precursor used in growth solution (VAu(g)), concentration of Au in seed solutions (cAu(s)), concentration of additional silver nitrate in growth (cAg(g)), different temperatures (T), pH, and different ligands (L). The data shows that by changing Vs, VAu(g) and cAg(g), only ~25 nm tunability in the wavelength of the plasmon peak maxima (λmax) can be achieved. Tunability of ~50 nm is possible by T and pH, suggesting an important role of protein structure (as these two parameters



strongly affect the structure of the protein) in determining the quality of synthesized NPs. The plasmon wavelength (λmax) of GNPs can be tuned until the near-infrared (NIR) range (~700 nm) using different ligands (L). However, the best popcorn shape of tunable size can be achieved by varying the seed GNP size (achieved by changing cAu(s)). In most of the cases, peaks below 550 nm turned out to be spherical GNPs instead of popcorn-shaped or branched GNPs. GNPC@BSA showed the highest stability and monodispersity among all protein-protected GNPCs, and hence, further studies were carried out with GNPC@BSA only. From our previous research with Ag NPs,25 we have found that protein structure plays a significant role in dictating the particle morphology. Therefore, studying the structural changes in BSA during the synthesis of GNPCs may reveal the mechanism behind the formation of such specific popcorn-shaped GNPs. Structural changes of BSA during the nanopopcorn formation were investigated using circular dichroism (CD) spectroscopy (Figure 3A).35 The CD spectra of GNPC@BSA were compared with those of native BSA at different pH (6.5 and 3.1). These pH values were chosen explicitly as to examine the CD spectroscopic behavior of natural BSA solution, which has a pH value of about 6.5, against that of an acidified solution of BSA (pH 3.5), which corresponds to the pH of the GNPC@BSA solution. BSA at almost neutral pH shows one spectroscopic band at 190 nm in the positive absorption side and two other bands at ca. 209 and 222 nm in the negative absorption side. The later peaks are characteristic for the α-helix structure of BSA (Figure 3A).36 Deconvolution of the spectrum37 (Figure 3A and Figure S5) suggests that BSA at neutral pH has 50.8% of α-helix, 3.1% of strand, 14.1% of turn, and 32% irregular structures. On the other hand, the intensity of all the corresponding bands in GNPC@BSA reduced drastically (Figure 3A). In this case, the protein structure contains only 9.9% of α-helix, 26.4% of strand, and 48% of irregular structures, suggesting the complete loss of the proteins' secondary structure. To understand at which stage such structural transformation is initiated, control measurements were carried out by investigating the CD spectral behavior of BSA in the presence of all the materials used in the growth step such as seeds, HAuCl4 and ascorbic acid (with the same concentrations used in the GNPC@BSA synthesis). In the presence of HAuCl4, the bands are almost disappeared, suggesting a strong interaction of HAuCl4 with BSA, resulting in breakdown of the α-helix structure of BSA, thus indicating BSA unfolding.38 It is worth noting that the interaction of BSA with either GNPC seeds or ascorbic acid does not induce significant changes in the CD spectra. Therefore, the observed structural changes of BSA in GNPC@BSA, majorly take place during HAuCl4 addition at the growth step and the subsequent BSA unfolding may provide an appropriate biotemplate, which in turn exerts control over GNP morphology, giving rise to the observed anisotropic GNP growth. Addition of HAuCl4 in the growth step decreases the pH of the solution, as well as Au3+ ions, can form complexes with proteins, which together help to unfold the protein’s secondary structure. Effect of temperature on protein structural change was negligible since the growth step was carried out at room temperature. To further investigate the structural aspects of BSA, fluorescence spectra were collected for all the samples. The intrinsic


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fluorescence of BSA is majorly due to the presence of two tryptophan (Trp) residues located on the bottom of hydrophobic pocket in domain II (Trp-213), while another one is located on the surface of the molecule in domain I (Trp-134).36 Structural changes could be probed by selectively exciting the Trp (λex = 280 nm), as any change in local environment of Trp could alter the emission intensity, as well as the emission wavelength of the fluorescence peak. Our studies showed a drastic change in fluorescence intensity and emission maximum in GNPC@BSA (λmax = 378 nm) as compared to native BSA (λmax = 350 nm) (Figure 3B and Figure S6). These results are consistent with the CD spectra and confirm the unfolding of the protein during the GNPC formation. This suggests that the change in protein structure occurs in the growth step through complexation with Au3+ ions, as well as due to the low pH environment, and then unfolded protein functionalities facilitate the growth of Au3+/Au1 to Au0 on the (111) and (200) facets to form the popcorn-shaped GNPs.

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Figure 3. Understanding the structural changes of BSA during GNPC synthesis: (A) CD spectra of GNPC@BSA and of different control samples such as BSA at pH 3.1 and 6.5, BSA with ascorbic acid (AA), HAuCl4, and seeds to understanding the structural difference of BSA during GNPC formation. The ellipticity has the unit of deg·cm2·d·mol-1, where d refers to the cuvette path length. To avoid the noise at the lower wavelength (190-200 nm), all spectra were smoothed using OriginPro 2018 (5 points, Savitzky-Golay). The inset shows a heat map specifying the contents of different secondary structure elements (% of the helical, strand, turn and irregular form) of protein as evaluated with an online tool mentioned in the SI. (B) Fluorescence intensity (Iλmax) (yellow bars) and maximum emission wavelength (λmax) (green bars) of different samples (outlined on the bottom) (excitation at λex = 280 nm).



Colloidal stability of GNPs in biological media is crucial for diverse biological applications.10 It has been found that many GNPs may lose their optical properties as soon as they are dispersed in biological media, mainly due to loss of their colloidal stability and strong protein corona formation.12 Since surface ligands play an active role in determining the major surface properties of NPs,39 three different types of NPs of almost similar sizes (Figure 4A) with different surface coatings, namely, citrate-protected gold nanospheres (GNP@Citrate), surfactant-free gold nanostars (GNS), and CTAB-protected gold nanopopcorns (GNPC@CTAB) have been synthesized using previously reported methods29, 40-41 (a detailed characterization is mentioned in Figure S7) for comparison. The colloidal stability (Figure S8) was evaluated in water; phosphate buffered saline (PBS), fetal bovine serum (FBS)-supplemented Dulbecco's Modified Eagle Medium (DMEM) and FBS-free DMEM. The hydrodynamic diameters (dh) of the GNPs were monitored up to 36 h in the respected media (Figure 4B, C, and Figure S8). In water, except for GNSs, all of the NPs

Figure 4. Effects of surface ligands on the colloidal stability of GNPs in biological media: (A) Comparative cartoon structures and TEM images of (a, b) GNPC@BSA, (c, d) GNP@Citrate, (e, f) GNS, and (g, h) GNPC@CTAB. (B) Colloidal stability monitored as a change in dh of GNPs over time (up to 36 h after incubation; data for longer incubation times for GNPC@BSA and GNPC@CTAB are shown in Figure S8) in DMEM supplemented with FBS. GNPC@BSA shows the highest colloidal stability. (C) Hydrodynamic diameter (dh) of different GNPs in water and DMEM supplemented with FBS (measured directly after addition, at t= 0, collected from the marked region of Figure 4B). The dh values drastically increased for GNPC@CTAB and GNS as


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compared to GNPC@BSA. (D) Proposed schematics of the corona formation for the four different GNPs. were stable, whereas, in DMEM supplemented with FBS (Figure 4), the stability order is the following; GNPC@BSA>GNP@Citrate>GNS>GNPC@CTAB (ordered according to their effective hydrodynamic diameter dh at 36 h, Figure 4B). Moreover, the effective hydrodynamic diameter of GNPC@CTAB and GNS changed significantly as soon as they were incubated with DMEM supplemented with FBS, suggesting a strong protein corona formation as well as aggregation of the GNPs (Figure 4C-D). The high positive charge (Figure S7C) of GNPC@CTAB, due to the CTAB molecules, facilitates strong interaction of proteins with the GNP surface. Proteins thus get adsorbed on the surface of GNPC@CTAB, leading to significant aggregation as soon as they are in contact with media. GNS is surfactant free; therefore, their surfaces are easily accessible, and their positive surface charge further helps to adsorb proteins, resulting in aggregated NPs and an increase in the net effective hydrodynamic diameter. Comparatively, less aggregation is observed in GNP@Citrate, which is probably due to the negative surface charge of these GNPs. GNPC@BSA almost remains intact (Figure 4), and only a slight change in effective hydrodynamic diameter (∆dh = 20 ± 10 nm) was seen while incubating with FBS- supplemented DMEM, suggesting high colloidal stability. Cytotoxicity of GNPC@BSA was evaluated and compared to that of the other GNPs using cancerous HeLa and non-cancerous MRC-5 cell lines. The results revealed that GNPC@CTAB is highly toxic at higher concentrations (CAu= 3.1-100 µg/mL) for both the cell lines. This is due to the CTAB coating, as CTAB has already been found to be toxic for cellular applications9, 25. In contrast, all other GNPs including GNPC@BSA exerted no acute reduction in the viability of HeLa as well as MRC-5 cells up to the maximum concentration used in this study (Figure 5B, E, Figure S9-10). Acute cytotoxicity was further probed using the standard lactate dehydrogenase (LDH) assay. LDH is an intracellular enzyme, which is rapidly released into the culture medium when the cell membrane is damaged. The LDH assay measures the cell membrane integrity and hence can be used to assess the cytotoxicity. To evaluate the cytotoxicity of the NPs, the percentage of LDH release RLDH into the culture medium of HeLa cells, as well as MRC-5 cells, after exposure to different concentrations of NPs, was measured (Figure 5C, F). After 24 h, exposure of HeLa cells as well as MRC-5 to relatively higher concentrations of GNPC@CTAB (CAu= 3.1-100 µg/mL) resulted in a significant increase in LDH release relative to the untreated cells in comparison to the same concentrations of GNP@Citrate, GNPC@BSA and GNS (Figure 5 and Figure S11). The LDH assay results are in good agreement with the findings from the resazurin assay, where the GNPC@CTAB at concentrations above CAu= 3.1 µg/mL) elicits cytotoxic effects and significantly diminishes the viability of HeLa and MRC-5 cells. These results showed that the cell membrane integrity of both cells was damaged when exposed to certain



concentrations of GNPC@CTAB, resulting in an overall increase in the permeability of the cell membrane giving rise to the subsequent leakage of the cytosolic LDH enzyme into the culture medium. Although GNP@Citrate and GNS did not induce acute toxicity, limitations of their colloidal stability in buffers (PBS) and media (DMEM, cf. Figure S8) make them less attractive for biological applications. V [%]

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GNPC@BSA GNPC@CTAB GNS GNP@Citrate

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Figure 5. Effects of surface ligands on the biocompatibility of GNPs: Concentration-dependent cytotoxicity assays of GNPC@BSA against different ligand-protected NPs after 24 h exposure. (A, D) Cartoon illustration of HeLa cells and MRC-5 cells, respectively, (B, E) Heat maps representing the dose-dependent reduction in cell viability V after 24 h exposure of HeLa and MRC-5 cells, respectively, to different NPs using the Resazurin assay42 (mean values only, a plot with error bars can be found in the SI) from three independent experiments. HeLa cells and MRC-5 cells were exposed to various concentrations of GNPs (CAu) in serum-supplemented media for 24 h. Due to colloidal instability, GNS and GNPC@BSA cannot be concentrated more beyond the star marks labeled in the plot. Hence, toxicity beyond this concentration was not carried out for these two NPs. (C, F) Effect of 24 h exposure of HeLa cells (C) and MRC-5 cells (F), respectively, to different NPs on LDH release as depicted by the dose-response curves. Full LDH release (RLDH = 100%) was ascribed to lysed cells, see the Supporting Information.

CONCLUSIONS In this work, we were able to develop a highly sustainable and facile route for the synthesis of biocompatible anisotropic GNPCs using BSA as a shape-directing agent. The optical properties


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of such GNPCs can be tuned over a broad spectral range (from the visible to the near IR regions). GNPC@BSA shows high colloidal stability in different biological media including cell culturing media (DMEM+FBS). Along with the detailed GNPs characterizations, changes in protein structure during the GNPC formation were evaluated to elucidate the role of proteins in directing the crystalline growth and dictating the shape of the GNPs. Our studies showed that during the GNPC formation, the BSA molecule is mostly unfolded and exhibits a low degree of helicity (i.e., low percentage of α-helix structure). Strong coordination with Au ions is the main reason for such changes in protein structure. Cytotoxicity data revealed that GNPC@BSA is highly biocompatible and does not elicit acute cytotoxic effects on HeLa cells as well as noncancerous MRC-5 cells at a comparable concentration range where corresponding CTABprotected nanopopcorn-shaped GNPs reduce the cells viability. EXPERIMENTAL SECTIONS Materials: Before use, all glassware was appropriately washed with aqua regia (3:1 (v/v) conc. HCl/conc. HNO3) followed by rinsing with copious amounts of Milli-Q water. All chemicals were used as received. For the syntheses processes, gold(III) chloride trihydrate (HAuCl4) was purchased from Alfa Aesar, while Sigma Aldrich was the supplier of polyvinylpyrrolidone (PVP), sodium citrate, L-glutathione, silver nitrate (AgNO3), ascorbic acid, sodium borohydride, hexadecyltrimethylammonium bromide (CTAB), bovine serum albumin (BSA), lysozyme from chicken egg white (Lys), catalase (Cat), pepsin of porcine gastric mucosa (Pep), betalactoglobulin from bovine milk (Blg), hemoglobin (Hem), myoglobin (Mb), human serum albumin (HSA), casein from bovine milk (Cas), L-alanine, L-leucine, L-phenylalanine, Larginine, L-asparagine, L-aspartic acid, L-glutamine, L-histidine, L-isoleucine, L-lysine, Lmethionine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, L-glutathione (GSH), Dulbecco's Modified Eagle Medium (DMEM), and Roswell Park Memorial Institute medium (RPMI) 1640. Sodium hydroxide, glycine, and hydrochloric acid were purchased from Roth, and glutamic acid was bought from Alfa Aesar. Methods: Synthesis of GNPCs: The synthesis of BSA-protected GNPCs involved a two-step seed-mediated route. First, the seeds were prepared by adding 10 mL milliQ water in a 40 mL glass vial, mixed with sodium citrate (100 mM, 25 µL), and kept under heating until the solution had reached near the boiling stage (~90 oC). Then HAuCl4 (100 mM, 25 µL) was added to the solution mixture along with freshly prepared NaBH4 (20 mM, 50 µL). The simultaneous addition was used to avoid the reduction of gold by the weak reducing agent sodium citrate, instead of the strong reducing agent NaBH4. The resulting seed NP solution was kept stirring for an additional 30 s, and then it was removed from the heater and was allowed to cool down to room temperature. The seed NPs must be stored in the fridge (4 oC) for 24 h before any further use. Then in a second step, 10 mL milliQ water was taken in a 40 mL glass vial and HAuCl4 (100 mM, 50 µL), BSA (750 µM, 60 µL), seed solution (300 µL), and ascorbic acid (100 mM, 60 µL) were added sequentially at room temperature under constant stirring (~800 revolutions per



minute (rpm)) for 30 min (Figure 1A). The resulting solution turned deep blue in color which confirmed the formation of GNPC@BSA. For the scale-up synthesis, the vial was replaced with a 250-mL flask, and the volumes of all solutions were increased by a factor of twenty-five. Similar methodologies were followed for other ligands used in this experiment. Instrumental details are mentioned in the supporting information (§SI1). ACKNOWLEDGEMENT This work was supported by the German Research Foundation (grant DFG PA 794/28-1). I.C. was supported by an Alexander von Humboldt fellowship. M.G. acknowledges The Ministry of Higher Education and Scientific Research (MHESR) of Egypt and the Deutscher Akademischer Austausdienst (DAAD) for his fellowship. The authors are grateful to Mrs. Marta Gallego (CIC Biomagune) and Dr. Andreas Kornowsky (Universität Hamburg) for the TEM images. We acknowledge CD instrumental support by the SPC facility at EMBL Hamburg. SUPPORTING INFORMATION The Supporting Information is available free of charge on the ACS Publications website: details of instrumentation characterization of seed NPs and GNPC@BSA, the effect of different synthetic parameters on UV-vis absorption spectra, structural analysis of the proteins in GNPC@BSA, understanding the importance of surface ligands. REFERENCES 1. Saha, K.; Agasti, S. S.; Kim, C.; Li, X.; Rotello, V. M., Gold nanoparticles in chemical and biological sensing. Chem. Rev. 2012, 112 (5), 2739-2779, DOI 10.1021/cr2001178. 2. Daniel, M.-C.; Astruc, D., Gold Nanoparticles: Assembly, Supramolecular Chemistry, Quantum-Size-Related Properties, and Applications toward Biology, Catalysis, and Nanotechnology. Chem. Rev. 2004, 104 (1), 293-346, DOI 10.1021/cr030698+. 3. Chakraborty, I.; Pradeep, T., Atomically Precise Clusters of Noble Metals: Emerging Link between Atoms and Nanoparticles. Chem. Rev. 2017, 117 (12), 8208-8271, DOI 10.1021/acs.chemrev.6b00769. 4. Grzelczak, M.; Perez-Juste, J.; Mulvaney, P.; Liz-Marzan, L. M., Shape control in gold nanoparticle synthesis. Chem. Soc. Rev. 2008, 37 (9), 1783-1791, DOI 10.1039/B711490G. 5. Li, N.; Zhao, P.; Astruc, D., Anisotropic Gold Nanoparticles: Synthesis, Properties, Applications, and Toxicity. Angew. Chem. Int. Ed. 2014, 53 (7), 1756-1789, DOI 10.1002/anie.201300441. 6. Giljohann, D. A.; Seferos, D. S.; Daniel, W. L.; Massich, M. D.; Patel, P. C.; Mirkin, C. A., Gold Nanoparticles for Biology and Medicine. Angew. Chem. Int. Ed. 2010, 49 (19), 328094, DOI 10.1002/anie.200904359. 7. Alvarez-Puebla, R. A.; Zubarev, E. R.; Kotov, N. A.; Liz-Marzan, L. M., Self-assembled nanorod supercrystals for ultrasensitive SERS diagnostics. Nano Today 2012, 7 (1), 6-9, DOI 10.1016/j.nantod.2011.11.001. 8. Nel, A.; Xia, T.; Madler, L.; Li, N., Toxic potential of materials at the nanolevel. Science 2006, 311 (5761), 622-627, DOI 10.1126/science.1114397.


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TOC Popcorn-shaped gold nanoparticles (GNPC) was synthesized through a sustainable route using protein as a ligand. GNPC@Protein shows high colloidal stability and biocompatibility compare to other GNPs of similar size.

Sustainable Route

Biocompatibility

Gold Nanopopcorn

High Colloidal Stability


Tunable Plasmon

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Sustainable Synthesis and Improved Colloidal Stability of Popcorn

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