Shape-Dependent Dissolution and Cellular Uptake of Silver

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Shape-Dependent Dissolution and Cellular Uptake of Silver Nanoparticles Christina Maria Graf, Daniel Nordmeyer, Christina Sengstock, Sebastian Ahlberg, Joerg Diendorf, Jörg Raabe, Matthias Epple, Manfred Köller, Juergen Lademann, Annika Vogt, Fiorenza Rancan, and Eckart Ruehl Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.7b03126 • Publication Date (Web): 22 Dec 2017 Downloaded from http://pubs.acs.org on December 25, 2017

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Shape-Dependent Dissolution and Cellular Uptake of Silver Nanoparticles Christina Graf,§*† Daniel Nordmeyer,§ Christina Sengstock,† Sebastian Ahlberg, Jörg Diendorf, Jörg Raabe, Matthias Epple, Manfred Köller,† Jürgen Lademann, Annika Vogt, Fiorenza Rancan,# and Eckart Rühl§# § Freie Universität Berlin, Physikalische und Theoretische Chemie, Institut für Chemie und Biochemie, Berlin, Germany. † Bergmannsheil University Hospital/Surgical Research, Ruhr-University Bochum, Bochum, Germany.  Charité-Universitätsmedizin Berlin, Clinical Research Center for Hair and Skin Science, Department of Dermatology and Allergy, Berlin, Germany  Inorganic Chemistry and Center for Nanointegration Duisburg-Essen (CeNIDE), University of Duisburg-Essen, Essen, Germany.  Paul Scherrer Institut, Swiss Light Source, Villigen, Switzerland.

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* Corresponding author: [email protected] Tel: +49 6151-16-38216 † Present address: Fachbereich Chemie- und Biotechnologie, Hochschule Darmstadt, Darmstadt, Germany # These authors contributed equally to this manuscript.

KEYWORDS Silver nanoparticles, nanoprisms, human mesenchymal stem cells, human keratinocytes, nanoparticle dissolution, cellular uptake, Scanning Transmission X-ray Microscopy

ABSTRACT

Cellular uptake and dissolution of trigonal silver nanoprisms (edge length: 42±15 nm, thickness: 8±1 nm) and mostly spherical silver nanoparticles (diameter: 70±25 nm) in human mesenchymal stem cells (hMSC) and human keratinocytes (HaCaT cells) were investigated. Both particles are stabilized by polyvinylpyrrolidone (PVP), the prisms additionally by citrate. The nanoprisms dissolved slightly in pure water but strongly in isotonic saline or at pH 4, corresponding to the lowest limit for the pH during cellular uptake. The tips of the prisms became rounded within minutes, due to their high surface energy. Afterward, the dissolution process slowed down due to the presence of both, PVP stabilizing Ag{100} sites and citrate blocking Ag{111} sites. Contrary, nanospheres, solely stabilized by PVP, dissolved within 24 h. These results correlate with the finding that particles in both cell types have lost >90% of their volume within 24 h. hMSC took up significantly more Ag from nanoprisms than from nanospheres, whereas HaCaT


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cells showed no preference for one particle shape. This can be rationalized by the large cellular interaction area of the platelet-like nanoprisms and the bending stiffness of the cell membranes. hMSC have a highly flexible cell membrane resulting in increased uptake of platelet-like particles. HaCaT cells have a membrane with a three orders of magnitude higher Young’s modulus than hMSC. Hence, the energy gain due to the larger interaction area of the nanoprisms is compensated by the higher energy needed for cell membrane deformation compared to spheres leading to no shape preference.

Introduction Silver nanoparticles are the most widespread manufactured nanomaterial, about 30% of consumer products that include engineered nanomaterials claim to contain silver nanoparticles.1 Their application in food, cosmetics, biomedicine, medicine, and other life sciences is based on their high antimicrobial, antiviral, and antifungal activities.2-3 Moreover, they have unique plasmonic and catalytic properties making them highly attractive for the development of advanced functional materials.4 Hence, the intense contact with human beings cannot be avoided. Albeit a wide range of studies has been carried using various Ag nanoparticles and cell systems, current knowledge on their adverse effects on biological systems is rather incomplete, and the reported toxicity widely varies depending on the tested system.5 The stability of silver nanoparticles and hence, their biological effects are governed by their size, shape, and capping agents.6 Conventional preparation routes, mostly used for nanosilver applied in consumer goods, yield products with a broad range of sizes and crystal shapes, including anisotropic structures, such as trigonal and hexagonal prisms and rods. Modern synthetic approaches allow for a precise


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control of their size and partially also their shape.7-9 For the latter, surface-directing agents are often employed, which block the growth of the nanocrystals on certain crystalline facets, while enabling the growth in other crystalline directions.7-8 Anisotropic nanoparticles are attractive because of the associated large near-field enhancements allowing their use in a variety of plasmonic applications and SERS (Surface Enhanced Raman Spectroscopy).10 Trigonal silver nanoprisms possessing highly tunable strong plasmon bands11 have a great potential for such usage.12-15 Moreover, anisotropic nanoparticles are important model systems to determine the shape-dependency of the uptake of nanoparticles into cells and tissues. 16-18 The cytotoxicity of Ag nanoparticles is largely associated with their dissolution in biological media and the release of toxic silver ions (Ag+).19 In turn, the release of Ag+ is a function of the pH value and the O2 concentration in the dispersion and is influenced by the presence of reducing sugars, sulfides, sulfur-containing components, and chlorides.20-22 The mechanism of dissolution of anisotropic Ag particles in biological media, such as cell culture media, and in cells, has hardly been investigated.23 Preliminary studies on the dissolution of Ag nanoprisms in NaCl solutions with concentrations (10 mM) far below isotonic concentrations (154 mM) indicate that even under such mild conditions a quick degradation of the particles occurs, where especially the tips of the prisms are prone to rapid dissolution.24 In general, the dissolution of particles depends on their surface functionalization. The formation of anisotropic nanoparticles requires the use of specific directing ligands so that it can become challenging to separate effects of the capping agent from those of the particle shape. Hence, the use of various ligands in different synthetic approaches may lead to diverging results on the dissolution processes and toxicity.25-26 Also, the particle shape may influence their internalization into cells. In general, various factors, such as material, size, surface coating, and zeta potential determine the cellular


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uptake of nanoparticles.27-30 For other nanomaterials, it has been shown that the particle shape influences both, the type of interaction with the cell membrane and the uptake mechanism. Changing of polystyrene nanoparticles from spherical to disk-like shapes enhances their cell surface binding, but reduces their internalization in several cell lines.31 In contrast, mammalian epithelial and immune cells preferentially take up disk-shaped polyethylene glycol-based hydrogel particles with high aspect ratios in contrast to nanorods and nanodiscs with a lower one.27 HeLa cells also preferably internalize high aspect ratio CaCO3 nanoparticles compared to those with a more isotropic shape.32 The cellular uptake of anisotropic Ag nanoparticles has barely been studied.23, 33 Especially, triangular Ag nanoprisms have not been investigated so far. For this reason, in the present work, the cellular uptake and dissolution processes of trigonal Ag nanoprisms are compared with those of Ag nanospheres. Both particle types are surfacestabilized by polyvinylpyrrolidone (PVP) and have a similar zeta potential so that the effects of the ligand shell and charge on their interaction with cells are comparable to each other. The dissolution of the nanoparticles is investigated in solvents of different pH and salt concentrations and cell media with or without fetal calf serum (FCS) supplement, using both microscopic (Scanning Transmission Electron Microscopy, STEM) and spectroscopic (Atomic Absorption Spectroscopy, AAS) methods. The uptake and degree of dissolution of particles were investigated in two different cell types. (1) HaCaT (human adult low calcium high temperature) cells, which are spontaneously transformed human keratinocytes, representing immortalized cells that have a high capacity for differentiation and proliferation.34 Since silver nanoparticles are widely applied in dermatology, in particular in wound dressings and gels,35 a proper risk assessment of the different type of Ag nanoparticles on skin and skin cells is highly relevant. 22 (2) Human mesenchymal stem cells (hMSC) are non-transformed, non-immortalized cells found


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in various tissues, such as bone marrow, fat, and muscle tissue. These fibroblast-like cells are also involved in the process of tissue regeneration during wound healing so that the probability is high that these cells interact with cytotoxic Ag nanoparticles after exposure.

36-37

((Scanning)

transmission electron microscopy ((S)TEM), scanning transmission X-ray microscopy (STXM), and AAS are applied for the quantitative, semi-quantitative, and qualitative investigation of the cellular uptake of the Ag nanoparticles. TEM allows the imaging of nanoparticles in biological samples with a high spatial resolution of about 1 nm. However, only sliced samples of < 70 nm thickness can be studied. In STXM, thick (up to 10 μm) and wet samples, i.e., complete cells, can be investigated with slightly lower (15 nm) resolution. Staining of cells is not required, which is contrary to TEM. Due of the limited number of available setups, STXM has been only applied in a small number of cell studies,22,

38-39

but the high potential of this method for the

investigation of biological samples has been proved in some recent studies on the uptake of drugs and nanocarriers in human skin.40-43

Results and Discussion Trigonal Ag nanoprisms were prepared by thermal reduction of AgNO3 in an aqueous dispersion of citrate, PVP, H2O2, and NaBH4 according to ref. 7. TEM analysis confirmed that these nanoprisms are not aggregated and have a uniform morphology but a rather polydisperse size distribution (Fig. 1 a). Particle characterization data are given in Table 1. The average thickness of these particles obtained from TEM images of stacks of vertically oriented Ag prisms is 8±1 nm, whereas the edge length is 42±15 nm. Spherical Ag nanoparticles were obtained from the reduction of AgNO3 with glucose in the presence of PVP as described before.44-45 These particles


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have a diameter in TEM of 70±25 nm. They are also not aggregated but less polydisperse and mostly spherical. A few triangular particles are also present (Fig. 1 b). DLS measurements confirmed that both particle species are colloidally stable in water (Table 1). The hydrodynamic diameter of the spherical particles is only slightly larger than the one obtained from TEM. In contrast, the equivalent spherical hydrodynamic diameter of the nanoprisms is significantly smaller than their edge length obtained from TEM because of their strongly anisotropic shape. Both particle types are mostly stabilized by PVP and have a negative zeta potential (Table 1). The zeta potential of the nanoprisms is slightly more negative, likely because these particles are also stabilized by citrate groups used during their synthesis.

a

b

100 nm

100 nm

Figure 1. TEM images of (a) trigonal silver nanoprisms with an edge length of 54±28 nm and (b) mostly spherical silver nanoparticles with a diameter of 70±25 nm.


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Table 1. Characterization of the silver nanoprisms and nanospheres in water. Sample Surface Edge Prism Diameter Hydro- PDI Zeta Surfacecoating length thickness (TEM) dynamic potential to-volume (TEM) (TEM) diameter ratio [nm] Silver nanoprisms

PVP (+citrate)

Silver PVP nanospheres

[nm]

[nm-1]

[nm]

[nm]

[mV]

42±15 8±1

-

27±1

0.39±0.04 -29±1

0.41±0.10

-

70±25

75±20

0.22±0.02 -25±1

0.09±0.02

-

Since it is assumed that the release of Ag+, going along with a size reduction is a major cause of the toxicity of Ag particles,19, 46 the dissolution process of the particles was studied in greater detail. The dissolution of spherical Ag nanoparticles in different media has already been investigated.19-20, 47-49 Most authors combine a filtration method to separate ions and particles, such as dialysis and ultrafiltration, with a spectroscopic ion detection method, such as AAS, inductively coupled plasma-mass spectrometry (ICP-MS), or inductively coupled plasma optical emission spectrometry (ICP-OES). Here, we focus on the less studied trigonal silver nanoprisms. The dissolution of trigonal Ag prisms with a concentration (cAg) of 25 mg/L was studied in four different media: ultrapure water, isotonic saline (ionic strength (I) = 154 mmol/L), Roswell Park Memorial Institute Medium 1640 (RPMI medium, I = 138 mmol / L), and RPMI medium with 10% fetal calf serum (RPMI/FCS) (I = 124 mmol/L). These media correspond in ascending order to solutions with higher ionic strength and protein content and are similar to in vivo extracellular fluids. In this way, the influence of the different components of the cell culture medium can be modeled. All experiments were carried out in the presence of ambient O2 with particles previously stored in O2-free water and under Ar. Centrifugal filter units were applied for the


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separation of released Ag+ from the Ag particles after defined time periods. The Ag+ concentration was determined by AAS. Filter units with a molecular weight cut-off (MWCO) of 3 kDa, corresponding to a pore size of 1-2 nm, were used to guarantee an accurate separation of small Ag particles from dissolved Ag+. With these filter units, the Ag particles could be separated within 15 min from Ag+. Since Ag+ can be adsorbed by the filter membrane, the percentage of Ag+ remaining in the pores was determined using AgNO3 solutions of different concentrations (see Table S1 in the Supporting Information). It turned out that only 3.60.3% of the nanoprisms in ultrapure H2O were dissolved as Ag+ after 48 h (Fig. S2). Already 2.60.3% of the Ag was dissolved in the first measurement, where the particles were immediately separated from the released ions. This initial amount of Ag+ is likely released during the particle preparation when after the formation of the nanoparticles, the reaction mixture is centrifuged for 2 h to remove excess reactants (see Supp. Inf.). If this initial amount of Ag+ is substracted, it results that only about 1.10.5% of the prisms are dissolved within 48 h in ultrapure H2O. In isotonic saline, all anions and in RPMI and RPMI/FCS 78% and 71%, respectively of the anions are Cl-. AgCl has a solubility of about 1.88 mg/L in H2O.50 Thus, it can be assumed that all excess free Ag+ binds directly after release to Cl- and precipitates as AgCl. Control experiments show that precipitated AgCl cannot diffuse through the filter membranes and thus, Ag which is bound as AgCl, cannot be detected by AAS. However, in isotonic saline still 2.30.2% of the Ag could be detected as released Ag+ by AAS (Fig. S2). This value did not change significantly over 48 h. In RPMI there are additionally to Cl- sulfur-containing amino acids (L-methionine and Lcysteine) and their dimers present, which are known to bind Ag+. In RPMI/FCS also proteins, such as bovine serum albumin, are present which can bind Ag+.51 As a result, almost all released Ag+ was bound or precipitated and, hence, in RPMI and RPMI/FCS no released Ag+ could be


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detected by AAS. It was recently shown that nanoparticulate AgCl, which is likely formed during this process has the same cytotoxicity as Ag+ towards eukaryotic cells.21 It was expected that Ag nanoprisms dissolve quicker and to a greater extent than Ag nanospheres. Due to the Gibbs–Thomson effect, a convex surface has a higher surface energy than a flat one in the same phase.11, 52-53 Thus, Ag atoms at the tips of the prisms should be especially prone to dissolution.24, 54

However, earlier studies showed that up to 15% of citrate-functionalized spherical Ag particles

and > 40% of PVP-coated spherical Ag particles are released as Ag+ within the same period as in the present case (48 h).19-20 Nevertheless, due to the synthesis procedure, the studied Ag prisms are most probably coated by a mixture of PVP and citrate. STEM was applied to monitor the temporal changes of Ag prisms in different aqueous media. Investigations in pure water (pH 7) in the presence of O2 show that within 24 h neither the shape nor the average size of the prisms significantly changes (Fig. 2 a, b), only a slight rounding of the tips is observed (Fig. 2 c). This finding agrees with AAS results indicating only minor dissolution in this period. The small (< 5 nm) gray spots in Fig. 2 c are not strongly shrunk Ag prisms, but likely originate from the small fraction of released Ag+ which reacts with atmospheric O2 and precipitates as Ag2O on the TEM grids.55 AgNO3 solutions (c = 0.125 and 1.25 mg/L), corresponding to the amount of Ag+ which is released when 0.5% and 5%, respectively, of the Ag prisms, dissolve, were dried on TEM grids. In both cases, similar gray spots in the same size range were observed (data not shown). The dissolution process in the different stages of cellular uptake was modeled by incubating the Ag prisms for 24 h in (1) aqueous acetate buffer at pH 4 (Fig. 2 d), (2) isotonic aqueous solution of NaCl at pH 7 (Fig. 2 e), and (3) isotonic aqueous solution of NaCl, buffered with acetate to pH 4 (Fig. 2 f). While the pH outside the cells is neutral, it decreases during the different stages of cellular endosomal


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uptake from pH ~ 6 in early endosomes to pH ~ 5 to 6 in late endosomes and pH ~ 4.5 to 5 in lysosomes.56 Hence, the acetate buffer at pH 4 represents the lowest limit for the pH during cellular uptake. At pH 4 the results still resemble those in ultrapure H2O (pH 7). Some particles still have a prismatic shape, but the tips are significantly more rounded than at pH 7 (Fig. 2 d). In contrast, the particles significantly shrink and turn into round platelets in isotonic saline at pH 7 (Fig. 2 e). Similar results are found for isotonic saline at pH 4 (Fig. 2 f). In both cases, the average diameter is after 24 h of incubation 16±4 nm, corresponding to a decrease of the mean area per particle of 32±18%. The significantly enhanced dissolution of Ag prisms in the presence of Cl- can be explained by the high affinity of Cl- for Ag+ (H0f = -127.01 kJ/mol).57 It was not possible to investigate the dissolution process in RPMI or RPMI/FCS using STEM, since several of the contained components, e. g. amino acids, proteins, and glucose, made it impossible to image the prisms accurately.


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a

b

100 nm

c

100 nm

e

d

100 nm

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25 nm

f

100 nm

100 nm

Figure 2. STEM images of initially triangular-prismatic particles after (a) 0.5 h and (b, c) 24 h of incubation in water saturated with air, as well as after 24 h of incubation in (d) acetate buffer at pH 4, (e) an isotonic aqueous solution of NaCl, and (f) an isotonic aqueous solution of NaCl, buffered to pH 4 with acetate.

Additional extinction measurements monitored the degradation of the Ag prisms in different media (Fig. 3). cAg was in all experiments 70 mg/L to reach a sufficient signal-to-noise ratio during the complete dissolution processes. The surface plasmon resonance spectrum of the Ag prisms remains nearly unchanged after incubation in ultrapure water. Only a slight blue-shift of the in-plane dipole plasmon resonance from 675±1 nm to 663±1 nm was observed after 24 h, which is associated with both, a reduction in edge lengths of the prisms and less sharp tips of the prisms.7, 53 This confirms the results from STEM that a slight rounding of the tips of the prisms takes place. The in-plane quadrupole resonance (331±1 nm) and the out-of-plane quadrupole resonance (391±1 nm) remain unchanged within 24 h, indicating that the thickness of the prisms remains unchanged.53 In contrast, Tang et al. observed that when Ag nanoprisms from an almost identical synthetic procedure are heated in water in the presence of O2, and a transition to round


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platelets takes place, the in-plane dipole plasmon resonance is blue-shifted, but the out-of-plane quadrupole plasmon resonance band is red-shifted, i.e., the thickness of the particles increases which was also confirmed by TEM. Thus, a shape transformation takes place, which is explained by a migration process of surface atoms of the Ag nanoplates from the tips to the triangular plane. In the present case, there is no evidence that Ag+ is reabsorbed at a new position on the particle, suggesting that all Ag+ is released into the solvent. This corresponds to the AAS results in pure water, indicating that minor dissolution takes place. In contrast, the extinction spectrum immediately changes when the pH is lowered to 4. A strong blue-shift (852 nm) of the in-plane dipole plasmon resonance occurs accompanied by a broadening of the spectrum. The latter indicates that some agglomeration occurs. After 24 h of incubation, the spectrum is not further shifted in wavelength, but the band is slightly narrowed. Likely, some agglomerates redisperse with time. Also at this pH, the in-plane and the out-of-plane quadrupole resonances did not shift within 24 h. This observation indicates that a dissolution process is taking only place at the tips and there is no significant release of Ag+ from the triangular faces. In isotonic saline, a rapid and marked decrease of the extinction spectrum is observed, and it is blue-shifted by about 280 nm and significantly broadened. It is impossible to identify the bands of the in-plane quadrupole and in-plane dipole plasmon resonance modes in this spectrum. These results suggest that rather polymorphous and polydisperse Ag particles and not mainly platelets are present, i.e., also the triangular faces of the prisms are degraded, which is in agreement with the results from STEM. Incubation for another 24 h in isotonic saline or a lowering of the pH to 4 does not induce a significant change of the surface plasmon resonance spectra. These observations show that the dissolution process takes place immediately after the particles are transferred into a medium with an isotonic NaCl concentration. The formation and precipitation of AgCl likely accelerate the


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dissolution process of the prism tips strongly, which was already observed in water and acetate buffer (see above). However, this process terminates quickly. Likely, this is because an Ag2O layer on the particle surface is formed.48 Thus, during cellular uptake experiments, the dissolution already takes place in the cell culture medium before the majority of the particles are taken up by the cells. Since a lowering of the pH in isotonic saline does not cause a significant further dissolution, it appears straightforward to assume, that during the different stages of the endocytic process no significant further degradation of the Ag prisms occurs. Similar experiments were carried out on Ag nanospheres. Extinction spectra were measured in the same four media which were also used for the prisms. cAg was doubled in comparison to the prisms because the extinction at cAg = 70 mg/L was too low for taking extinction spectra with a reasonable signal-to-noise ratio. As observed for the Ag prisms, in ultrapure water only negligible changes in extinction spectra of the Ag nanospheres were found within 24 h. In contrast to nanoprisms, Ag nanospheres dissolve much slower in acetate buffer and isotonic saline. The shape of their extinction spectra changes only slightly with time in contrast to those of the silver nanoprisms (see a comparison of both particle types in Figure S1 in the Supporting Information) because the particle shape of the silver nanospheres remains preserved. Remarkably, the spectra are not significantly blue-shifted, although the particles shrink significantly in size (see STEM images in Figure 5). This can be explained by the rather weak size-dependence of the surface plasmon band of smaller silver nanoparticles,58 the rather broad size distribution of the samples and likely a superimposed aggregation effect causing a red-shift of the spectra. At pH 4 and in isotonic saline a tailing at higher wavelengths is observed, indicating some slight aggregation.


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The kinetics of the dissolution processes of the silver nanospheres can be monitored by measuring the decay of the intensity at the extinction maximum as a function of time (Fig. 4). The dissolution of the Ag nanospheres in acetate buffer (pH 4) can be well described by a simple first-order kinetics (Eq. 1 with A2 = 0 and k2 = 0). Their dissolution in saline solutions cannot be fitted by a monoexponential function, but rather by a biexponential function, i.e., multiple processes with significantly different rate constants take place (Eq. 1). This can be rationalized because in salt-containing solvents (isotonic saline and isotonic acetate buffer), in addition to the proton-driven dissolution process, precipitation of Ag+ with Cl- occurs.

I(t)  I   A1e k1t  A 2 e k2t

(1)

Here, I(t) is the time-dependent extinction, I(t) the extinction for t→, A1 and A2 the amplitudes and k1 and k2 the rate constants of the process. The results of the fits are summarized in the Supporting Information (Table S3). STEM images of Ag nanospheres after 24 h of incubation in ultrapure water (pH 7) or acetate buffer (pH 4) confirm the results from UV-VIS spectroscopy: Ag spheres in ultrapure water are hardly dissolved after 24 h (Fig. 5 a), while those in acetate buffer (Fig. 5 b) are significantly more dissolved also in respect to the Ag prisms in the same medium (Fig. 2 d). STEM images of Ag spheres that were incubated in isotonic saline or isotonic acetate buffer for 24 h could not be made since large amounts of NaCl crystallized on the TEM grids.


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pH 7, 0 h pH 7, 24 h pH 4, 0 h pH 4, 24 h NaCl, pH 7, 0 h NaCl, pH 7, 24 h NaCl, pH 4, 0 h NaCl, pH 4, 24 h

Extinction [arb. units]

1.0 0.8 0.6 0.4 0.2 0.0 300

400

500

600

700

800

Wavelength [nm]

Figure 3. Extinction spectra of Ag nanoprisms immediately after transfer (solid lines) and after 24 h of incubation (dotted lines) in water saturated with air at pH 7 (black lines), aqueous acetate buffer at pH 4 (red lines), isotonic aqueous solution of NaCl at pH 7 (blue lines), and isotonic aqueous solution of NaCl, buffered with acetate to pH 4 (green lines).

1.0

Extinction [arb. units]

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Pure water (pH 7) Acetate buffer (pH 4) Isotonic saline (pH 7) Isotonic acetate buffer (pH 4)

0.8 0.6 0.4 0.2 0

5

10

15

20

25

Time [h]

Figure 4. Maximum of the extinction spectra as a function of time for dispersions of Ag nanospheres (c = 140 mg/L) in water saturated with air (black dots), an aqueous buffer at pH 4 (red dots), an isotonic aqueous solution of NaCl (blue dots), and an isotonic aqueous solution of NaCl, buffered at pH 4 (pink dots). The dotted lines are exponential fits according to Eq. 1. The fit

parameters

are

summarized

in

Table

S3

in


the

Supporting

Information.

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a

b

100 nm

100 nm

Figure 5. STEM images of initially spherical silver nanoparticles after 24 h of incubation in (a) water and (b) an aqueous buffer at pH 4, both liquid samples were saturated with air.

The partially dissolved Ag nanospheres have a diameter of 16 ± 5 nm after 24 h of incubation in acetate buffer. Thus, they lose 991% of their original volume, assuming that the particles are approximately isotropic. The Ag prisms lose only 74±3% of their original volume under the same conditions, assuming from the results of the UV-VIS measurements that their thickness remains unchanged. Even in isotonic saline at pH 4, the Ag prisms lose 87±3% of their volume within 24 h (see Fig. 2), assuming their thickness decreases to the same degree as the lateral extension. Under these conditions, it was not possible to estimate their thickness from the surface plasmon resonance spectra. This degree of shrinkage is likely overestimated since the bases of the prisms are highly stabilized by citrate, and hence, their thickness barely decreases (see below). Although the nanoprisms start to dissolve extremely quickly within the first minutes after their transfer into the medium, they release fewer Ag+/volume within 24 h than the spheres. The extent of the dissolution of silver nanoprims and silver nanospheres in different media is compared in Fig. 6.


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Degree of Dissolution [%]

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120 100

Page 18 of 52

pH4 NaCl, pH7 NaCl, pH4

80 60 40 20 0

Prisms

Spheres

Figure 6. Degree of dissolution of silver nanoprims and silver nanospheres after 24 h of incubation in aqueous acetate buffer at pH 4, isotonic aqueous solution of NaCl at pH 7, and isotonic aqueous solution of NaCl, buffered with acetate to pH 4, according to STEM measurements (see Fig. 2 and 5).

The Ag nanoprisms used in the present study are stabilized by citrate and PVP. In contrast, the Ag nanospheres are only stabilized by PVP. The twofold stabilization of the prisms is a consequence of their growth process which is based on the concept that citrate preferably adsorbs onto the {111} faces of an initially hexagonal crystal plate to direct the formation of the final triangular shape (Fig. 7 a).7, 59-61 According to a model of Aherne et al. based on high-resolution transmission electron microscopy studies, the lateral faces of these lamellar-hexagonal crystals consist of {111} or {100} faces with a hexagonally-packed (hcp) (or defect-rich) region sandwiched in between.60 Thus, three of the lateral faces have a larger {100} plane than {111}


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plane while it is vice versa for the other three ones (Fig. 7 a).60 The hexagonal bases of these prisms are also {111} faces. The {100} faces are less stabilized by citrate than the {111} faces so that a directed growth in three directions and a trigonal-prismatic shape result (Fig. 7 a, b). Since particles stabilized by citrate are only temporary stable, PVP is added as an additional stabilizer. According to several studies modeling PVP binding energies to Ag,62-64 PVP binds preferably to {100} faces compared to {111} faces.59 The initial fast dissolution of the apices of the Ag prisms can be explained by their high surface energy (see above). Most of the Ag nanospheres do not show such prominent convex structures (Fig. 7 b) and hence, dissolve much slower. After this initial process, the former triangular prisms are converted into truncated triangular prisms with rounded tips (Fig. 7 b and Fig. 2 b-d). The further dissolution process is, therefore, likely no more governed by local high surface energies due to their geometrical shape, but rather by the different stabilization of the different crystal faces. Calculations based on density functional theory (DFT) revealed that the binding energies of citrate groups on Ag{111} and Ag{100} surfaces are 57.9 and 15.4 kJ/mol, respectively, because four ligand-surface bonds are formed with Ag{111} but only two bonds with Ag{100}.65 This difference in binding energy corresponds to six orders of magnitude higher binding constant of citrate on Ag{111}.65 The truncated triangular prisms formed after the initial dissolution process consist mainly of {111} surfaces, which are stabilized by citrate o not dissolve not or dissolve extremely slowly even at low pH and high concentration of chloride (Fig. 7 c). PVP is, in contrast to citrate, a polymeric capping agent and hence binds polyvalently to Ag. This makes an estimation of its binding energies, especially a quantitative comparison with other molecules, more complicated.66 Experimental data and calculations show (see above) that PVP stabilizes Ag{100} facets more efficiently than Ag{111} facets.

59, 62-64, 66-67

Moreover, citrate also acts as a reducing agent.68


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Therefore, the remaining {100} facets of the truncated prisms are likely highly stabilized, and consequently, highly stable structures result which hardly dissolve even at lower pH or in the presence of Cl- (Fig.7 c). The surface of Ag nanospheres consists of a mixture of {111} and {100} facets. The use of ligands in the synthesis of Ag nanoparticles that preferably adsorb to one of these two surfaces orientations, lead to the formation of nanocrystals where the more stabilized crystal orientation predominates, usually resulting in the formation of certain anisotropic structures, e. g. nanocubes in the case of PVP.66 The silver particles referred to as “silver spheres” in the present study are polymorphic but mostly spherical. i.e., rather isotropically grown, besides the presence of a few prisms (Fig. 1 b). This suggests that no strongly surface-directing process took place during their growth. According to molecular dynamics (MD) simulations69 as well as calculations using a thermodynamic model based on a geometric summation of the specific Gibbs free energy of an entire nanoparticle,70 the most stable structure for a silver particle in the size range of the spheres studied in the present work is a truncated octahedron consisting of {111} and {100} facets (see Figure 6 d). A significant part of the surface of such a particle are consequently {111} facets, which are less effectively stabilized. Thus, in this case, a situation where all facets are highly stabilized, and the dissolution process almost stops, is not quickly reached (see Figure 6 d and e).


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Ag Ag+ Ag+ + Ag Ag+

+

{111}

Ag+

X

{111}

Ag+

{111}

Ag+ Ag+ Ag+

Ag+

Ag+

Ag+

Ag+ + Ag+ Ag

Ag+

Ag+

Ag+ Ag+

(a)

(b) Ag+ Ag+

(c) Ag+

Ag+

Ag+

Ag+

(d)

(e)

Figure 7. (a) Growth of silver prisms from hexagonal platelets in the presence of citrate according to ref. 60; (b) quick initial dissolution of the tips of the prisms occurs because of their high surface energy, according to the Gibbs-Thomson effect; (c) slow dissolution of the silver platelets after the dissolution of their tips. The dissolution occurs likely preferably from the {100} facets and possible interjacent layers with hcp structure, which is due to the high binding energy of citrate to {111} facets. (d) Silver particles denoted as spheres are faceted crystals consisting of {100} and {111} facets, in the present size range likely truncated octahedrons.69-70 (e) Since {111} facets are less effectively stabilized by PVP than {100} facets, a situation where all facets are highly stabilized, and the dissolution process strongly decelerates is not quickly reached.


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In the present study, the silver spheres are larger than the silver prisms. The effect of the particle size on the dissolution of spherical silver nanoparticles has been investigated in earlier studies.23, 71-72

In particular, the size dependence of the dissolution of PVP-coated silver nanospheres

prepared by the same method as in the present work has been investigated.23 It was shown that the dissolution process occurs by the same mechanism for spherical Ag particles with different diameters and that the dissolution rate is proportional to the specific surface area of the spheres.

The dissolution of citrate and PVP-stabilized Ag nanoparticles in different media have been studied before. Some authors found that citrate-coated nanocrystals are more stable,19, 73 whereas others observed that PVP-stabilized particles dissolve less than the citrate stabilized ones74-75 or no significant differences between both coating agents were found.76 In all these cases, Ag particles denoted as “spherical” are studied, i.e., facetted crystals with an approximately isotropic shape, where the exact ratio of {111}:{100} facets is unknown. Since this ratio determines if citrate or PVP is a more effective stabilizer, no clear trend can be assigned. In earlier studies, a much higher release of Ag+ from Ag nanoparticles was observed in water than in the present study.19-20 This might also be explained by the use of dialysis membranes or centrifugation filter with membranes with large pores compared to the particle size for the separation of Ag particles from the released Ag+ before the determination of the Ag+ concentration by AAS or ICP-OES (9 nm pores in ref. 19, 1-2 nm pores for 4.81.6 nm diameter particles in ref. 20). Consequently, small Ag particles may have contributed to the signal assigned to Ag+. In the present study, a smaller membrane pore size in comparison to the particle size was chosen so that the diffusion of Ag particles through the filter membrane can be largely excluded. The moderate dissolution of the particles under the used conditions is well confirmed by the STEM and UV-VIS


22

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spectroscopy results, which are independent of a proper separation of nanoparticles and released ions.

STXM, TEM, and AAS were used to study the qualitative, semi-quantitative, and quantitative uptake and dissolution of prismatic and spherical Ag nanoparticles (cAg= 10 and 25 mg/L, respectively) in hMSC and HaCaT cells. For STXM experiments, hMSC and HaCaT cells were grown on windows with a collagen-coated Si3N4 membrane and incubated for 24 h with dispersions (cAg = 25 mg/L) of the Ag nanoparticles in RPMI/FCS. After incubation, cells were washed and fixed with 2% paraformaldehyde. A second window was glued on the first one to form a wet chamber. The STXM experiments were carried out in a He atmosphere (p = 5104 Pa) and the measurements were done in the water window at E = 510 eV below the O K edge. At this photon energy, the chemical contrast of the organic material is optimal since the absorption of proteins is considerably higher than the absorption of water. Slicing or contrasting of the cells is not required for these experiments. STXM imaging of hMSC incubated with Ag spheres shows that large amounts of these particles are taken up in the perinuclear region (Fig. 8 a, b). The particles cluster or at least associate to larger units in the cells, so that the spatial extent of the observed Ag structures is much larger than the diameter of the single particles (70±25 nm, s. Fig. 8 b). In contrast, only a few Ag structures are found in the STXM images of the hMSC incubated with Ag prisms (Fig. 8 d-f). These aggregates are significantly smaller than those of the Ag spheres and, hence, have a lower-contrast. Please note that the large dark structures in Fig. 8 d are artifacts, likely from salt crystals in the cell culture medium and not Ag particles. This suggestion is supported by imaging at the Ag M5,4 edges, yielding no Ag signal for this kind of large structures in the range of micrometers. Much fewer Ag spheres were detected in HaCaT


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Page 24 of 52

cells than in hMSC (Fig. 8 c, the large dark structures are again salt crystals). It was not possible to image any Ag nanoprisms or aggregates thereof in HaCaT cells. Possibly this is because the Ag particles are taken up by HaCaT cells to a lower extent or because they could not be detected by STXM due to less aggregation in HaCaT cells than in hMSC. Alternatively, these findings might be explained by the fact that Ag particles dissolve, once taken up by HaCaT cells and thus cannot be detected. The resolution of STXM in these experiments was about 15 nm, which is well below the edge length (42±15 nm) of the initial prisms. It has to be considered that the thickness of the prisms is only 8±1 nm. Thus, when the X-rays impinge perpendicular to one of the three side surfaces of the prisms, single prisms cannot be easily identified by their characteristic shape using STXM in cells.

a

c

5 µm

5 µm

b

1 µm

d

e

2 µm f 5 µm

500 nm


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Figure 8. STXM images at 510 eV of (a, b) hMSC and (c) HaCaT cells after 24 h of incubation with Ag nanospheres and (d-f) hMSC after 24 h of incubation with Ag prisms. For additional examples, s. Fig. S3 in the Supporting Information.

TEM has a much higher resolution than STXM so that even single upright Ag prisms can be easily imaged. The TEM micrographs indicate that significant amounts of Ag nanoprisms (Fig. 9 a-c), as well as Ag nanospheres (Fig. 9 d-f), are taken up into many of the HaCaT cells. Although, none or few of these particles could be identified by STXM in HaCaT cells. However, also with TEM, in some cell sections, no particles were found (Fig. S3 d). A size analysis of the Ag prisms in the HaCaT cells yields an average diameter of only 7±4 nm. This value contains a correction considering that during the slicing of the cells also randomly oriented nanoparticles are cut through. Thus, the particles lose > 95% of their original volume. For simplicity, a spherical shape of the particles was taken as an estimate for the volume loss. The TEM images (Fig. 9 and Fig. S4 in the Supporting Information) and the results of the in vitro dissolution experiments (Figs. 2 and 3a) suggest that prismatic particles are no longer present, i. e. the particles are no more trigonal prismatic but rather round shaped (Fig. 9 c). In the case of the Ag spheres, the average diameter is also markedly decreased to an average value of 13±7 nm. However, the loss in volume is less (> 90%) than for the prisms (Fig. 9 f and Fig. S5 d in the Supporting Information). The Ag spheres form denser aggregates in the HaCaT cells than the former prisms so that still structures with up to several 100 nm are observed (Fig. 9 e, f, and Fig. S5 d), which can also be pictured by STXM (Fig. 8 c). In contrast, most structures formed by the Ag prisms in HaCaT cells are smaller and less electron dense than the spheres (Fig. 9 c),


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and hence, they are too small for STXM imaging. Both types of particles are found exclusively in vesicles, supporting the assumption that the particles are taken up via endocytosis.77-78 Endosomal escape was not observed in TEM. From the in vitro dissolution studies in isotonic saline, it is assumed that, when the particles come in contact with the cell culture medium or the cytoplasm, large quantities of Ag+ are released, which explains the strong reduction in the volume of both particles species. A comparison of the sample with control cells (Fig. S6 in the Supporting Information) shows that only particle-treated cells have bubbles on their surface. This morphological change is an indication of acute toxicity and induction of apoptotic cell death.79

a

d

N N

2000 nm

2000 nm e

b

1000 nm 1000 nm f

c

100 nm

100 nm


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Figure 9. TEM images of HaCaT cells after (a-c) 24 h of incubation with Ag prisms and (d-f) 24 h of incubation with Ag spheres. The insets show particles of the same batches before their incubation with cells at the same magnification. For additional examples see Fig. S4 and S5.

Since hMSC have a larger spatial extent than HaCaT cells and become faster confluent, it was sufficient to sow approximately 100,000 cells/well to obtain a confluent monolayer of cells while in the case of HaCaT cells 250,000 cells/well were needed. For this reason, c Ag was adjusted to 10 mg/L for the hMSC to keep the amount of incubated Ag per cell constant. In a second experiment, 100,000 hMSC/well were incubated with a silver particle dispersion with cAg = 25 mg/L. Thus, in this case, the concentration was the same as in the experiments on HaCaT cells, while the number of particles/cell was 2.5 times larger. The TEM images of hMSC (Fig. 10 a-c) indicate that these cells take up significantly more Ag prisms than HaCaT cells (see representative images in Fig. 9 a-c), although cAg was only 10 mg/L. The (former) prisms in the hMSC are much smaller than the initial nano-particles and have a round shape. This decrease in size is slightly less pronounced than in the case of the HaCaT cells (Table 2). The hMSC, which were incubated with Ag nanospheres (10 mg/L), took up significantly fewer particles than hMSC incubated with equally concentrated dispersions of Ag prisms (s. representative images in Fig. 9 d-f), which agrees with the findings obtained from STXM experiments. The diameter of the Ag spheres is significantly decreased after 24 h of incubation, slightly more than in the case of the HaCaT cells (Table 2). Also in the case of hMSC, both types of Ag particles are located only in the endosomes/lysosomes. About 50% of the investigated cells, where particles are found, have bubbles on the cell surface indicating the


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beginning of apoptosis (Fig. 10), which is not observed in untreated control cells (Fig. S6). In none of the analyzed cells (hMSC and HaCaT cells) particles are found in the nucleus (N).

a

d

2000 nm

N

1000 nm b

e

100 nm c

100 nm f

50 nm

100 nm

Figure 10. TEM images of hMSC after (a-c) 24 h of incubation with Ag nanoprisms (c = 10 mg/L) and (d-f) 24 h of incubation with Ag nanospheres (c = 10 mg/L).


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Table 2. Diameters of silver nanoparticles after 24 h of incubation in cells. Cell type HaCaT cells Mesenchymal stem Particles

Triangularprismatic nanoparticles

Spherical nanoparticles

Triangularprismatic nanoparticles

Spherical nanoparticles

Diameter

7±4

13±7

9±6

11±7

[nm]

50 cells (HaCaT cells or hMSC) in TEM sections per sample were analyzed whether they contain nanoparticles, or not. It was not differentiated if a cell section contained many or few particles (Fig. 11). However, either the cells had internalized nanoparticles in large amounts (at least 50 particles per cell in a TEM section), or no nanoparticles were found at all. It has to be considered that only TEM sections of 70 nm thickness and no complete cells were analyzed. The obtained data reveal that significantly more hMSC take up trigonal silver nanoprisms than HaCaT cells. On the contrary, silver spheres are taken-up by a higher percentage of HaCaT cells as compared to hMSC, at least when identical Ag particle to cell ratios are evaluated, i.e., 10 mg/L for hMSC and 25 mg/L for HaCaT cells.


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Cells with Ag nanoparticles [%]

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hMSC, 10 mg/L hMSC, 25 mg/L HaCaT, 25 mg/L

120 100 80 60 40 20 0

Prisms

Spheres

Figure 11. Percentage of cells in TEM sections containing nanoparticles. cAg during the incubation for 24 h was 25 mg/L for HaCaT cells and 10 mg/L and 25 mg/L, respectively for hMSC.

HaCaT cells and hMSC with incorporated Ag nanoprisms contain endosomes/lysosomes with a larger spatial than those observed in cells that have been incubated with Ag nanospheres. This points to possible different uptake mechanisms for prisms and spheres. HaCaT cells and hMSC incubated with Ag nanoprisms have approximately the same number of endosomes/lysosomes per cell section. Contrary to this, the number of endosomes/lysosomes filled with nanoparticles significantly varies in cells which have been incubated with Ag nanospheres. In some sections of HaCaT cells about 30 endosomes/lysosomes with incorporated spheres are found (Fig. 8 d-f), whereas in other cell sections only 3 endosomes/lysosomes are present (Fig. S5). These differences are probably due to the degree of cell differentiation. The sections of the hMSC incubated with spheres contain on average 3±2 endosomes/lysosomes per cell (Table 3).


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Table 3. Number of endosomes/lysosomes per cell and diameter of endosomes/lysosomes in hMSC and HaCaT cells. cAg during the incubation for 24 h was 25 mg/L for HaCaT cells and 10 mg/L for hMSC. Number of endosomes/ lysosomes per cell

Diameter of endosomes/lysosomes [nm]

HaCaT

hMSC

HaCaT

hMSC

Prisms

Spheres

Prisms

Spheres

Prisms

Spheres

Prisms

Spheres

6±4

14±18*

7±4

3±2

700±500

330±130

770±170

490±170

* n = 3 cells (2, 5, and 35 endosomes per cell contain nanoparticles)

AAS was employed for a quantitative analysis of the uptake of Ag nanoparticles into HaCaT cells and hMSC (Fig. 12). For this purpose, the cells were incubated with the nanoparticles for 24 h. Subsequently, the cells were detached by trypsinization, and all nanoparticles which had not been taken up were removed. The remaining cells were treated with aqua regia for 2 h, until no significant scattering was observed in DLS, indicating that nanoparticles, cells, and subcellular structures are completely dissolved. The AAS data reveal that the Ag concentrations in hMSC increase hyperlinearly when they are incubated with 25 mg/L instead of 10 mg/L Ag nanoparticles. This increase is for the spheres (31 times higher) much more important than for the prisms (1.360.09 times higher). Such hyperlinear increase has been observed for the uptake of Ag nanoparticles in other cell lines, such as Chinese hamster ovary cell line CHO-K1.80 hMSC and HaCaT cells, which are incubated with equal amounts of Ag particles per cell, i.e., 25 mg/L in the case of HaCaT cells and 10 mg/L for the significantly larger hMSC, present a different shape-dependent uptake of


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nanoparticles. HaCaT cells take up the same quantity of Ag after incubation with both the nanospheres or the nanoprisms, hMSC (cAg = 10 mg/L) take up 6.1±1.8 times more Ag from prisms than from spheres. For cAg = 25 mg/L this difference is not as strong, but still, a ratio of 3.5±0.1 was found between cells incubated with prisms and cells incubated with spheres.

Ag concentration [mg/L]

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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8

hMSC, 10 mg/L hMSC, 25 mg/L HaCaT, 25 mg/L

6 4 2 0

Prisms Spheres Figure 12. Silver concentration in the different cell samples, obtained from an AAS analysis of cell lysates. cAg during the incubation for 24 h was 25 mg/L for the HaCaT cells and 10 mg/L and 25 mg/L for the hMSC, respectively.

For the hMSC, the AAS results correlate well with those from STXM and TEM for cAg = 10 mg/L where hMSC prefer Ag prisms compared to Ag spheres. At the higher concentration (c Ag = 25 mg/L) almost all cells (94-100%) contain particles, so that in Fig. 11, where only the


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percentage of cells with and without particles is shown, no major differences are found, while the AAS data confirm a strong preference for the uptake of Ag prisms compared to spheres. In the case of the HaCaT cells, the percentage of cells that have taken up spheres is significantly higher than that of cells with Ag prisms (Fig. 11). However, the total amount of absorbed silver hardly differs for both particle species (Fig. 12). Possibly, because some HaCaT cells contain many particles, while others contain only a few (s. above), i.e., if a cell takes up Ag prisms at all then this happens to a rather large extent compared to the uptake of Ag spheres. As shown above (Table 3), the endosomes/lysosomes found after incubation with prisms are larger than those observed for spherical particles. The internalization of nanoparticles depends in general on various factors, such as the physicochemical properties of the particles and their material, surface functionalization, zeta potential, shape, size, as well as the contact surface of the nanoparticles with the cells 27-30 and the state of the individual cell in the cell cycle. Also, sedimentation of the nanoparticles due to gravity during the cellular uptake experiment has to be considered,81 and it has to be distinguished whether mainly Ag+ or Ag nanoparticles are taken up.46, 82-83 In the present study, the material of the nanoparticle cores is identical, and the zeta potential of both particle types differs only slightly (Table 1). Both particle species are coated with PVP with a not too different molar mass (prisms: 29,000 g/mol, spheres 40,000 g/mol). Due to the preparation process, the prisms are also coated with citrate that stabilizes especially the {111} facets of the prisms, i.e., the bases and part of the side faces (Fig. 7). Citrate-stabilized Ag nanoparticles have an about one magnitude higher affinity for the adsorption of proteins, such as albumin,22 which might influence nanoparticle uptake mechanism. Several authors have compared the uptake of citrate and PVP-coated Ag nanospheres with diameters between 10-75 nm in various cell lines (bronchoalveolar


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carcinoma,84 colon adenocarcinoma,84 liver cancer,85 and epithelial cells5, 86). They found either no difference between both coatings5,

84

or a slightly higher uptake of citrate-stabilized

nanoparticles (< 20%)85 or a slight preference for small PVP-coated Ag spheres.86 Regarding these results, it is rather unlikely that a certain fraction of citrate in the ligand shell of the prismatic nanoparticles causes several times higher uptake of these particles into hMSC compared to the spherical ones (see Fig. 12). Solely citrate-stabilized Ag nanospheres often tend to cluster in cell culture media in contrast to PVP-stabilized ones,83-84 including those studied in this work.77 However, the Ag prisms with a PVP/citrate-coating in the present study show no tendency for aggregation in the cell culture medium and form even significantly less clustered structures inside HaCaT cells than their spherical counterparts (Fig. 9 and Fig. S4 and S5). The Ag nanospheres are at least at the beginning of the cell experiments significantly larger and hence, their gravitational force (1.810-17 N) is higher than that of the Ag nanoprisms (6.310-19 N). i. e. the spheres initially sediment faster (~ 0.09 mm/h) than the prisms (~ 0.01 mm/h) which leads to a higher local concentration of the spherical particles at the cell surface. 66 The gravitational force in the interaction zone between the cell and nanoparticles is negligibly small compared to the forces acting due to specific and non-specific binding (10-12 compared to 10-9 N). As a result, this difference does not explain why spheres are preferably taken up by HaCaT cells and prisms in hMSC.81, 87 The most significant difference between the nanoparticles in this study is evidently their shape. The model studies in the various media suggest that the tips of the Ag nanoprisms are dissolved quickly, but their plate-like shape is preserved, so that thin, flat nanoparticles interact with the cells. Thus, they have a large contact area with the cells, in contrast, to the Ag nanospheres (or more accurately: facetted crystals with an approximately spherical shape45 see Fig. 1 B). It has been shown before for negatively charged polystyrene


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spheres compared to two-dimensional disk-shaped particles of the same size, that a more flat, disk-like shape promotes cell surface binding.31 Similar results were found for polyelectrolyte coated Ca(CO3)2 particles with different aspect ratios32 as well as for discoidal and rod-shaped polyethylene glycol (PEG)-based hydrogel nanoparticles of equivalent volume.27 This enhanced contact surface enables increased multivalent interactions with cell membranes27, 88-89 resulting in larger adhesion forces27 and, hence, in a more or less increased cellular internalization, depending on the cell type.27, 32 For an idealized nanosphere, the corresponding interactions are minimal, due to the low contact.27 However, solely the higher surface contact area cannot explain the differences in the uptake by HaCaT cells and hMSC. According to Chithrani et al., the rate of cellular uptake can be regarded as a consequence of the competition between the thermodynamic driving force for wrapping the nanoparticles and the receptor diffusion kinetics.83,

90

In this

model, the thermodynamic driving force corresponds to the amount of free energy which is needed to drag the nanoparticles into the cell, and the receptor diffusion kinetics corresponds to the recruitment of receptors to the docking site.83, 90 Besides the particle-cell adhesion, the strain energy, which is needed for membrane stretching and membrane bending around nanoparticles, determines this thermodynamic driving force,27 which is proposed to be a reason for the differences found for the uptake of spherical and anisotropic nanoparticles in different cell lines.27 The bending stiffness of cells determines their interaction with materials in the exoplasm.91 This suggests that the bending stiffness of the cell membrane plays a critical role in shape discrimination during the cellular uptake of nanoparticles. HaCaT cells have a high bending stiffness with a corresponding Young's modulus of about 0.1 MPa,92 whereas hMSC are much more flexible with a Young's modulus of about 10-4 MPa.93 Obviously, the internalization of platelet-like nanoparticles is enhanced compared to nanospheres when the cell has a rather


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flexible membrane like the hMSC. Presumably, a flat particle with an extended interaction area is more easily internalized than a spherical one when the cell membrane is flexible so that a favorable energy balance results from the energy gain due to the significant increase in particlecell adhesion and a not too high strain energy. On the contrary, if the cell membrane is stiff, as, in the case of the HaCaT cells, a particularly high deformation energy is required for the uptake of an extended, rigid solid particle. Hence, despite the energy gain due to the large interaction area, the uptake of platelet-like particles is not favored compared to that of nanospheres. Such considerations apply mainly to rigid nanoparticles. In other studies, on the shape-dependence of cellular internalization, polymeric or hydrogel nanoparticles were used,27,

31

which are

significantly softer than the crystalline metal nanoparticles investigated in this work. Hence, these can adjust their shape when they interact with the cell membrane. In summary, anisotropy and membrane bending explain well why we found so large differences in the uptake of different nanoparticles by different cell types, which has not been observed before.

Conclusions The dissolution process and cellular uptake of PVP-stabilized trigonal Ag nanoprisms from a synthesis with citrate as a reducing agent are compared to those of PVP-stabilized, mostly spherical Ag nanoparticles obtained by reduction with glucose. Ag nanoprisms and nanospheres dissolve only slightly in pure water but strongly degrade in isotonic saline or at lower pH corresponding to the situation in lysosomes after endocytosis. The tips of the nanoprisms round within a few minutes because of their high surface energy. Subsequently, the dissolution process slows significantly down, which can be explained by the presence of PVP that effectively


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stabilizes Ag{100} and of citrate that blocks the Ag{111} facets resulting in a high overall stability. The platelet-like shape of the nanocrystals remains retained even after long exposure to these conditions. In contrast, the spherical particles dissolve more constantly within 24 h, which can be explained by their stabilization solely with PVP. These data derived from the cell medium correlate well with the results found in both cells types, where the particles lose more than 90% of their original volume within 24 h of incubation with cells. AAS and TEM analyses reveal that hMSC take up significantly more Ag when incubated with nanoprisms than with nanospheres. On the contrary, HaCaT cells ingest the same quantity of Ag after incubation with both particle types. This difference can be rationalized by the large interaction area of the platelet-like particles (former nanoprisms) with the cell surface and the bending stiffness of the cell membrane. In the case of hMSC, which have a highly flexible cell membrane, the uptake of platelet-like particles is strongly enhanced. HaCaT cells have a rather stiff membrane with an about three orders of magnitude higher Young’s modulus than hMSC. Thus, the energy gain due to the larger interaction area of the former nanoprisms compensates the higher energy cost which is required for the deformation of the cell membrane for the endocytosis of a stiff platelet compared to a more isotropic nanoparticle which results in no specific shape preference. Most metal and metal oxide nanoparticles are faceted crystals, which are inherently never perfect spheres, i.e., they have a certain anisotropy. For most applications in consumer goods, the nanoparticle morphology is not controlled in the preparation process. Moreover, for plasmonic applications, anisotropically shaped nanoparticles are needed. Given this, these results are highly relevant for a comprehensive understanding of the biological effects of nanoparticles: The crystalline shape can be an important factor determining the extent of cellular uptake by specific


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cell types as well as their dissolution process. This information should be considered in a proper risk assessment of such nanomaterials.

ASSOCIATED CONTENT. Supporting Information Available. Details of the synthesis of the particles and their physicochemical characterization, the microscopy studies, and the cell experiments are given. Further information on the dissolution of the Ag nanoprisms and Ag nanospheres, as well as additional extinction spectra, STXM and TEM images, are presented, as well. This material is available free of charge via the Internet at http://pubs.acs.org.

Acknowledgments This work was supported by the Deutsche Forschungsgemeinschaft (DFG) (SPP 1313 Bio-NanoResponses) and Freie Universität Berlin. The authors thank P. Schrade (Multi-User Unit Electron Microscopy, Charité-Universitätsmedizin Berlin) for the preparation of cell samples for TEM, A. Schindler (Physikalische Chemie, Freie Universität Berlin) for the recording of various STEM images, and Dr. H. Renz and Prof. Dr. R. J. Radlanski (Department of Craniofacial Developmental Biology, Charité - Universitätsmedizin Berlin) for the opportunity to use their electron microscope.


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Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

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For Table of Contents only:

Silver spheres

hMSC

Silver prisms

Preferential uptake of prisms HaCat hMSC cells

HaCat cells


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Shape-Dependent Dissolution and Cellular Uptake of Silver

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