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Cytotoxicity of gold nanoparticles with different structures and surface-anchored chiral polymers
Accepted Manuscript Cytotoxicity of gold nanoparticles with different structures and surface-anchored chiral polymers Jun Deng, Mengyun Yao, Changyou Gao PII: DOI: Reference:
S1742-7061(17)30088-0 http://dx.doi.org/10.1016/j.actbio.2017.01.082 ACTBIO 4717
To appear in:
Acta Biomaterialia
Received Date: Revised Date: Accepted Date:
20 September 2016 24 January 2017 30 January 2017
Please cite this article as: Deng, J., Yao, M., Gao, C., Cytotoxicity of gold nanoparticles with different structures and surface-anchored chiral polymers, Acta Biomaterialia (2017), doi: http://dx.doi.org/10.1016/j.actbio. 2017.01.082
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Cytotoxicity of gold nanoparticles with different structures and surface-anchored chiral polymers Jun Deng1, Mengyun Yao1, Changyou Gao* MOE Key Laboratory of Macromolecular Synthesis and Functionalization, Department of Polymer Science and Engineering, Zhejiang University, Hangzhou 310027, China. 1
co-first author, *Corresponding author.
Email: [email protected] Fax: +86-571-87951108
1
Abstract Nanoparticles (NPs) can have profound effects on cell biology. However, the potential adverse effects of gold nanoparticles (AuNPs) with different surface chirality and structures have not been elucidated. In this study, monolayers of poly(acryloyl-L(D)-valine (L(D)-PAV) chiral molecules were anchored on the surfaces of gold nanocubes (AuNCs) and nanooctahedras (AuNOs), respectively. The L-PAV-AuNCs and D-PAV-AuNCs, or the L-PAV-AuNOs and D-PAV-AuNOs, had identical physicochemical properties in terms of size, morphology and ligand density except of the reverse molecular chirality on the particle surfaces, respectively. The L-PAV capped AuNCs and AuNOs exhibited larger cytotoxicity to A549 cells than the D-PAV coated ones, and the PAV-AuNOs had larger cytotoxicity than PAV-AuNCs when being capped with the same type of enantiomers, respectively. The cytotoxicity was positively correlated with the cellular uptake amount, and thereby the production of intracellular reactive oxygen species (ROS). Keywords:
Gold
nanoparticles;
nanoparticle
structure;
cytotoxicity.
2
poly(acryloyl-L(D)-valine;
chirality;
Introduction Nanoparticles (NPs) hold great promise for biomedical applications such as imaging [1], diagnostics [1], drug delivery [2], and sensing [3]. There is, however, a large knowledge gap on the biological mechanisms arising from NPs interactions with human systems. These uncertainties aggravate concerns especially when NPs are found to adversely affect cell viability or functions [4, 5]. Therefore, it is urgent to elucidate the interactions of NPs with biological systems including the living cells and tissues, which play a predominant role in determining the efficacy [6] and toxicity of NPs in biological and environmental systems [7]. The most interesting properties of NPs are their size-, morphologyand surface-induced effects. So far the effects of NPs size [8], shape [9], surface charge [10], and chemical compositions [9] on intracellular trafficking, toxicity, and gene expression have been extensively investigated. NPs are often considered as an idealized shape (e.g. spheres or rods), while they are actually nanocrystals with complex facet and defect structures [11, 12]. The NPs surface may become unstable because of their large surface curvature, and large fractions of edge and corner regions, resulting in high surface energy [13]. The potentially unstable surface of the NPs is usually coated by chemisorbed ligands. These properties of the NPs are significantly influenced by the NPs’ topography, which in turn dramatically influences on how the nanoparticles interact with their surrounding environment [14]. However, most of the previous studies focus on the interactions of biological systems with idealized NPs (e.g. spheres and rods [15], spheres and bowl [16]). It is urgent to research the effects of shape with large fractions of edge and corner regions on cytotoxicity and cell behaviors. Biological systems respond strongly to NP surfaces. NPs can interact with cellular components such as DNAs, proteins and lipids. Interactions of surface-functionalized NPs with cells can damage the
3
integrity of membrane structure, with the possibility of leakage of cytosol depending on the NP surface chemistry [17]. For example, cationic molecules-modified NPs show higher toxicity than their anionic analogues [18]. Although many studies have demonstrated that surface modification of NPs with biomolecules could endow them with various biological functionalities [19, 20], the impact of chirality of the surface biomolecules on living organisms has been largely neglected. Chirality is an important phenomenon in living systems, and most of the biological molecules are homochiral to take functions [21]. It is known that all amino acids in proteins (except of glycine) and phospholipids in cell membrane are “left-handed”, whereas all sugars in DNAs and RNAs are “right-handed” [22, 23]. Different chiral properties of biomolecules may determine their ability to interact with other biomolecules, and thereby modulate a range of downstream processes. Although some previous works show how surface chirality affects the cellular uptake [24], protein adsorption [25] and functions of the NPs (e.g. cell imaging ability of gold nanoclusters [26] and quantum dots (QDs) [27]), little is known about the effect of surface chirality at the nanoscale on the subsequent cytotoxicity, genotoxicity and cell functions such as adhesion and migration. Gold nanoparticles (AuNPs) have gained much popularity recently because of their unique chemical and physical properties (e.g. size- and shape-dependent optical and electronic features, high surface-to-volume ratio, excellent biocompatibility and chemical stability) [27, 28]. Although AuNPs are considered as inert and biocompatible, there are contradictory results concerning their cytotoxicity [29]. Therefore, in this study AuNPs are served as an ideal platform to investigate the surface chirality at the nanoscale on cytotoxicity, genotoxicity and cell functions. Valine is one of the eight essential amino acids of human body, and plays essential roles in a wide variety of physiological processes [30, 31]. Our previous work found that the gold nanospheres
4
surface-coated by 2-mercaptoacetyl-L(D)-valine (L(D)-MAV) has a chirality-independent cellular uptake behavior. However, the difference in internalization of AuNPs is prominent by anchoring poly(acryloyl-L(D)-valine) with enhanced chirality-selectivity [24]. Moreover, gold spheres with surface-anchored chiral poly(acryloyl-L(D)-valine) induce differential response on mesenchymal stem cell osteogenesis [32]. Therefore, the L- and D-valine are selected as the chiral centers, and poly(acryloyl-L-valine) (L-PAV) and poly(acryloyl-D-valine) (D-PAV) are synthesized via reversible addition-fragmentation chain transfer polymerization to enhance the chiral effect [24]. Two different shapes of AuNPs, i.e. gold nanocubes (AuNCs) and gold nanooctahedras (AuNOs) are synthesized and coated with either L-PAV or D-PAV via the thiocarbonylthio-Au bond, respectively (Scheme 1). Lung is one of the major organs that NPs accumulate when they enter the body. Therefore, lung cells, i.e. A549 cells are widely used in vitro as a model to study cell-NPs interactions [33, 34]. The chirality-associated and shape-dependent regulation of cytotoxicity and genotoxicity highlight the important role of conformation of the stabilizers and shapes of NPs, and has important implications for the design of novel AuNPs possibly applied in biological field. 2 Materials and methods 2.1 Materials Gold (III) chloride hydrate (HAuCl4) were purchased from Sinopharm group Co. Ld. 1,5-Pentanediol (96 %) (PD) was obtained from Aladdin Company. Polyvinylpyrrolidone (PVP, Mw ~55 kDa) and 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from
Sigma.
Poly(acryloyl-L-valine)
(L-PAV,
Mw
3580
Da,
polydispersity
1.14)
and
poly(acryloyl-D-valine) (D-PAV, Mw 3611 Da, polydispersity 1.15) were synthesized according our previously reported method [24]. A549 cells were purchased from the Cell Bank of Typical Culture
5
Collection of Chinese Academy of Sciences, and all reagents for the cell culture were used directly. All other chemicals were of analytical grade and used without further treatment if not specially mentioned. The MilliQ water was used throughout the experiments. For gold nanoparticle synthesis, all glasswares used were cleaned by freshly prepared aqua regia solution (HCl/HNO3, 3:1). 2.2 NPs synthesis and surface functionalization Gold nanocubes (AuNCs) and gold nanooctahedras (AuNOs) were synthesized using a high temperature polyol process [35]. The AuNCs or AuNOs solution was obtained by centrifugation at 500 rpm for 5 min at 10 oC, and the supernatant was decanted into a second tube to remove any larger particles or aggregates. The supernatant solution was washed 5 times with ethanol by centrifugation (4000 rpm, 30 min, and 10 oC) to remove excess PVP, and the concentrated AuNCs or AuNOs solution was collected as a stock solution. The surface of the obtained AuNCs and AuNOs was functionalized with L-PAV and D-PAV using a ligand exchange protocol, respectively. To ensure complete exchange, the stock NP solution was incubated with a large amount of 1 mg·mL-1 L-PAV and D-PAV solution under heavily shaking at room temperature overnight, respectively. The solution was then sonicated and centrifuged to remove the excess PAV molecules. The NPs were re-dispersed in ethanol solution with 1 mg·mL-1 L-PAV and D-PAV, and the solution was stirred for another 12 h, respectively. Then the NPs were washed with ethanol solution 3 times. Finally, the NPs were re-dispersed in water for further characterizations. 2.3 NP characterization The morphology and dimensions of NPs were obtained from transmission electron microscopy (TEM, H-7650) and scanning electron microscopy (SEM, SIRION-100, FEI), respectively. The TEM samples were prepared by depositing 10 µ L of diluted sample solution on a carbon-coated copper grid, and
6
dried under atmosphere overnight. The SEM samples were prepared by depositing diluted NP solutions on silica wafers, and dried under atmosphere overnight. The size distribution of NPs was obtained from both TEM and SEM micrographs using the Image J software, with at least 500 particles counted per sample, respectively. Nanocube (NC) dimensions were measured by averaging two side lengths of each particle (Double arrow in Figure 1 a1, b1), and nanooctahedra (NO) dimensions were determined from 1 to 2 side measurements per particle (Double arrow in Figure 1 c1, d1). SEM micrographs at several tilt angles were also taken for AuNCs and AuNOs to determine the nanoparticles’ height. The gold concentration of AuNCs (mc, g·mL-1) or AuNOs (mo, g·mL-1) solutions was measured by using inductively coupled plasma mass spectrometry (ICP-MS, Thermo Elemental Corporation of USA, XSeries II), respectively. To determine the NP concentration, individual nanoparticle mass was calculated by using the particle volume calculated with the NPs’ edge length with assumption of “perfect” morphology. The NCs’ volume was determined simply to be ac3, where ac is the edge length of the cube. NOs’ volume was determined to be [2/(31/2)]ao3 where ao is the edge length of the octahedron. Individual particle mass was calculated by using the particle volume (V) and the gold density (ρ, 19.3 g.cm-3). The particle number per milliliter (mL) was obtained by dividing the Au concentration by the individual particle mass. With these parameters, the molar concentration of gold NPs (AuNCs or AuNOs) per mL was calculated by c=1000 m/ρVNA. The circular dichroism (CD) spectrum was obtained by using a JASCO-820 spectropolarimeter equipped with a thermostatically controlled cell holder. The temperature of the sample was controlled at 25 oC. The UV region was scanned between 190 and 260 nm with an average of 3 scans. The concentrations of PAV-AuNCs and PAV-AuNOs were kept at 50 pM. The final spectra (∆θ) of
7
PAV-AuNCs and PAV-AuNOs were obtained by subtracting the spectrum of gold NPs (without PAV grafting) solution of same concentration, respectively. 2.4 Cell experiment NPs distribution The intracellular NPs distribution was measured using confocal laser scanning microscopy (CLSM, Leica TCS SP5). Briefly, the cells were seeded on a glass bottom cell culture dish (diameter, 20 mm) at a density of 5×103 cells/cm2 , and allowed to attach for 24 h. Then, the cells were treated with PAV-AuNCs and PAV-AuNOs at a NP concentration of 4 pM for another 24 h, respectively. After 5 washes with phosphate buffered saline (PBS), the cells were fixed with 0.4 % paraformaldehyde at 37 o
C overnight, and washed with PBS 3 times. They were further treated in 0.5% (v/v) Triton X-100
(Sigma-Aldrich) for 10 min at 37 °C to enhance the permeability of cell membrane. After being blocked
with
1%
BSA/PBS
at
37
o
C
for
2
h,
the
samples
were
stained
with
4’,6-diamidino-2-phenylindole (DAPI, sigma, 1:50) at 37 oC for 30 min, following with 5 washes with PBS. Cellular uptake The internalized amount of PAV-AuNCs and PAV-AuNOs was determined by ICP-MS, respectively. Briefly, the cells were seeded on a 12-well plate at a density of 5× 104 cells/cm2, and allowed to attach for 24 h. The medium was replaced with the fresh one containing PAV-AuNCs and PAV-AuNOs with a NP concentration of 4 pM, respectively. After 24 h, the plates were washed 5 times with PBS to remove free NPs. After the cells were harvested by trypsinization, their numbers were quantified by a cell counter. The samples were treated with aqua regia solution (HCl: HNO3 = 1:3, volume ratio) for 2
8
h. The obtained solution was diluted to determine the Au concentration by ICP-MS. The Au amount per 104 cells from ICP-MS analysis is presented as mean ± standard deviation (n=4). Cell adhesion and migration To determine the cell adhesion and migration, the A549 cells were seeded on a glass bottom cell culture dish (diameter, 20 mm) at a density of 5×103 cells/cm2, and allowed to attach for 6 h. Then, the cells were treated with PAV-AuNCs and PAV-AuNOs with a NP concentration of 4 pM, respectively. Then the cell adhesion and migration were measued using a time-lapse phase-contrast microscope (DMI 6000B, Leica). The cell spreading areas were obtained by analyzing the optical images of cells using the image J software. The migration rate was obtained using the previously reported protocols [36, 37]. Cell viability To determine the cell viability, A549 cells were plated at a density of 5×104 cells/cm2 in a 24-well plate and cultured for 24 h. The medium was replaced with fresh one containing the PAV-AuNCs or PAV-AuNOs (0.9 mL for each well) with different NP concentration from 0.5 to 8 pM. After treatment for another 24 h, 100 µL 5 mg·mL-1 MTT solution was added to each well, and the cells were further cultured at 37 °C for 3 h. The dark blue formazan crystals generated by the mitochondria dehydrogenase in live cells were dissolved with dimethyl sulfoxide (DMSO, Sigma-Aldrich). After the sample was centrifuged at 12000 g/min for 5 min, the absorbance of supernatant was measured by a microplate reader (MODEL 680, Bio Rad) at 570 nm. Measurement of reactive oxygen species (ROS) To measure the intracellular generation of ROS, the oxidant-sensitive dye 2′,7′-dichlorofluorescin diacetate (DCFH-DA, sigma) was used as described previously [38]. The generation of ROS was
9
measured by incubating one million cells with 4 pM PAV-AuNCs or PAV-AuNOs for 24 h, followed by staining with DCFH-DA for 20 min at 37 °C. A549 cells being treated with and without hydrogen peroxide (H2O2, 10 mM) were served as the positive and negative controls, respectively. Cells were then washed 3 times in serum-free medium and analyzed using flow cytometry (FACs Calibur, BD) at an excitation wavelength of 488 nm and emission wavelength of 530 nm. The concentration of the PAV-AuNCs or PAV-AuNOs was chosen based on the viability data. For each sample, 1×104 cells were collected, and data were analyzed using the WinMDI 2.8 software. Comet assay in DNA fragmentation The comet assay was used to monitor DNA damage in the cells, based on the method described previously [39]. The A549 cells were harvested and washed twice in PBS before re-suspending in Hank’s balance salt solution (HBSS, Sigma-Aldrich, St. Louis, MO) with 10 % DMSO and ethylene diamine tetraacetic acid (EDTA, 1st Base, Singapore). The cells being treated with H2O2 and without any treatment were served as the positive and negative controls, respectively. The cells were embedded in 0.7 % (w/v) low melting agarose (LMPA, Argoros) on comet slides (Trevigen, Gaithersburg, MD), and then lysed in prechilled lysis solution (2.5 M NaCl, 0.1 M EDTA, 10 mM Tris base, pH 10) containing 1 % Triton-X 100 for 1 h at 4 °C. Cells were then subjected to denaturation in alkaline buffer (0.3 M NaCl, 1 mM EDTA) for 40 min in dark at room temperature. Electrophoresis was performed at 24 V and 300 mA for 16 min. The slides were immersed in neutralization buffer (0.5 M Tris-HCl, pH 7.4) for 15 min, followed by dehydration in 70 % ethanol. The slides were air dried and stained with ethidium bromide (EB) fluorescent dye (0.2 % w/v). The tail moments of the nuclei were measured under a fluorescence microscope (Zeiss Axiovert 200), which reveal the DNA damage. Analysis
was
done
using
the
comet
imager
10
OpenComet
v1.3
(Download
from
http://www.opencomet.org/). At least 50 comets were analyzed per concentration. The comet evaluation was carried out based on the percentage of the DNA in the tail. Statistical Analysis. Four to five biological replicates and two technical repeats were performed in all the experiments. The experimental data are expressed as mean ± standard deviation, and the significant difference between groups was analyzed using one-way analysis of variance (ANOVA) (for two groups) and two-way ANOVA (for more than two groups) in the Origin software. The statistical significance was set as p < 0.05 and p < 0.01. 3 Results and discussion 3.1 Nanoparticle characterization Transmission electron microscopy (TEM) and scanning electron microscopy (SEM) observation demonstrate that the PAV-AuNCs and PAV-AuNOs were successfully synthesized with the desired morphology (Figure 1) and narrow distribution (Figure S1). Table 1 shows that the L-PAV-AuNCs and D-PAV-AuNCs had identical physicochemical properties in terms of size (~165 nm on each side) and surface molecular density (~2.0 molecules/nm2 ) determined from SEM and thermogravimetric analysis (TGA), respectively (Figure S2). The L-PAV-AuNOs and D-PAV-AuNOs had the same properties in terms of size (~110 nm in edge) and surface molecular density (~1.9 molecules/nm2) as well. By contrast, the L-PAV-AuNCs and D-PAV-AuNCs, or the L-PAV-AuNOs and D-PAV-AuNOs, showed essential mirror images of CD spectra in the region of 190 to 260 nm (Figure 1 e, f ). The CD signal of PAV-AuNCs was stronger than that of PAV-AuNOs, suggesting a larger number of PAV molecules on the PAV-AuNCs (Figure 1 e, f). The NPs’ stability was monitored through absorption measurement of the particle surface plasmon resonance (SPR), a parameter determined by coherent oscillation of conduction electrons in the gold
11
NP surface that is exquisitely sensitive to their local dielectric environment [40]. During the ligand exchange process there was no shift in the SPR peaks, which were located at 566 nm for PAV-AuNCs (Figure S3a) and 568 nm for PAV-AuNOs (Figure S3b), respectively. These results demonstrate the good colloidal stability, and the similar geometric features of the PAV-AuNCs and PAV-AuNOs. Peak broadening to some extent was evident, probably conveying a slight rounding of the nanoparticle vertices (Figure S3) as the SPR is very sensitive to the relative sharpness of the corners in anisotropic particles [41]. Moreover, no significant change of SPR was found in cell culture medium containing 10% fetal bovine serum (10% FBS/DMEM) (Figure S3), suggesting the good colloidal stability of the PAV-AuNCs and PAV-AuNOs under the cell culture conditions. In summary, the L-PAV-AuNCs and D-PAV-AuNCs, or the L-PAV-AuNOs and D-PAV-AuNOs, have identical physicochemical properties except of the reverse molecular chirality on the particle surface, allowing the correlation of cellular behaviors of PAV-AuNCs or PAV-AuNOs with the molecular chirality on their surface, respectively. 3.2 NPs distribution and cellular uptake The uptake amount and intracellular distribution of NPs are tied intrinsically with the nanotoxicity to cells. In this regard, the A549 cells were co-cultured with the PAV-AuNCs and PAV-AuNOs at a NP concentration of 4 pM, and their intracellular distribution and internalized amount were monitored by CLSM and ICP-MS, respectively. According to literatures and our experiences, the highest cellular loading of NPs is usually achieved at 24 h [34, 42], which was adopted in this study. The blue color shows the nuclei of cells stained by DAPI, while the red color corresponds to the PAV-capped AuNPs (PAV-AuNCs or PAV-AuNOs). As shown in Figure 2A, the PAV-AuNCs and PAV-AuNOs were mainly distributed in the cytoplasm around nuclei. No signal was detected in the nuclei of A549 cells
12
regardless of their geometric feature and surface chirality. Comparatively, the L-PAV capped AuNCs and AuNOs were internalized with significantly larger amount than the D-PAV capped ones, respectively (Figure 2A), which was further confirmed by quantitative analysis with ICP-MS (Figure 2B). This result is different with our previous observation when enantiomer PAV-capped spherical AuNPs were co-cultured with A549 and HepG2 cells, in which the D-PAV-AuNPs are preferentially internalized [24]. Although the intrinsic reasons are not clear so far, they are likely related with the difference in particle morphology and size, and thereby the surface protein adsorption. Indeed, the impact of surface chirality on uptake of NPs is strongly protein adsorption-dependent, and the adsorption of proteins such as albumin could weaken or even screen the influence of surface chirality [25]. Moreover, the adhesion mediators (fibrinogen, vitronectin, fibronectin and laminin) are adsorbed with larger amount on the L- enantiomer capped gold NPs, enhancing their uptake by mesenchymal stem cells [32]. For the same enantiomer of L-PAV coated NPs, the AuNOs were more significantly taken up than the AuNCs (p<0.05, p>0.01) (Figure 2B). This is not the case for the same D-PAV coated NPs, e.g. the AuNOs was internalized with a slight larger amount than the AuNCs (p>0.05). Because the volume of AuNCs was larger than that of AuNOs, the Au concentration of AuNCs was larger than that of AuNOs at the same mole concentration of NPs (Here 4 pM). Therefore, the A549 cells would prefer to internalize the AuNOs than the AuNCs of the same enantiomers coated, suggesting the significant role of particle shape on the cellular internalization. 3.3 Effect of PAV-AuNCs and PAV-AuNOs on cellular morphology The first and most readily noticeable effect following exposure of cells to nanomaterials (NMs) is the alteration in cell adhesion and migration in a monolayer culture [43, 44]. However, little is known on these effects induced by particle structure and surface chirality at the nanoscale. After being treated
13
with PAV-AuNCs or PAV-AuNOs at a concentration of 4 pM for 24 h, the spreading area of A549 cells was reduced to different extents depending on the shape and surface chirality of NPs (Figure 3A). The L-PAV capped AuNCs or AuNOs caused more severe inhibition on spreading of A549 cells compared to the NPs-free control (p<0.01), whereas the D-PAV coated ones did not significantly reduce the cell spreading (p>0.05) (Figure 3B Left). Moreover, no significant difference (p>0.05) was found between the PAV-AuNCs and PAV-AuNOs with the same enantiomers (Figure 3B Left), respectively. Therefore, the spreading of A549 cells is mainly governed by the surface chirality rather than the shape of gold NPs. Similar phenomena were observed for cell migration (Figure 3B Right). The PAV-AuNCs significantly reduced the migration rate of A549 cells (p<0.01) compared with the particle-free control in a chirality-dependent manner. In particular, the L-PAV-AuNCs slowed down significantly the mobility of A549 cells than the D-PAV-AuNCs (p<0.01). By contrast, the PAV-AuNOs showed no significant influence on cell migration compared to the particle-free control (p>0.05) regardless of their surface chirality (Figure 3B Right). Therefore, the geometric structure of NPs influences significantly the migration ability of A549 cells, and the effect can be enhanced by the surface anchored L-PAV enantiomers. The cell migration is a complex multifaceted biological process that is regulated by an integrated network of biochemical and biomechanical signals. It contains the turnover of cell-substrate adhesion sites, the actin cytoskeletons that pull the cell at the front, and the microtubule networks responsible for cellular rear retraction in a coordinated sequence [45]. From a biomechanical viewpoint, cells need active generation of traction force on the underlying extracellular matrix (ECM) to drive their migration [46]. Thus, the direct mechanistic explanation of the inhibition of cell migration by NPs is still unknown. Although several works have observed inhibition of cell migration
14
upon NP exposure, this is the first observation that cell migration is hampered in a NP shape- and surface chirality-dependent manner. 3.4 Effect of PAV-AuNCs and PAV-AuNOs on cell viability and ROS generation To explore the effect of surface chirality and shape of NPs on A549 cell viability, MTT assay was performed, in which cytotoxicity was evaluated according to the activity of enzymes to reduce MTT to formazan dyes with a purple color [47]. As shown in Figure 4a, both L- and D-PAV-AuNCs, or L- and D-PAV-AuNOs, showed concentration-dependent cytotoxicity with almost linear regimes vs particle concentration, but the extents were different depending on the AuNPs shape and surface chirality. At a given concentration, the PAV-AuNOs showed larger cytotoxicity than PAV-AuNCs regardless of their surface chirality. For the same geometric structure of particles, the L-PAV capped AuNCs and AuNOs exhibited larger cytotoxicity than the D-PAV capped ones (especially at 8 pM), respectively (Figure 4a). Nonetheless, the viability of A549 cells at 8 pM was remained nearly 76% and 87% of that of the particle-free control, respectively (Figure 4a), showing that these AuNPs represent low toxicity to cells. The PAV-AuNC or PAV-AuNO consists of a metal core surrounded by an organic shell. In order to evaluate the effect of organic shell (PAV) on the cell viability, the A549 cells were treated with PAV molecules only at the same concentrations of PAV molecules in 8 pM PAV-AuNCs or PAV-AuNOs. As shown in Figure S5, the viability of A549 cells remained nearly 95%. Hence, one can conclude that the PAV-AuNCs and PAV-AuNOs as a whole, rather than the surface-coated PAV molecules, should be responsible for the slight cytotoxicity. Oxidative stress has been reported as one of the most important and common mechanisms of toxicity related to NP exposure [48, 49]. Early works have emphasized the role of oxidative stress in NPs toxicity [50], leading to oxidative damage to proteins and DNAs [51]. In the presence of ROS,
15
fluorescent intensity of cells stained with dyes increased, resulting in a red shift of the emission maximum [52]. Hence, the generation of ROS was studied to determine its contribution to the toxicity of PAV-AuNCs and PAV-AuNOs. The A549 cells without treatment and treated with H2O2 were used as the negative and positive controls, respectively. Compared to the negative control, ROS was significantly produced in cells being treated with H2 O2 and PAV-AuNCs or PAV-AuNOs at a NP concentration of 4 pM for 24 h, showing a chirality- and shape-dependent manner too (Figure 4b). By contrast, the PAV molecules at the same concentrations as that in 4 pM PAV-AuNCs or PAV-AuNOs generated a similar level of ROS with that of the negative control regardless of the chirality, which was significantly smaller than that of PAV-AuNCs or PAV-AuNOs. This result correlates well with the viability assay (Figure S5). Comparatively, the L-PAV-AuNOs and D-PAV-AuNOs generated more ROS than L-PAV-AuNCs and D-PAV-AuNCs (p<0.01), respectively (Figure 4b). Moreover, the L-PAV capped NPs (L-PAV-AuNCs or L-PAV-AuNOs) generated significantly more ROS than the D-PAV capped ones (p<0.05, p>0.01) (Figure 4b). Taking into account all these results, the cytotoxicity influenced by the surface chirality at the nanoscale is correlated very well with the chirality-dependent cellular uptake behaviors. Therefore, the surface chirality-dependent cytotoxicity of NPs and associated cell behaviors are basically governed mainly, if not entirely, by their difference in cellular uptake. However, the cytotoxicity affected by shape of the NPs coated with the same enantiomers is different from their internalized amount. For example, the cytotoxicity of D-PAV-AuNOs was larger than that of D-PAV-AuNCs, although their intracellular Au levels were similar. Hence, it is reasonable to assume that multiple mechanisms associated with NPs cytotoxicity may exist, in which the intracellular dose of NPs plays a major role. 3.5 DNA Damage
16
The comet assay was used to determine the DNA damage as a result of cellular uptake of the PAV-capped AuNPs (Figure 5). The cells exposed to the AuNPs showed an increase in tail momentum compared to those particle-free controls, but their extents were apparently weaker than that of the positive controls (Figure 5A). A comet-like tail implies presence of a damaged DNA strand that lags behind. The length of the tail increases with the extent of DNA damage. Figure 5B shows the shapeand chirality-dependent DNA fragmentation of the A549 cells being exposed to the PAV-AuNCs or PAV-AuNOs in terms of the percentage of DNA in the tail. Comparatively, A549 cells treated with PAV-AuNOs had higher DNA fragmentation than with PAV-AuNCs regardless of their surface chirality (Figure 5B). Moreover, the L-PAV coated AuNCs and AuNOs showed higher DNA fragmentation than the D-PAV coated ones, respectively (Figure 5B). This outcome is consistent with the formation of ROS in cells (Figure 4b), since ROS is considered to be the major source of spontaneous damage to DNAs [53]. Oxidative attack on the DNA results in mutagenic structures such as 8-hydroxyadenine and 8-hydroxyguanine, which induces instability of repetitive sequences [38]. The chemical reactions that bring about such mutations are based on the formation of highly reactive and short lived hydroxyl radical (•OH) in close proximity to DNAs [54]. It has been observed that metal oxide NPs can induce the ROS-mediated genotoxicity [38, 55]. Therefore, the DNA damage of A549 cells is most possibly caused by the generation of ROS induced by intracellular localization of PAV-AuNCs and PAV-AuNOs. The intracellular ROS causes injury to various cellular constituents such as lipids, proteins and DNAs, leading to decrease of cell viability, DNA damage and thereby cell functions such as migration. However, the reason why the ROS generation can be mediated by the surface chirality and structure of the NPs is still unclear, which requires delicate study to disclose various signaling transduction pathways.
17
Nonetheless, it was reported that chiral polymer brush films based on L(D)-valine units show different abilities in modulating the interactions of proteins and materials’ surface [56, 57]. Furthermore, the chirality could modulate the interactions of other biomolecules and surface too. For example, Wei et al. observed that surface chirality can modulate insulin assembly, and determine the cellular effect of insulin on neuronal PC12 cells growth and differentiation. Insulin retains its bioactivity on the D-surface, but forms amyloid fibrils with lost bioactivity on the L-surface. PC12 cells show high proliferation and a high differentiation rate on the insulin-adsorbed D-surface, whereas represent neither proliferation nor differentiation on the insulin-adsorbed L-surface [58]. Our previous work found that D-surface prefers to interact with the L-phospholipids of the cell membrane [24]. Although the inert gold core material is considered to reduce the potential for toxicity issues arising from particle degradation, more and more evidence shows that the ultra-small AuNPs elicit significant cytotoxicity. Apiwat Chompoosor et al. observed that 2 nm AuNP are capable of generating endogenous ROS and further causing DNA damage, even at AuNP concentrations where 100% cell viability is observed [59]. K T Butterworth et al. reported that 1.5 nm AuNPs can cause significant levels of cell type specific cytotoxicity, apoptosis and increased oxidative stress [60]. Moreover, Stefania Sabella et al. found that AuNPs (4 nm) release Au+ ions in lysosomes, and generate significant endogenous ROS [61]. However, the AuNCs (the side of the cube: 165 nm) and AuNOs (the edge of the octahedra: 110 nm) used here are much larger than the AuNPs (the diameter of sphere: 1-4 nm) mentioned above. Thus, one possible mechanism is that different configuration of the surface ligands and the shape of the NPs can interact with the receptors of cell membranes or proteins located inside cells, and then activate certain pathways to produce ROS. 4. Conclusions
18
Two types of AuNPs, i.e. AuNCs and AuNOs were synthesized and capped with chiral L-PAV and D-PAV, respectively. The L-PAV-AuNCs and D-PAV-AuNCs, or the L-PAV-AuNOs and D-PAV-AuNOs, had identical physicochemical properties except of the reverse molecular chirality on the particle surface, allowing the correlation of cellular behaviors of PAV-AuNCs or PAV-AuNOs with the molecular chirality on their surface, respectively. The cytotoxicity of A549 cells exposed to these NPs was strongly surface chirality- and shape-dependent. The L-PAV capped AuNCs and AuNOs exhibited larger cytotoxicity to A549 cells than the D-PAV coated ones, and the PAV-AuNOs had larger cytotoxicity than PAV-AuNCs when being capped with the same type of enantiomers, respectively. The cytotoxicity is in positive correlation with the intracellular amount of AuNPs, and thereby the level of ROS. Clarification of these effects provides important insight for design of more biocompatible surface coatings by using biomolecules and structures, and may open a new avenue for further development of AuNPs for biomedical applications. Acknowledgment: This study is financially supported by the Natural Science Foundation of China (21374097, 21434006), and the Key Science Technology Innovation Team of Zhejiang Province (2013TD02).
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Table 1. Characterization of chiral PAV-modified gold nanoparticles. Length a (nm)
Chain density
Nanoparticles (chains/nm2) L-PAV-AuNCs
164±35
2.93
D-PAV-AuNCs
165±34
2.94
L-PAV-AuNOs
112±18
0.58
D-PAV-AuNOs
113±16
0.60
a Length refers to the edge length in the case of octahedra or cubes.
27
Scheme 1. Schematic illustration to show the structure of chiral PAV-modified AuNOs and AuNCs. Figure 1. Representative TEM images (a1-d1) and SEM images (a2-d2) of (a1, a2) L-PAV-AuNCs , (b1, b2) D-PAV-AuNCs, (c1, c2) L-PAV-AuNOs, and (d1, d2) D-PAV-AuNOs. Double arrow indicates the edge length of the NPs. CD spectra (e, f) of (e) L-PAV-AuNCs and D-PAV-AuNCs, and (f) L-PAV-AuNOs and D-PAV-AuNOs. Figure 2. (A) Merged bright field and fluorescence CLSM images of A549 cells being treated with L-PAV-AuNCs, D-PAV-AuNCs, L-PAV-AuNOs and D-PAV-AuNOs for 24 h, respectively. Nuclei were stained with DAPI (blue), and intrinsic fluorescence of gold nanoparticles showed red color. (B) Cellular uptake of PAV-AuNCs and PAV-AuNOs quantified by ICP-MS, respectively. Error bars represent mean ± standard deviation (SD, n =4). * and ** denotes significant difference at p < 0.05 and p <0.01 level, respectively. Figure 3. (A) Representative images of A549 cells after being co-cultured with PAV-AuNCs and PAV-AuNOs, respectively. (a) Control, (b) L-PAV-AuNCs, (c) D-PAV-AuNCs, (d) L-PAV-AuNOs, (e) D-PAV-AuNOs. The scar bar represents 25 µm. (B) Spreading area (Left) and migration rate (Right) of A549 cells in the presence and absence of PAV-AuNCs and PAV-AuNOs, respectively. Error bars represent mean ± standard deviation (SD, n =4). * and ** denote significant difference at p < 0.05 and p < 0.01 levels, respectively. Figure 4. (a) Cytotoxicity of A549 cells which were incubated with PAV-AuNCs and PAV-AuNOs with different NP concentration of 0.5-8 pM for 24 h, respectively. (b) Effect of the PAV-AuNCs and PAV-AuNOs with a NP concentration of 4 pM on ROS generation after exposure for 24 h, respectively. Free PAV molecules at the same concentrations as that in 4 pM PAV-AuNCs or PAV-AuNOs were also incubated with A549 cells to examine their ability to produce ROS. (+) Represents the cells pretreated
28
with H2O2 (positive control); (-) represents the untreated cells (negative control). * and ** denote significant difference at p < 0.05 and p <0.01 level, respectively. Figure 5. Comet analysis. (A) Typical comet images from untreated (negative control), H2O2 treated (10 mM, positive control), and L-PAV-AuNCs, D-PAV-AuNCs, L-PAV-AuNOs and D-PAV-AuNOs treated A549 cells, respectively. The A549 cells were stained with ethidium bromide (0.2 %, w/v). (B) DNA damage expressed by percentage of DNA in tail. Error bars represent mean ± standard deviation (SD, n =4). * and ** denote significant difference at p<0.05 and p<0.01 levels, respectively.
29
*
*
*
*
* Chiral center
L-PAV-AuNCs
D-PAV-AuNCs
L-PAV-AuNOs
D-PAV-AuNOs
S
PAV
*
Chiral * center
C4H9 S
S
= Gold binding motif
Scheme 1 30
(a1)
(a2)
(b1)
(c1)
(c2)
(b2)
(d1)
(d2)
200 nm
Figure 1 31
30 L-PAV-AuNCs D-PAV-AuNCs
10
15
Elipticity (mdeg)
Ellipticity (mdeg)
(e)
0
-15
L-PAV-AuNOs D-PAV-AuNOs
(f)
5 0 -5 -10
-30 200
220
240
260
200
Wavelength (nm)
220 240 Wavelength (nm)
260
Figure 1 32
(A)
6
Au (µ g/104 cells)
L D
*
**
4
** 2
Figure 2
0 PAV-AuNCs
PAV-AuNOs
33
(A)
Control
(a)
(b)
(c)
(d)
(e)
Control L D
1.0
50
**
** *
*
0.5
Cell migration rate (µ m/h)
Relative spreading area vs control
(B)
0.0
40
Control L D
30
** **
* ** *
20 10 0
Control
PAV-AuNCs
PAV-AuNOs
Control
PAV-AuNCs
PAV-AuNOs
Figure 3 34
Relative cell viability (%)
(a) 100
L-PAV-AuNCs D-PAV-AuNCs L-PAV-AuNOs D-PAV-AuNOs
90
80
Fluorescent inensity (O.D)
110 200
150
4 2 1 Au concentration (pM)
(b)
** * ** **
100
** * ** **
50
0
0.5
Positive control Negative control L-PAV D-PAV L-PAV (without NPs) D-PAV (without NPs)
8
+
-
AuNCs
AuNOs
Figure 4
35
(A) Negative control
Positive control
(B)
L-PAV-AuNCs
L-PAV-AuNOs
D-PAV-AuNCs
DNA damage expressed by percentage of DNA in tail
100 Positive control Negative control L D
80 60
*
40
*
20
*
*
D-PAV-AuNOs
0
+
-
PAV-AuNCs PAV-AuNOs
Figure 5 36
Graphical abstract
* * * Chiral
* *
center
L-PAV-AuNCs
D-PAV-AuNCs
L-PAV-AuNOs
D-PAV-AuNOs
ROS
Nucleus
DNA damage
37
Significance:
Gold nanoparticles with different structure and surface chirality are fabricated.
The structure and surface chirality at the nanoscale can influence cytotoxicity and genotoxicity.
A new perspective on designing nanoparticles for drug delivery, bioimaging and diagnosis.
38
S1742-7061(17)30088-0 http://dx.doi.org/10.1016/j.actbio.2017.01.082 ACTBIO 4717
To appear in:
Acta Biomaterialia
Received Date: Revised Date: Accepted Date:
20 September 2016 24 January 2017 30 January 2017
Please cite this article as: Deng, J., Yao, M., Gao, C., Cytotoxicity of gold nanoparticles with different structures and surface-anchored chiral polymers, Acta Biomaterialia (2017), doi: http://dx.doi.org/10.1016/j.actbio. 2017.01.082
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Cytotoxicity of gold nanoparticles with different structures and surface-anchored chiral polymers Jun Deng1, Mengyun Yao1, Changyou Gao* MOE Key Laboratory of Macromolecular Synthesis and Functionalization, Department of Polymer Science and Engineering, Zhejiang University, Hangzhou 310027, China. 1
co-first author, *Corresponding author.
Email: [email protected] Fax: +86-571-87951108
1
Abstract Nanoparticles (NPs) can have profound effects on cell biology. However, the potential adverse effects of gold nanoparticles (AuNPs) with different surface chirality and structures have not been elucidated. In this study, monolayers of poly(acryloyl-L(D)-valine (L(D)-PAV) chiral molecules were anchored on the surfaces of gold nanocubes (AuNCs) and nanooctahedras (AuNOs), respectively. The L-PAV-AuNCs and D-PAV-AuNCs, or the L-PAV-AuNOs and D-PAV-AuNOs, had identical physicochemical properties in terms of size, morphology and ligand density except of the reverse molecular chirality on the particle surfaces, respectively. The L-PAV capped AuNCs and AuNOs exhibited larger cytotoxicity to A549 cells than the D-PAV coated ones, and the PAV-AuNOs had larger cytotoxicity than PAV-AuNCs when being capped with the same type of enantiomers, respectively. The cytotoxicity was positively correlated with the cellular uptake amount, and thereby the production of intracellular reactive oxygen species (ROS). Keywords:
Gold
nanoparticles;
nanoparticle
structure;
cytotoxicity.
2
poly(acryloyl-L(D)-valine;
chirality;
Introduction Nanoparticles (NPs) hold great promise for biomedical applications such as imaging [1], diagnostics [1], drug delivery [2], and sensing [3]. There is, however, a large knowledge gap on the biological mechanisms arising from NPs interactions with human systems. These uncertainties aggravate concerns especially when NPs are found to adversely affect cell viability or functions [4, 5]. Therefore, it is urgent to elucidate the interactions of NPs with biological systems including the living cells and tissues, which play a predominant role in determining the efficacy [6] and toxicity of NPs in biological and environmental systems [7]. The most interesting properties of NPs are their size-, morphologyand surface-induced effects. So far the effects of NPs size [8], shape [9], surface charge [10], and chemical compositions [9] on intracellular trafficking, toxicity, and gene expression have been extensively investigated. NPs are often considered as an idealized shape (e.g. spheres or rods), while they are actually nanocrystals with complex facet and defect structures [11, 12]. The NPs surface may become unstable because of their large surface curvature, and large fractions of edge and corner regions, resulting in high surface energy [13]. The potentially unstable surface of the NPs is usually coated by chemisorbed ligands. These properties of the NPs are significantly influenced by the NPs’ topography, which in turn dramatically influences on how the nanoparticles interact with their surrounding environment [14]. However, most of the previous studies focus on the interactions of biological systems with idealized NPs (e.g. spheres and rods [15], spheres and bowl [16]). It is urgent to research the effects of shape with large fractions of edge and corner regions on cytotoxicity and cell behaviors. Biological systems respond strongly to NP surfaces. NPs can interact with cellular components such as DNAs, proteins and lipids. Interactions of surface-functionalized NPs with cells can damage the
3
integrity of membrane structure, with the possibility of leakage of cytosol depending on the NP surface chemistry [17]. For example, cationic molecules-modified NPs show higher toxicity than their anionic analogues [18]. Although many studies have demonstrated that surface modification of NPs with biomolecules could endow them with various biological functionalities [19, 20], the impact of chirality of the surface biomolecules on living organisms has been largely neglected. Chirality is an important phenomenon in living systems, and most of the biological molecules are homochiral to take functions [21]. It is known that all amino acids in proteins (except of glycine) and phospholipids in cell membrane are “left-handed”, whereas all sugars in DNAs and RNAs are “right-handed” [22, 23]. Different chiral properties of biomolecules may determine their ability to interact with other biomolecules, and thereby modulate a range of downstream processes. Although some previous works show how surface chirality affects the cellular uptake [24], protein adsorption [25] and functions of the NPs (e.g. cell imaging ability of gold nanoclusters [26] and quantum dots (QDs) [27]), little is known about the effect of surface chirality at the nanoscale on the subsequent cytotoxicity, genotoxicity and cell functions such as adhesion and migration. Gold nanoparticles (AuNPs) have gained much popularity recently because of their unique chemical and physical properties (e.g. size- and shape-dependent optical and electronic features, high surface-to-volume ratio, excellent biocompatibility and chemical stability) [27, 28]. Although AuNPs are considered as inert and biocompatible, there are contradictory results concerning their cytotoxicity [29]. Therefore, in this study AuNPs are served as an ideal platform to investigate the surface chirality at the nanoscale on cytotoxicity, genotoxicity and cell functions. Valine is one of the eight essential amino acids of human body, and plays essential roles in a wide variety of physiological processes [30, 31]. Our previous work found that the gold nanospheres
4
surface-coated by 2-mercaptoacetyl-L(D)-valine (L(D)-MAV) has a chirality-independent cellular uptake behavior. However, the difference in internalization of AuNPs is prominent by anchoring poly(acryloyl-L(D)-valine) with enhanced chirality-selectivity [24]. Moreover, gold spheres with surface-anchored chiral poly(acryloyl-L(D)-valine) induce differential response on mesenchymal stem cell osteogenesis [32]. Therefore, the L- and D-valine are selected as the chiral centers, and poly(acryloyl-L-valine) (L-PAV) and poly(acryloyl-D-valine) (D-PAV) are synthesized via reversible addition-fragmentation chain transfer polymerization to enhance the chiral effect [24]. Two different shapes of AuNPs, i.e. gold nanocubes (AuNCs) and gold nanooctahedras (AuNOs) are synthesized and coated with either L-PAV or D-PAV via the thiocarbonylthio-Au bond, respectively (Scheme 1). Lung is one of the major organs that NPs accumulate when they enter the body. Therefore, lung cells, i.e. A549 cells are widely used in vitro as a model to study cell-NPs interactions [33, 34]. The chirality-associated and shape-dependent regulation of cytotoxicity and genotoxicity highlight the important role of conformation of the stabilizers and shapes of NPs, and has important implications for the design of novel AuNPs possibly applied in biological field. 2 Materials and methods 2.1 Materials Gold (III) chloride hydrate (HAuCl4) were purchased from Sinopharm group Co. Ld. 1,5-Pentanediol (96 %) (PD) was obtained from Aladdin Company. Polyvinylpyrrolidone (PVP, Mw ~55 kDa) and 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from
Sigma.
Poly(acryloyl-L-valine)
(L-PAV,
Mw
3580
Da,
polydispersity
1.14)
and
poly(acryloyl-D-valine) (D-PAV, Mw 3611 Da, polydispersity 1.15) were synthesized according our previously reported method [24]. A549 cells were purchased from the Cell Bank of Typical Culture
5
Collection of Chinese Academy of Sciences, and all reagents for the cell culture were used directly. All other chemicals were of analytical grade and used without further treatment if not specially mentioned. The MilliQ water was used throughout the experiments. For gold nanoparticle synthesis, all glasswares used were cleaned by freshly prepared aqua regia solution (HCl/HNO3, 3:1). 2.2 NPs synthesis and surface functionalization Gold nanocubes (AuNCs) and gold nanooctahedras (AuNOs) were synthesized using a high temperature polyol process [35]. The AuNCs or AuNOs solution was obtained by centrifugation at 500 rpm for 5 min at 10 oC, and the supernatant was decanted into a second tube to remove any larger particles or aggregates. The supernatant solution was washed 5 times with ethanol by centrifugation (4000 rpm, 30 min, and 10 oC) to remove excess PVP, and the concentrated AuNCs or AuNOs solution was collected as a stock solution. The surface of the obtained AuNCs and AuNOs was functionalized with L-PAV and D-PAV using a ligand exchange protocol, respectively. To ensure complete exchange, the stock NP solution was incubated with a large amount of 1 mg·mL-1 L-PAV and D-PAV solution under heavily shaking at room temperature overnight, respectively. The solution was then sonicated and centrifuged to remove the excess PAV molecules. The NPs were re-dispersed in ethanol solution with 1 mg·mL-1 L-PAV and D-PAV, and the solution was stirred for another 12 h, respectively. Then the NPs were washed with ethanol solution 3 times. Finally, the NPs were re-dispersed in water for further characterizations. 2.3 NP characterization The morphology and dimensions of NPs were obtained from transmission electron microscopy (TEM, H-7650) and scanning electron microscopy (SEM, SIRION-100, FEI), respectively. The TEM samples were prepared by depositing 10 µ L of diluted sample solution on a carbon-coated copper grid, and
6
dried under atmosphere overnight. The SEM samples were prepared by depositing diluted NP solutions on silica wafers, and dried under atmosphere overnight. The size distribution of NPs was obtained from both TEM and SEM micrographs using the Image J software, with at least 500 particles counted per sample, respectively. Nanocube (NC) dimensions were measured by averaging two side lengths of each particle (Double arrow in Figure 1 a1, b1), and nanooctahedra (NO) dimensions were determined from 1 to 2 side measurements per particle (Double arrow in Figure 1 c1, d1). SEM micrographs at several tilt angles were also taken for AuNCs and AuNOs to determine the nanoparticles’ height. The gold concentration of AuNCs (mc, g·mL-1) or AuNOs (mo, g·mL-1) solutions was measured by using inductively coupled plasma mass spectrometry (ICP-MS, Thermo Elemental Corporation of USA, XSeries II), respectively. To determine the NP concentration, individual nanoparticle mass was calculated by using the particle volume calculated with the NPs’ edge length with assumption of “perfect” morphology. The NCs’ volume was determined simply to be ac3, where ac is the edge length of the cube. NOs’ volume was determined to be [2/(31/2)]ao3 where ao is the edge length of the octahedron. Individual particle mass was calculated by using the particle volume (V) and the gold density (ρ, 19.3 g.cm-3). The particle number per milliliter (mL) was obtained by dividing the Au concentration by the individual particle mass. With these parameters, the molar concentration of gold NPs (AuNCs or AuNOs) per mL was calculated by c=1000 m/ρVNA. The circular dichroism (CD) spectrum was obtained by using a JASCO-820 spectropolarimeter equipped with a thermostatically controlled cell holder. The temperature of the sample was controlled at 25 oC. The UV region was scanned between 190 and 260 nm with an average of 3 scans. The concentrations of PAV-AuNCs and PAV-AuNOs were kept at 50 pM. The final spectra (∆θ) of
7
PAV-AuNCs and PAV-AuNOs were obtained by subtracting the spectrum of gold NPs (without PAV grafting) solution of same concentration, respectively. 2.4 Cell experiment NPs distribution The intracellular NPs distribution was measured using confocal laser scanning microscopy (CLSM, Leica TCS SP5). Briefly, the cells were seeded on a glass bottom cell culture dish (diameter, 20 mm) at a density of 5×103 cells/cm2 , and allowed to attach for 24 h. Then, the cells were treated with PAV-AuNCs and PAV-AuNOs at a NP concentration of 4 pM for another 24 h, respectively. After 5 washes with phosphate buffered saline (PBS), the cells were fixed with 0.4 % paraformaldehyde at 37 o
C overnight, and washed with PBS 3 times. They were further treated in 0.5% (v/v) Triton X-100
(Sigma-Aldrich) for 10 min at 37 °C to enhance the permeability of cell membrane. After being blocked
with
1%
BSA/PBS
at
37
o
C
for
2
h,
the
samples
were
stained
with
4’,6-diamidino-2-phenylindole (DAPI, sigma, 1:50) at 37 oC for 30 min, following with 5 washes with PBS. Cellular uptake The internalized amount of PAV-AuNCs and PAV-AuNOs was determined by ICP-MS, respectively. Briefly, the cells were seeded on a 12-well plate at a density of 5× 104 cells/cm2, and allowed to attach for 24 h. The medium was replaced with the fresh one containing PAV-AuNCs and PAV-AuNOs with a NP concentration of 4 pM, respectively. After 24 h, the plates were washed 5 times with PBS to remove free NPs. After the cells were harvested by trypsinization, their numbers were quantified by a cell counter. The samples were treated with aqua regia solution (HCl: HNO3 = 1:3, volume ratio) for 2
8
h. The obtained solution was diluted to determine the Au concentration by ICP-MS. The Au amount per 104 cells from ICP-MS analysis is presented as mean ± standard deviation (n=4). Cell adhesion and migration To determine the cell adhesion and migration, the A549 cells were seeded on a glass bottom cell culture dish (diameter, 20 mm) at a density of 5×103 cells/cm2, and allowed to attach for 6 h. Then, the cells were treated with PAV-AuNCs and PAV-AuNOs with a NP concentration of 4 pM, respectively. Then the cell adhesion and migration were measued using a time-lapse phase-contrast microscope (DMI 6000B, Leica). The cell spreading areas were obtained by analyzing the optical images of cells using the image J software. The migration rate was obtained using the previously reported protocols [36, 37]. Cell viability To determine the cell viability, A549 cells were plated at a density of 5×104 cells/cm2 in a 24-well plate and cultured for 24 h. The medium was replaced with fresh one containing the PAV-AuNCs or PAV-AuNOs (0.9 mL for each well) with different NP concentration from 0.5 to 8 pM. After treatment for another 24 h, 100 µL 5 mg·mL-1 MTT solution was added to each well, and the cells were further cultured at 37 °C for 3 h. The dark blue formazan crystals generated by the mitochondria dehydrogenase in live cells were dissolved with dimethyl sulfoxide (DMSO, Sigma-Aldrich). After the sample was centrifuged at 12000 g/min for 5 min, the absorbance of supernatant was measured by a microplate reader (MODEL 680, Bio Rad) at 570 nm. Measurement of reactive oxygen species (ROS) To measure the intracellular generation of ROS, the oxidant-sensitive dye 2′,7′-dichlorofluorescin diacetate (DCFH-DA, sigma) was used as described previously [38]. The generation of ROS was
9
measured by incubating one million cells with 4 pM PAV-AuNCs or PAV-AuNOs for 24 h, followed by staining with DCFH-DA for 20 min at 37 °C. A549 cells being treated with and without hydrogen peroxide (H2O2, 10 mM) were served as the positive and negative controls, respectively. Cells were then washed 3 times in serum-free medium and analyzed using flow cytometry (FACs Calibur, BD) at an excitation wavelength of 488 nm and emission wavelength of 530 nm. The concentration of the PAV-AuNCs or PAV-AuNOs was chosen based on the viability data. For each sample, 1×104 cells were collected, and data were analyzed using the WinMDI 2.8 software. Comet assay in DNA fragmentation The comet assay was used to monitor DNA damage in the cells, based on the method described previously [39]. The A549 cells were harvested and washed twice in PBS before re-suspending in Hank’s balance salt solution (HBSS, Sigma-Aldrich, St. Louis, MO) with 10 % DMSO and ethylene diamine tetraacetic acid (EDTA, 1st Base, Singapore). The cells being treated with H2O2 and without any treatment were served as the positive and negative controls, respectively. The cells were embedded in 0.7 % (w/v) low melting agarose (LMPA, Argoros) on comet slides (Trevigen, Gaithersburg, MD), and then lysed in prechilled lysis solution (2.5 M NaCl, 0.1 M EDTA, 10 mM Tris base, pH 10) containing 1 % Triton-X 100 for 1 h at 4 °C. Cells were then subjected to denaturation in alkaline buffer (0.3 M NaCl, 1 mM EDTA) for 40 min in dark at room temperature. Electrophoresis was performed at 24 V and 300 mA for 16 min. The slides were immersed in neutralization buffer (0.5 M Tris-HCl, pH 7.4) for 15 min, followed by dehydration in 70 % ethanol. The slides were air dried and stained with ethidium bromide (EB) fluorescent dye (0.2 % w/v). The tail moments of the nuclei were measured under a fluorescence microscope (Zeiss Axiovert 200), which reveal the DNA damage. Analysis
was
done
using
the
comet
imager
10
OpenComet
v1.3
(Download
from
http://www.opencomet.org/). At least 50 comets were analyzed per concentration. The comet evaluation was carried out based on the percentage of the DNA in the tail. Statistical Analysis. Four to five biological replicates and two technical repeats were performed in all the experiments. The experimental data are expressed as mean ± standard deviation, and the significant difference between groups was analyzed using one-way analysis of variance (ANOVA) (for two groups) and two-way ANOVA (for more than two groups) in the Origin software. The statistical significance was set as p < 0.05 and p < 0.01. 3 Results and discussion 3.1 Nanoparticle characterization Transmission electron microscopy (TEM) and scanning electron microscopy (SEM) observation demonstrate that the PAV-AuNCs and PAV-AuNOs were successfully synthesized with the desired morphology (Figure 1) and narrow distribution (Figure S1). Table 1 shows that the L-PAV-AuNCs and D-PAV-AuNCs had identical physicochemical properties in terms of size (~165 nm on each side) and surface molecular density (~2.0 molecules/nm2 ) determined from SEM and thermogravimetric analysis (TGA), respectively (Figure S2). The L-PAV-AuNOs and D-PAV-AuNOs had the same properties in terms of size (~110 nm in edge) and surface molecular density (~1.9 molecules/nm2) as well. By contrast, the L-PAV-AuNCs and D-PAV-AuNCs, or the L-PAV-AuNOs and D-PAV-AuNOs, showed essential mirror images of CD spectra in the region of 190 to 260 nm (Figure 1 e, f ). The CD signal of PAV-AuNCs was stronger than that of PAV-AuNOs, suggesting a larger number of PAV molecules on the PAV-AuNCs (Figure 1 e, f). The NPs’ stability was monitored through absorption measurement of the particle surface plasmon resonance (SPR), a parameter determined by coherent oscillation of conduction electrons in the gold
11
NP surface that is exquisitely sensitive to their local dielectric environment [40]. During the ligand exchange process there was no shift in the SPR peaks, which were located at 566 nm for PAV-AuNCs (Figure S3a) and 568 nm for PAV-AuNOs (Figure S3b), respectively. These results demonstrate the good colloidal stability, and the similar geometric features of the PAV-AuNCs and PAV-AuNOs. Peak broadening to some extent was evident, probably conveying a slight rounding of the nanoparticle vertices (Figure S3) as the SPR is very sensitive to the relative sharpness of the corners in anisotropic particles [41]. Moreover, no significant change of SPR was found in cell culture medium containing 10% fetal bovine serum (10% FBS/DMEM) (Figure S3), suggesting the good colloidal stability of the PAV-AuNCs and PAV-AuNOs under the cell culture conditions. In summary, the L-PAV-AuNCs and D-PAV-AuNCs, or the L-PAV-AuNOs and D-PAV-AuNOs, have identical physicochemical properties except of the reverse molecular chirality on the particle surface, allowing the correlation of cellular behaviors of PAV-AuNCs or PAV-AuNOs with the molecular chirality on their surface, respectively. 3.2 NPs distribution and cellular uptake The uptake amount and intracellular distribution of NPs are tied intrinsically with the nanotoxicity to cells. In this regard, the A549 cells were co-cultured with the PAV-AuNCs and PAV-AuNOs at a NP concentration of 4 pM, and their intracellular distribution and internalized amount were monitored by CLSM and ICP-MS, respectively. According to literatures and our experiences, the highest cellular loading of NPs is usually achieved at 24 h [34, 42], which was adopted in this study. The blue color shows the nuclei of cells stained by DAPI, while the red color corresponds to the PAV-capped AuNPs (PAV-AuNCs or PAV-AuNOs). As shown in Figure 2A, the PAV-AuNCs and PAV-AuNOs were mainly distributed in the cytoplasm around nuclei. No signal was detected in the nuclei of A549 cells
12
regardless of their geometric feature and surface chirality. Comparatively, the L-PAV capped AuNCs and AuNOs were internalized with significantly larger amount than the D-PAV capped ones, respectively (Figure 2A), which was further confirmed by quantitative analysis with ICP-MS (Figure 2B). This result is different with our previous observation when enantiomer PAV-capped spherical AuNPs were co-cultured with A549 and HepG2 cells, in which the D-PAV-AuNPs are preferentially internalized [24]. Although the intrinsic reasons are not clear so far, they are likely related with the difference in particle morphology and size, and thereby the surface protein adsorption. Indeed, the impact of surface chirality on uptake of NPs is strongly protein adsorption-dependent, and the adsorption of proteins such as albumin could weaken or even screen the influence of surface chirality [25]. Moreover, the adhesion mediators (fibrinogen, vitronectin, fibronectin and laminin) are adsorbed with larger amount on the L- enantiomer capped gold NPs, enhancing their uptake by mesenchymal stem cells [32]. For the same enantiomer of L-PAV coated NPs, the AuNOs were more significantly taken up than the AuNCs (p<0.05, p>0.01) (Figure 2B). This is not the case for the same D-PAV coated NPs, e.g. the AuNOs was internalized with a slight larger amount than the AuNCs (p>0.05). Because the volume of AuNCs was larger than that of AuNOs, the Au concentration of AuNCs was larger than that of AuNOs at the same mole concentration of NPs (Here 4 pM). Therefore, the A549 cells would prefer to internalize the AuNOs than the AuNCs of the same enantiomers coated, suggesting the significant role of particle shape on the cellular internalization. 3.3 Effect of PAV-AuNCs and PAV-AuNOs on cellular morphology The first and most readily noticeable effect following exposure of cells to nanomaterials (NMs) is the alteration in cell adhesion and migration in a monolayer culture [43, 44]. However, little is known on these effects induced by particle structure and surface chirality at the nanoscale. After being treated
13
with PAV-AuNCs or PAV-AuNOs at a concentration of 4 pM for 24 h, the spreading area of A549 cells was reduced to different extents depending on the shape and surface chirality of NPs (Figure 3A). The L-PAV capped AuNCs or AuNOs caused more severe inhibition on spreading of A549 cells compared to the NPs-free control (p<0.01), whereas the D-PAV coated ones did not significantly reduce the cell spreading (p>0.05) (Figure 3B Left). Moreover, no significant difference (p>0.05) was found between the PAV-AuNCs and PAV-AuNOs with the same enantiomers (Figure 3B Left), respectively. Therefore, the spreading of A549 cells is mainly governed by the surface chirality rather than the shape of gold NPs. Similar phenomena were observed for cell migration (Figure 3B Right). The PAV-AuNCs significantly reduced the migration rate of A549 cells (p<0.01) compared with the particle-free control in a chirality-dependent manner. In particular, the L-PAV-AuNCs slowed down significantly the mobility of A549 cells than the D-PAV-AuNCs (p<0.01). By contrast, the PAV-AuNOs showed no significant influence on cell migration compared to the particle-free control (p>0.05) regardless of their surface chirality (Figure 3B Right). Therefore, the geometric structure of NPs influences significantly the migration ability of A549 cells, and the effect can be enhanced by the surface anchored L-PAV enantiomers. The cell migration is a complex multifaceted biological process that is regulated by an integrated network of biochemical and biomechanical signals. It contains the turnover of cell-substrate adhesion sites, the actin cytoskeletons that pull the cell at the front, and the microtubule networks responsible for cellular rear retraction in a coordinated sequence [45]. From a biomechanical viewpoint, cells need active generation of traction force on the underlying extracellular matrix (ECM) to drive their migration [46]. Thus, the direct mechanistic explanation of the inhibition of cell migration by NPs is still unknown. Although several works have observed inhibition of cell migration
14
upon NP exposure, this is the first observation that cell migration is hampered in a NP shape- and surface chirality-dependent manner. 3.4 Effect of PAV-AuNCs and PAV-AuNOs on cell viability and ROS generation To explore the effect of surface chirality and shape of NPs on A549 cell viability, MTT assay was performed, in which cytotoxicity was evaluated according to the activity of enzymes to reduce MTT to formazan dyes with a purple color [47]. As shown in Figure 4a, both L- and D-PAV-AuNCs, or L- and D-PAV-AuNOs, showed concentration-dependent cytotoxicity with almost linear regimes vs particle concentration, but the extents were different depending on the AuNPs shape and surface chirality. At a given concentration, the PAV-AuNOs showed larger cytotoxicity than PAV-AuNCs regardless of their surface chirality. For the same geometric structure of particles, the L-PAV capped AuNCs and AuNOs exhibited larger cytotoxicity than the D-PAV capped ones (especially at 8 pM), respectively (Figure 4a). Nonetheless, the viability of A549 cells at 8 pM was remained nearly 76% and 87% of that of the particle-free control, respectively (Figure 4a), showing that these AuNPs represent low toxicity to cells. The PAV-AuNC or PAV-AuNO consists of a metal core surrounded by an organic shell. In order to evaluate the effect of organic shell (PAV) on the cell viability, the A549 cells were treated with PAV molecules only at the same concentrations of PAV molecules in 8 pM PAV-AuNCs or PAV-AuNOs. As shown in Figure S5, the viability of A549 cells remained nearly 95%. Hence, one can conclude that the PAV-AuNCs and PAV-AuNOs as a whole, rather than the surface-coated PAV molecules, should be responsible for the slight cytotoxicity. Oxidative stress has been reported as one of the most important and common mechanisms of toxicity related to NP exposure [48, 49]. Early works have emphasized the role of oxidative stress in NPs toxicity [50], leading to oxidative damage to proteins and DNAs [51]. In the presence of ROS,
15
fluorescent intensity of cells stained with dyes increased, resulting in a red shift of the emission maximum [52]. Hence, the generation of ROS was studied to determine its contribution to the toxicity of PAV-AuNCs and PAV-AuNOs. The A549 cells without treatment and treated with H2O2 were used as the negative and positive controls, respectively. Compared to the negative control, ROS was significantly produced in cells being treated with H2 O2 and PAV-AuNCs or PAV-AuNOs at a NP concentration of 4 pM for 24 h, showing a chirality- and shape-dependent manner too (Figure 4b). By contrast, the PAV molecules at the same concentrations as that in 4 pM PAV-AuNCs or PAV-AuNOs generated a similar level of ROS with that of the negative control regardless of the chirality, which was significantly smaller than that of PAV-AuNCs or PAV-AuNOs. This result correlates well with the viability assay (Figure S5). Comparatively, the L-PAV-AuNOs and D-PAV-AuNOs generated more ROS than L-PAV-AuNCs and D-PAV-AuNCs (p<0.01), respectively (Figure 4b). Moreover, the L-PAV capped NPs (L-PAV-AuNCs or L-PAV-AuNOs) generated significantly more ROS than the D-PAV capped ones (p<0.05, p>0.01) (Figure 4b). Taking into account all these results, the cytotoxicity influenced by the surface chirality at the nanoscale is correlated very well with the chirality-dependent cellular uptake behaviors. Therefore, the surface chirality-dependent cytotoxicity of NPs and associated cell behaviors are basically governed mainly, if not entirely, by their difference in cellular uptake. However, the cytotoxicity affected by shape of the NPs coated with the same enantiomers is different from their internalized amount. For example, the cytotoxicity of D-PAV-AuNOs was larger than that of D-PAV-AuNCs, although their intracellular Au levels were similar. Hence, it is reasonable to assume that multiple mechanisms associated with NPs cytotoxicity may exist, in which the intracellular dose of NPs plays a major role. 3.5 DNA Damage
16
The comet assay was used to determine the DNA damage as a result of cellular uptake of the PAV-capped AuNPs (Figure 5). The cells exposed to the AuNPs showed an increase in tail momentum compared to those particle-free controls, but their extents were apparently weaker than that of the positive controls (Figure 5A). A comet-like tail implies presence of a damaged DNA strand that lags behind. The length of the tail increases with the extent of DNA damage. Figure 5B shows the shapeand chirality-dependent DNA fragmentation of the A549 cells being exposed to the PAV-AuNCs or PAV-AuNOs in terms of the percentage of DNA in the tail. Comparatively, A549 cells treated with PAV-AuNOs had higher DNA fragmentation than with PAV-AuNCs regardless of their surface chirality (Figure 5B). Moreover, the L-PAV coated AuNCs and AuNOs showed higher DNA fragmentation than the D-PAV coated ones, respectively (Figure 5B). This outcome is consistent with the formation of ROS in cells (Figure 4b), since ROS is considered to be the major source of spontaneous damage to DNAs [53]. Oxidative attack on the DNA results in mutagenic structures such as 8-hydroxyadenine and 8-hydroxyguanine, which induces instability of repetitive sequences [38]. The chemical reactions that bring about such mutations are based on the formation of highly reactive and short lived hydroxyl radical (•OH) in close proximity to DNAs [54]. It has been observed that metal oxide NPs can induce the ROS-mediated genotoxicity [38, 55]. Therefore, the DNA damage of A549 cells is most possibly caused by the generation of ROS induced by intracellular localization of PAV-AuNCs and PAV-AuNOs. The intracellular ROS causes injury to various cellular constituents such as lipids, proteins and DNAs, leading to decrease of cell viability, DNA damage and thereby cell functions such as migration. However, the reason why the ROS generation can be mediated by the surface chirality and structure of the NPs is still unclear, which requires delicate study to disclose various signaling transduction pathways.
17
Nonetheless, it was reported that chiral polymer brush films based on L(D)-valine units show different abilities in modulating the interactions of proteins and materials’ surface [56, 57]. Furthermore, the chirality could modulate the interactions of other biomolecules and surface too. For example, Wei et al. observed that surface chirality can modulate insulin assembly, and determine the cellular effect of insulin on neuronal PC12 cells growth and differentiation. Insulin retains its bioactivity on the D-surface, but forms amyloid fibrils with lost bioactivity on the L-surface. PC12 cells show high proliferation and a high differentiation rate on the insulin-adsorbed D-surface, whereas represent neither proliferation nor differentiation on the insulin-adsorbed L-surface [58]. Our previous work found that D-surface prefers to interact with the L-phospholipids of the cell membrane [24]. Although the inert gold core material is considered to reduce the potential for toxicity issues arising from particle degradation, more and more evidence shows that the ultra-small AuNPs elicit significant cytotoxicity. Apiwat Chompoosor et al. observed that 2 nm AuNP are capable of generating endogenous ROS and further causing DNA damage, even at AuNP concentrations where 100% cell viability is observed [59]. K T Butterworth et al. reported that 1.5 nm AuNPs can cause significant levels of cell type specific cytotoxicity, apoptosis and increased oxidative stress [60]. Moreover, Stefania Sabella et al. found that AuNPs (4 nm) release Au+ ions in lysosomes, and generate significant endogenous ROS [61]. However, the AuNCs (the side of the cube: 165 nm) and AuNOs (the edge of the octahedra: 110 nm) used here are much larger than the AuNPs (the diameter of sphere: 1-4 nm) mentioned above. Thus, one possible mechanism is that different configuration of the surface ligands and the shape of the NPs can interact with the receptors of cell membranes or proteins located inside cells, and then activate certain pathways to produce ROS. 4. Conclusions
18
Two types of AuNPs, i.e. AuNCs and AuNOs were synthesized and capped with chiral L-PAV and D-PAV, respectively. The L-PAV-AuNCs and D-PAV-AuNCs, or the L-PAV-AuNOs and D-PAV-AuNOs, had identical physicochemical properties except of the reverse molecular chirality on the particle surface, allowing the correlation of cellular behaviors of PAV-AuNCs or PAV-AuNOs with the molecular chirality on their surface, respectively. The cytotoxicity of A549 cells exposed to these NPs was strongly surface chirality- and shape-dependent. The L-PAV capped AuNCs and AuNOs exhibited larger cytotoxicity to A549 cells than the D-PAV coated ones, and the PAV-AuNOs had larger cytotoxicity than PAV-AuNCs when being capped with the same type of enantiomers, respectively. The cytotoxicity is in positive correlation with the intracellular amount of AuNPs, and thereby the level of ROS. Clarification of these effects provides important insight for design of more biocompatible surface coatings by using biomolecules and structures, and may open a new avenue for further development of AuNPs for biomedical applications. Acknowledgment: This study is financially supported by the Natural Science Foundation of China (21374097, 21434006), and the Key Science Technology Innovation Team of Zhejiang Province (2013TD02).
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Table 1. Characterization of chiral PAV-modified gold nanoparticles. Length a (nm)
Chain density
Nanoparticles (chains/nm2) L-PAV-AuNCs
164±35
2.93
D-PAV-AuNCs
165±34
2.94
L-PAV-AuNOs
112±18
0.58
D-PAV-AuNOs
113±16
0.60
a Length refers to the edge length in the case of octahedra or cubes.
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Scheme 1. Schematic illustration to show the structure of chiral PAV-modified AuNOs and AuNCs. Figure 1. Representative TEM images (a1-d1) and SEM images (a2-d2) of (a1, a2) L-PAV-AuNCs , (b1, b2) D-PAV-AuNCs, (c1, c2) L-PAV-AuNOs, and (d1, d2) D-PAV-AuNOs. Double arrow indicates the edge length of the NPs. CD spectra (e, f) of (e) L-PAV-AuNCs and D-PAV-AuNCs, and (f) L-PAV-AuNOs and D-PAV-AuNOs. Figure 2. (A) Merged bright field and fluorescence CLSM images of A549 cells being treated with L-PAV-AuNCs, D-PAV-AuNCs, L-PAV-AuNOs and D-PAV-AuNOs for 24 h, respectively. Nuclei were stained with DAPI (blue), and intrinsic fluorescence of gold nanoparticles showed red color. (B) Cellular uptake of PAV-AuNCs and PAV-AuNOs quantified by ICP-MS, respectively. Error bars represent mean ± standard deviation (SD, n =4). * and ** denotes significant difference at p < 0.05 and p <0.01 level, respectively. Figure 3. (A) Representative images of A549 cells after being co-cultured with PAV-AuNCs and PAV-AuNOs, respectively. (a) Control, (b) L-PAV-AuNCs, (c) D-PAV-AuNCs, (d) L-PAV-AuNOs, (e) D-PAV-AuNOs. The scar bar represents 25 µm. (B) Spreading area (Left) and migration rate (Right) of A549 cells in the presence and absence of PAV-AuNCs and PAV-AuNOs, respectively. Error bars represent mean ± standard deviation (SD, n =4). * and ** denote significant difference at p < 0.05 and p < 0.01 levels, respectively. Figure 4. (a) Cytotoxicity of A549 cells which were incubated with PAV-AuNCs and PAV-AuNOs with different NP concentration of 0.5-8 pM for 24 h, respectively. (b) Effect of the PAV-AuNCs and PAV-AuNOs with a NP concentration of 4 pM on ROS generation after exposure for 24 h, respectively. Free PAV molecules at the same concentrations as that in 4 pM PAV-AuNCs or PAV-AuNOs were also incubated with A549 cells to examine their ability to produce ROS. (+) Represents the cells pretreated
28
with H2O2 (positive control); (-) represents the untreated cells (negative control). * and ** denote significant difference at p < 0.05 and p <0.01 level, respectively. Figure 5. Comet analysis. (A) Typical comet images from untreated (negative control), H2O2 treated (10 mM, positive control), and L-PAV-AuNCs, D-PAV-AuNCs, L-PAV-AuNOs and D-PAV-AuNOs treated A549 cells, respectively. The A549 cells were stained with ethidium bromide (0.2 %, w/v). (B) DNA damage expressed by percentage of DNA in tail. Error bars represent mean ± standard deviation (SD, n =4). * and ** denote significant difference at p<0.05 and p<0.01 levels, respectively.
29
*
*
*
*
* Chiral center
L-PAV-AuNCs
D-PAV-AuNCs
L-PAV-AuNOs
D-PAV-AuNOs
S
PAV
*
Chiral * center
C4H9 S
S
= Gold binding motif
Scheme 1 30
(a1)
(a2)
(b1)
(c1)
(c2)
(b2)
(d1)
(d2)
200 nm
Figure 1 31
30 L-PAV-AuNCs D-PAV-AuNCs
10
15
Elipticity (mdeg)
Ellipticity (mdeg)
(e)
0
-15
L-PAV-AuNOs D-PAV-AuNOs
(f)
5 0 -5 -10
-30 200
220
240
260
200
Wavelength (nm)
220 240 Wavelength (nm)
260
Figure 1 32
(A)
6
Au (µ g/104 cells)
L D
*
**
4
** 2
Figure 2
0 PAV-AuNCs
PAV-AuNOs
33
(A)
Control
(a)
(b)
(c)
(d)
(e)
Control L D
1.0
50
**
** *
*
0.5
Cell migration rate (µ m/h)
Relative spreading area vs control
(B)
0.0
40
Control L D
30
** **
* ** *
20 10 0
Control
PAV-AuNCs
PAV-AuNOs
Control
PAV-AuNCs
PAV-AuNOs
Figure 3 34
Relative cell viability (%)
(a) 100
L-PAV-AuNCs D-PAV-AuNCs L-PAV-AuNOs D-PAV-AuNOs
90
80
Fluorescent inensity (O.D)
110 200
150
4 2 1 Au concentration (pM)
(b)
** * ** **
100
** * ** **
50
0
0.5
Positive control Negative control L-PAV D-PAV L-PAV (without NPs) D-PAV (without NPs)
8
+
-
AuNCs
AuNOs
Figure 4
35
(A) Negative control
Positive control
(B)
L-PAV-AuNCs
L-PAV-AuNOs
D-PAV-AuNCs
DNA damage expressed by percentage of DNA in tail
100 Positive control Negative control L D
80 60
*
40
*
20
*
*
D-PAV-AuNOs
0
+
-
PAV-AuNCs PAV-AuNOs
Figure 5 36
Graphical abstract
* * * Chiral
* *
center
L-PAV-AuNCs
D-PAV-AuNCs
L-PAV-AuNOs
D-PAV-AuNOs
ROS
Nucleus
DNA damage
37
Significance:
Gold nanoparticles with different structure and surface chirality are fabricated.
The structure and surface chirality at the nanoscale can influence cytotoxicity and genotoxicity.
A new perspective on designing nanoparticles for drug delivery, bioimaging and diagnosis.
38