Gold Nanoparticle Enantiomers and Their Chiral-Morphology Dependence of Cellular Uptake

Abstract

Open AccessCCS ChemistryRESEARCH ARTICLE1 Feb 2022Gold Nanoparticle Enantiomers and Their Chiral-Morphology Dependence of Cellular Uptake Ning-Ning Zhang†, Hao-Ran Sun†, Shuhan Liu, Yu-Chen Xing, Jun Lu, Fei Peng, Cheng-Long Han, Zhonglin Wei, Tianmeng Sun, Bai Yang and Kun Liu Ning-Ning Zhang† State Key Laboratory of Supramolecular Structure and Materials, College of Chemistry, Jilin University, Changchun 130012 , Hao-Ran Sun† State Key Laboratory of Supramolecular Structure and Materials, College of Chemistry, Jilin University, Changchun 130012 , Shuhan Liu Key Laboratory of Organ Regeneration and Transplantation of Ministry of Education, Institute of Immunology, The First Hospital, Jilin University, Changchun 130012 National-Local Joint Engineering Laboratory of Animal Models for Human Diseases, Changchun 130012 , Yu-Chen Xing State Key Laboratory of Supramolecular Structure and Materials, College of Chemistry, Jilin University, Changchun 130012 , Jun Lu Department of Chemical Engineering, University of Michigan, Ann Arbor, MI 48109 , Fei Peng State Key Laboratory of Supramolecular Structure and Materials, College of Chemistry, Jilin University, Changchun 130012 , Cheng-Long Han State Key Laboratory of Supramolecular Structure and Materials, College of Chemistry, Jilin University, Changchun 130012 , Zhonglin Wei Department of Organic Chemistry, College of Chemistry, Jilin University, Changchun 130012 , Tianmeng Sun Key Laboratory of Organ Regeneration and Transplantation of Ministry of Education, Institute of Immunology, The First Hospital, Jilin University, Changchun 130012 National-Local Joint Engineering Laboratory of Animal Models for Human Diseases, Changchun 130012 International Center of Future Science, Jilin University, Changchun 130012 , Bai Yang State Key Laboratory of Supramolecular Structure and Materials, College of Chemistry, Jilin University, Changchun 130012 and Kun Liu *Corresponding author: E-mail Address: [email protected] State Key Laboratory of Supramolecular Structure and Materials, College of Chemistry, Jilin University, Changchun 130012 Chiral Nanomaterial Research Center, International Center of Future Science, Jilin University, Changchun 130012 https://doi.org/10.31635/ccschem.021.202000637 SectionsSupplemental MaterialAboutAbstractPDF ToolsAdd to favoritesDownload CitationsTrack Citations ShareFacebookTwitterLinked InEmail Chiral molecules are widely prevalent in nature and biological systems, and artificial chiral nanoparticles have drawn enormous interest owing to their unique optical and physical properties. However, nanoparticles with chiral morphologies and their potential role in biology have been rarely explored. Herein, we report a seed-mediated synthesis of enantiomorphic Au nanooctopods (NOPs) and their chiral-morphology dependence of cellular uptake. With a high yield (∼80%), the chiral NOPs possess eight uniform arms that bend from ⟨111⟩ to ⟨100⟩ directions, like a propeller structure. The chiral NOPs synthesized with l- or d-glutathione (GSH) have opposite handedness, resulting in opposite circular dichroism signals, which is consistent with finite-difference time-domain simulations. d-GSH NOPs demonstrate greater than 30% (ca. 15%) enhanced cellular uptake in GL261 and bEnd.3 cells compared with l-GSH NOPs (racemic NOPs). Moreover, d-GSH NOPs modified with poly(ethylene glycol) or l-GSH are also preferred by the cells, proving the chiral-morphology dependence of cellular uptake. Our study develops the exploration of the chiral-specific interaction in biological systems, providing potential applications for drug delivery, biosensing, and tumor detection. Download figure Download PowerPoint Introduction Chirality is a ubiquitous phenomenon in nature.1,2 Nature's biological systems select l-handed amino acids and d-handed sugars and exclude their enantiomers.3,4 Even for artificial chiral molecules, the natural biological system shows specific chiral recognition, that is, one enantiomer can perform important functions, while the other could be inactive or even toxic.5,6 For instance, the (R)-thalidomide exerts a sedative effect, while its enantiomer (S)-thalidomide has embryotoxic and teratogenic effects.7 Recently, the fascinating functions of chiral nanoparticles (NPs) have attracted extensive research, due to the structural parallels between them and biomaterials as well as potential biomedical applications.8–12 The generation of chiral NPs involves three general strategies: (type I) achiral NPs with chiral surface molecular ligands,13–15 (type II) assembly of achiral NPs with chiral spatial arrangement normally induced by chiral molecular linkers,16–25 and (type III) single NPs with chiral morphologies generally synthesized with chiral surface ligands.26–28 The cellular interactions of achiral NPs with chiral surface ligands (type I) have been extensively studied.29–33 Biomolecules (proteins, peptides, and aptamers) on cell membranes show specific recognition of the chiral ligands on NP surfaces, resulting in chirality-dependent cellular uptake. The interaction of chiral assemblies of NPs (type II) with biological systems has also been demonstrated.34–36 For example, the Kuang group directly observed the selective autophagy induction in breast cancer cells by chiral tetrahedral assemblies of NPs linked by DNA. After entering the cells, the chiral assemblies are disassembled by the enzymatic digestion, which reduces the intracellular circular dichroism (CD) signal.37 In contrast to type I and II chiral NPs, single NPs with chiral morphology (type III) can maintain their chirality even after the removal or loss of their chiral surface ligands or linkers,38–40 which can illustrate the effect of nanoscale chiral morphologies on cellular interactions. Recently, Nam and co-workers41 reported the amino acid- and peptide-directed synthesis of well-defined chiral-morphology Au NPs with a size close to 200 nm, and the enantiomorphic Au NPs have the potential for the development of biologically responsive and tunable metamaterials. For cellular uptake, however, inorganic NPs within the 2–100 mm size range are preferred.42–45 Therefore, to illustrate the biological properties of inorganic NP enantiomers, it is of great significance to synthesize small size NPs with well-defined opposite chiral morphologies. Herein, we demonstrate a seed-mediated synthesis of enantiomorphic Au nanooctopods (NOPs) with the addition of l-/d-glutathione (GSH) and their chiral-morphology dependence of cellular uptake. The chiral NOPs possess eight bent arms with a propeller-like structure. With the assistance of finite-difference time-domain (FDTD) simulations, we explored the relationship between chiral NOP morphologies and their chiral plasmonic properties. Importantly, we studied the differences in cellular uptake efficiency of l-GSH, d-GSH, and racemic NOPs, and confirmed the chiral morphology-dependent cellular uptake with a series of control experiments. Experimental Methods Synthesis of chiral NOPs In a typical experiment, the aqueous solutions of hexadecyltrimethylammonium bromide (CTAB; 4.8 mL, 0.017 mol/L), gold(III) chloride (HAuCl4; 5.0 μL, 15 mmol/L), 1-methylpyrrolidine (1-MP; 0.030 mL, 1.0 mol/L), ascorbic acid (AA; 0.475 mL, 0.10 mol/L), and GSH (4.0 μL, 10 mmol/L) were added in a glass vial. Subsequently, the NOP (see Supporting Information for details) seed solution (50 μL, 10 nmol/L) was added and mixed quickly. After 15 min of reaction at 30 °C, the HAuCl4 in the growth solution had completely reacted, and then HAuCl4 (5.0 μL, 15 mmol/L) was supplemented to continue growth. The whole growth process requires eight-step growth at 30 °C, which means that HAuCl4 (5.0 μL, 15 mmol/L) must be supplemented seven times, and every step takes 15 min to complete. Finally, the gray-violet chiral NOP solution (0.10 nmol/L) was obtained. Cellular uptake efficiency Cellular uptake of chiral NOPs and their control groups were performed by quantifying the Au concentration in the cells at 2 h. First, GL261 cells (1.2 × 105 cells/well) and bEnd.3 cells (1.2 × 105 cells/well) were seeded in 24-well plates for 24 h. Then the culture medium was replaced with 100 μL Dulbecco's modified Eagle's medium (DMEM) containing Au NPs (final concentration, 3.3 nmol/L) and incubated at 37 °C for 2 h. Finally, the Au NPs that had not been taken up by the cells were removed, and then the cells were collected. The cell cytoskeleton was eliminated by trypsin, and the Au NPs in cells were dissolved by aqua regia. The Au concentrations in the cells were measured by inductively coupled plasma mass spectrometry (ICP-MS). Results and Discussion The chiral Au NOPs were prepared by the regrowth of single-crystalline Au NOP seeds synthesized with a method previously reported ( Supporting Information Figure S1).46,47 In a typical synthesis of chiral NOPs, the regrowth of NOP seeds was achieved by reducing HAuCl4 through a mixture of coreducing agents, that is, 1-MP and AA in a CTAB aqueous solution. GSH was used as a chiral additive for the regrowth of NOP seeds to chiral NOPs. The prepared chiral NOPs were characterized by scanning electron microscopy (SEM) and CD spectroscopy, as shown in Figure 1. Figure 1 | Characterization of chiral NOPs. (a) The extinction and (b) CD spectra of chiral Au NOPs in the vis–NIR region. (c) SEM images of l-GSH NOPs. (d) Models and corresponding SEM images of (left) l- and (right) d-GSH NOPs. Download figure Download PowerPoint The SEM image shows the l-GSH NOP consisted of eight bent arms (Figure 1c). The initial NOP seeds possess eight straight arms with a uniform arm length of 19.5 ± 1.4 nm and width of 9.4 ± 1.0 nm, corresponding to a longitudinal surface plasmon resonance (LSPR) at 705 nm and a transverse surface plasmon resonance (TSPR) at 515 nm, respectively, as shown in its extinction spectrum ( Supporting Information Figure S1a). After the regrowth, the chiral NOPs (with a bent arm length of 42.6 ± 4.6 nm and width of 19.1 ± 2.0 nm) show a strong LSPR peak at 810 nm and a weak TSPR peak at 540 nm (Figure 1a and Supporting Information Figure S2a). Their CD spectrum displayed two bisignate waves, of which the zero-cross points perfectly matched with the peak positions of the LSPR and TSPR bands (Figure 1b). The effect of the split TSPR and LSPR bands on the corresponding CD spectrum is agreement with the Cotton effect.48 In addition, the exact opposite CD signal can be obtained if the chiral NOPs are synthesized under the same conditions except using d-GSH, the enantiomer of l-GSH ( Supporting Information Figure S2). The synthesized l- and d-GSH NOPs are twisted in opposite directions depending on the l- and d-GSH (Figure 1d). This result indicates the chirality of Au NOPs is controlled by the chirality of GSH. The anisotropic factors (g-factor) of chiral NOPs, which are the ratio of their molar CD to molar extinction, can reach to 0.002 at both 620 and 780 nm ( Supporting Information Figure S2d). In addition, the surface adsorption of l-GSH on achiral NOP seeds can only induce negligible CD signals (⟨1 mdeg) in the plasmon band positions ( Supporting Information Figure S3). This result indicates the large CD signals of chiral NOPs are mainly attributed to the twisted morphology of chiral NOPs rather than chiral surface ligands. It is worth mentioning that compared with other branched Au NPs reported in the literature,49,50 the chiral NOPs show excellent long-term stability in aqueous solution at room temperature and in serum at 37 °C ( Supporting Information Figure S4), which is important for them to be used in biomedicine applications. We further studied the regrowth process of chiral NOPs from the NOP seeds induced by l-GSH. The regrowth was conducted in a step-growth process in which the Au precursor (HAuCl4) was added every 15 min for eight steps. In contrast, if all Au precursor was introduced in a single step, cubic Au NPs with a negligible CD signal were obtained ( Supporting Information Figure S5). We monitored the regrowth process at the end of each step by using transmission electron microscopy (TEM), UV–vis, and CD spectroscopies (Figure 2). During each step, the precursor was completely consumed after 15 min, as proven by the fact that the extinction values at 400 nm were proportional to the amount of Au precursor added.51 Figure 2 | Regrowth process of chiral l-GSH NOPs. (a–h) TEM images of Au nanocrystals at representative stages of the synthesis of l-GSH NOPs: (a) 0, (b) 5, (c) 15, (d) 30, (e) 45, (f) 60, (g) 75, and (h) 120 min, respectively. (i) Extinction and (j) CD spectra of l-GSH NOPs at different reaction times. Download figure Download PowerPoint TEM study shows that the tips of the arms started to grow asymmetrically after the second step (Figures 2a–2d). Meanwhile, the corresponding CD spectrum (blue line) shows a clear bisignate CD wave with a negative peak at 520 nm and a positive peak at 727 nm (Figure 2j), indicating asymmetric growth of the arms forming a chiral structure (Figure 2d). By the third step, the arm bent significantly (Figure 2e), and the arm width was increased from 9.9 to 14.2 nm, resulting in a new TSPR peak at 550 nm (Figure 2i). Consequently, the bisignate CD wave was separated into two opposite bisignate waves with a left-handed wave (the zero-cross point at 570 nm) at the TSPR band and a right-handed wave (the zero-cross point at 800 nm) at the LSPR band. As shown in the sequential images of the growth process (Figures 2f–2h), the sizes of arms gradually increased, and the edges of bent arms continued to sharpen and evolve into the final morphology. Both the extinction and CD spectra of l-GSH NOPs are consistent with their morphology changes in the TEM images. The above results reveal the arms of NOP seeds bend during the epitaxial growth, forming the final chiral NOPs. The structure of l-GSH NOP was further characterized by SEM, which shows the chiral NOP is composed of eight bent arms with a yield of around 80% (Figure 1c). Additionally, close inspection of the l-GSH NOP along the [001], [111], and [110] views reveals that the roots of bent arms are still along the ⟨111⟩ directions of the seed arms, and the tops of the bent arms are toward the ⟨100⟩ directions (Figure 3a). The high-resolution TEM (HRTEM) image in the [100] view confirms the arms are bent from ⟨111⟩ to ⟨100⟩ directions (Figure 3b). As shown in Figure 3c, the TEM analysis of over 100 chiral NOPs reveals that the bent angles between the root and top parts of the arms were ca. 120°, which is consistent with the angle between ⟨111⟩ and ⟨100⟩ of 125.3°. Based on the above TEM and SEM image analysis, we constructed an ideal three-dimensional (3D) model of the l-GSH NOP (Figure 3d). In the top view, the four arms bend from ⟨111⟩ to ⟨100⟩ in the (001) face, so that arranged in a clockwise direction, forming a structure similar to a four-bladed propeller (Figure 3e). In the bottom view, there are the other four bent arms also arranged in a clockwise direction. Therefore, the chiral NOP consists of two opposite four-bladed propellers, which is confirmed in the 3D electron tomography reconstruction of l-GSH NOP (Figures 3f and 3g). Figure 3 | Morphology of l-GSH NOPs. (a) The SEM images of individual l-GSH NOPs, as viewed along the [001], [111], and [110] directions, respectively. (b) HRTEM image of a bent arm, its corresponding fast Fourier transform pattern is shown in the inset. Scale bar: 5 nm. (c) TEM image of l-GSH NOPs, its inset highlights the bent angle of arms. (d) Schematic model of the bent directions of l-GSH NOP arms. (e) Photographs of the four-bladed propeller. (f) 3D electron tomography reconstruction of individual l-GSH NOPs. (g) Overlay of a NOP seed and l-GSH NOP model aligned in the same direction. Download figure Download PowerPoint Close inspection of the TEM and SEM images (Figures 3a and 3c) reveals the chiral NOPs possess extremely complicated configurations because the arms from ⟨111⟩ can bend into three possible ⟨100⟩ directions. For example, as shown in Figure 4a, the [111] arm can bend into [100] (red), [010] (blue), and [001] (green) at vertex I, corresponding to the clockwise, anticlockwise, and perpendicular directions viewed in the (001) face. It should be noted that even when the four arms are arranged in a clockwise direction (marked in red frame) in the top (001) and bottom ( 00 1 ¯ ) faces, they form in an anticlockwise direction (marked in blue frame) in the other four side faces, as shown in Figure 4b. In this case, if the [111] arm bends to [001], the six {100} faces show five different configurations, including clockwise, anticlockwise, and achiral arrangements (Figure 4c). These results indicate that when viewed in the different {100} faces, the bent arms exhibit various configurations with different chirality contributions. Figure 4 | Complicated configurations of l-GSH NOPs. (a) Schematic model of the bending directions of arms. (b) Schematic model of the l-GSH NOP with the four arms arranged in a clockwise direction both in the top and bottom faces. (c) The schematic model of l-GSH NOP when the [111] arm in (b) bends to [001]. The cases of models viewed from different {100} faces are listed in right. (d) Schematic models of {100} faces when there are 0 (0⊥), 1 (1⊥), 2 (separation) (2′⊥), 2 (adjacent) (2″⊥), 3 (3⊥), and 4 (4⊥) arms perpendicular to {100} faces. Statistical results of the {100} faces of (e) all possible l-GSH NOP models and (f) l-GSH NOPs in SEM images. Download figure Download PowerPoint To understand the complicated configurations and their chirality contributions, we make a category for different {100} faces based on the arm arrangement. We named the faces as 0⊥, 1⊥, 2′⊥, 2″⊥, 3⊥, and 4⊥ for 0, 1, 2 (separation), 2 (adjacent), 3, and 4 arms perpendicular to the {100} faces, respectively (Figure 4d and Supporting Information Figure S6). The fraction of each type of face for all possible chiral NOP models ( Supporting Information Figure S7) is summarized in Figure 4e. The result shows that 1⊥ is the most frequent face with a probability of 42.5%, and the second and third most frequent faces are 2′⊥ and 0⊥, respectively. The theoretical statistical results are consistent with the statistical results based on the SEM image analysis of l-GSH NOPs (Figure 4f). This result indicates that the l-GSH NOPs have complex and diverse chiral structures. Importantly, both the theoretical and experimental statistic results show that the clockwise configurations are dominated for the chiral NOPs synthesized with l-GSH (Figures 4e and 4f), which is consistent with the right-handed bisignate CD spectrum of their ensemble solution (Figure 1b). We performed the FDTD simulation to understand the relationship between 3D morphologies and chiral plasmonic properties of chiral Au NOPs. We considered the random orientations of chiral NOPs in solution and used orientational averaging to generate the ensemble spectra ( Supporting Information Figure S8), and the simulated extinction and CD spectra (Figures 5a and 5b, respectively) of l- and d-GSH NOP models are in good agreement with the corresponding experimental spectra (Figures 1a and 1b). The CD spectra of the chiral NOP models for l- and d-GSH display opposite signals, both of which show two bisignate waves. Particularly, the CD spectrum of l-GSH NOP displays a left-handed bisignate wave with a negative peak at 520 nm and a right-handed bisignate wave with a positive peak at 750 nm, which correspond to the TSPR and LSPR bands, respectively. Furthermore, we simulated the nearfield distribution of electric and magnetic fields of chiral NOP models under left circularly polarized (LCP) light and right circularly polarized (RCP) light at 520 and 750 nm, respectively (Figures 5c and 5d and Supporting Information Figure S9). The different distributions of electric and magnetic moments indicate the rotation of arms induces a CD response in the vis–near-infrared (NIR) region. Figure 5 | FDTD simulation of the optical properties of chiral Au NOPs. (a) Simulated extinction and (b) CD spectra of the models of l- and d-GSH NOPs. (c) Electric- and (d) magnetic-field intensities on l-GSH NOP upon normal incidence of LCP and RCP light at different wavelengths. Download figure Download PowerPoint The desirable stability and suitable size of chiral NOPs allow them to exhibit considerable superiorities in biomedical applications. To study the effects of chiral morphologies of chiral NOPs on their transport and biological effects in biological systems, we compared the cellular uptake efficiency of l- and d-GSH NOPs in mouse glioblastoma (GL261) cells and mouse brain endothelial (bEnd.3) cells after 2 h of incubation. Surprisingly, the cell uptake efficiencies of the d-GSH NOPs for GL261 and bEnd.3 cells were about 40% and 30% more than those of l-GSH NOPs, respectively (Figures 6a and 6b). The cellular uptake efficiency of racemic NOPs was between that of l- and d-GSH NOPs (Figure 6b). The intracellular localization of Cy5-labeled chiral NOPs was further visualized by confocal microscope imaging. Figure 6c shows a noticeably higher number of d-GSH NOPs on the cell membrane and cytoplasm of GL261 and bEnd.3 cells compared with l-GSH and racemic NOPs. The results suggest the d-GSH NOPs had stronger adhesion for cell membranes, which contributed to their higher cellular uptake These results indicate the l- and d-GSH NOPs show a in their cellular uptake efficiency for both normal and tumor Figure | Chiral morphology-dependent cellular uptake. (a) Schematic of the chiral morphology-dependent cellular uptake. (b) Cellular uptake efficiencies of chiral and racemic NOPs in GL261 and bEnd.3 cells for 2 h (c) microscope images of GL261 and bEnd.3 cells are the cytoskeleton is with Cy5-labeled l-GSH, and d-GSH NOPs for 2 h. Scale bar: (d) measured the molecular of the of l-GSH, and d-GSH NOPs in Cellular uptake of chiral NOPs, chiral [email chiral NOPs with opposite chiral GSH ligands [email is d-GSH [email is NOP [email and [email protected] in (e) GL261 and (f) bEnd.3 indicate were considered when Download figure Download PowerPoint Chiral NOPs possess chiral morphologies and chiral ligands both of which can the cellular uptake efficiency of chiral NOPs. To the role of chiral we modified the surface of chiral NOPs with to cellular interactions of chiral surface ligands. The results indicate that chiral NOP show to cellular there was still about a 30% in cellular uptake of d-GSH NOPs compared with l-GSH NOPs. Furthermore, we modified chiral NOPs with opposite chiral GSH to d-GSH NOPs with l-GSH ligands [email and l-GSH NOPs with d-GSH ligands [email The results reveal that the cellular uptake efficiency of d-GSH [email was higher than l-GSH [email (Figures and These results the GL261 and bEnd.3 cells preferred to the d-GSH NOPs that is, the chiral morphologies dominated the uptake of chiral NOPs. In addition, the in cellular uptake of l- and d-GSH ligands modified achiral NPs seeds and was negligible (Figures and The results further confirmed that chiral morphology a more important role in cell interactions compared with chiral surface ligands. to of l-GSH, surface of chiral NPs by l-GSH the dependence of cellular uptake. In NPs with chiral morphologies could their functions by the chiral in NPs can also with chiral surface with Additionally, we the on chiral and racemic NOPs in cell culture medium (DMEM) containing serum by three of NOPs small of with negligible differences (Figure The result the surface properties of different chiral NOPs were similar entering cells, which the of surface differences and further confirmed the chiral-morphology dependence of cellular uptake. we measured the potential of NOP ( Supporting Information Figure which shows have similar surface NOPs: ± d-GSH NOPs: ± The result that the in cellular uptake is mainly by the different chiral morphologies rather than their surface the of the interaction between the cell membrane and chiral morphology NOPs must be further to the of this the cellular uptake of chiral-morphology single NPs for the We have the synthesis of enantiomorphic Au NOPs and their chiral-morphology dependence on cellular uptake. With a we the arms of NOP seeds were bent during epitaxial growth, resulting in chiral morphology NOPs with a propeller-like structure. Furthermore, we confirmed the arms are bent from ⟨111⟩ to ⟨100⟩ directions with complicated FDTD of chiral plasmonic properties of chiral NOPs indicate their chiral morphologies can induce a CD response in the which are consistent with the experimental d-GSH NOPs exhibit cell uptake for GL261 and bEnd.3 cells compared with the l-GSH and racemic NOPs. Chiral morphology a more role in cell interactions compared with chiral surface ligands. the chiral morphology NOPs have great potential in the interactions between chiral and This a new for further development of chiral Supporting Information Supporting Information is and of NOP TEM images of d-GSH NOPs, the stability of chiral NOPs, a of growth for different {100} faces based on arm arrangement of all possible cases of l-GSH NOPs, and FDTD simulation of is of interest to the of and for and the of the First of Jilin University This is also by the for and Research 1. Chirality and the on and on the of Chirality of and in 45, Chirality and Chirality to The of Chiral 1,