Nanocrystals

Internalization Pathways of Anisotropic Disc-Shaped Zeolite L Nanocrystals with Different Surface Properties in HeLa Cancer Cells

Zhen Li , Jana Hüve , Christina Krampe , Gianluigi Luppi , Manuel Tsotsalas , Jürgen Klingauf , Luisa De Cola , * and Kristina Riehemann *

Information about the mechanisms underlying the interactions of nanoparticles with living cells is crucial for their medical application and also provides indications of the putative toxicity of such materials. Here the uptake and intracellular delivery of disc-shaped zeolite L nanocrystals as porous aminosilicates with well-defi ned crystal structure, uncoated as well as with COOH-, NH 2 -, polyethyleneglycol (PEG)- and polyallylamine hydrochloride (PAH) surface coatings are reported. HeLa cells are used as a model system to demonstrate the relation between these particles and cancer cells. Interactions are studied in terms of their fates under diverse in vitro cell culture conditions. Differently charged coatings demonstrated dissimilar behavior in terms of agglomeration in media, serum protein adsorption, nanoparticle cytotoxicity and cell internalization. It is also found that functionalized disc-shaped zeolite L particles enter the cancer cells via different, partly not yet characterized, pathways. These in vitro results provide additional insight about low-aspect ratio anisotropic nanoparticle interactions with cancer cells and demonstrate the possibility to manipulate the interactions of nanoparticles and cells by surface coating for the use of nanoparticles in medical applications.

1. Introduction

Nanotechnology in medicine, also termed ‘nanomedicine’,

has expanded as a research area for the last decade, with

© 2013 Wiley-VCH Verlag Gmb

DOI: 10.1002/smll.201201702

Dr. Z. Li, Dr. G. Luppi, Dr. M. Tsotsalas, Prof. L. De ColaCenter for Nanotechnology (CeNTech) Heisenbergstr. 11, 48149 Muenster, Germany E-mail: decola@uni-muenster.de

C. Krampe, Dr. K. RiehemannInsitute of Physics University of Muenster Wilhelm-Klemm-Straße 10, Center for Nanotechnology (CeNTech) Heisenbergstr. 11, 48149 Muenster, Germany E-mail: K.Riehemann@uni-muenster.de

Dr. J. Hüve, Prof. J. KlingaufFluorescence Microscopy Facility Münster Institute of Medical Physics and Biophysics Center for Nanotechnology (CeNTech) Heisenbergstr. 11, 48149 Muenster, Germany

small 2013, 9, No. 9–10, 1809–1820

more and more attention from both scientifi c and social

societies. It includes the development of nanoparticles, sur-

faces with nanostructures, and nanoanalytical techniques

for medical diagnostics, therapeutic treatment, monitoring,

and follow-ups. [ 1 ] As nanoparticle research rapidly develops,

nanoparticles have been applied widely in biomedicine and

biotechnology, including as carriers for drug delivery, [ 2–4 ] as

probes for spectroscopy and microscopy, [ 5 , 6 ] and as contrast

agents for magnetic resonance imaging (MRI). [ 7–13 ] As the

fundamental properties of nanoparticles, size and shape are

widely discussed, especially the sphere geometry and particle

sizes less than 100 nm.

As the interest in this area continues to grow, the size limits

of particles are increased to 1000 nm or even bigger when

mentioned in bionanotechnology, and anisotropic shapes like

cylinders, UFOs, or plates are also considered. [ 14 , 15 ] Apart

from the physical properties of the nano-objects, the cellular

uptake effi ciency will be infl uenced by the surface function-

ality of nanoparticles as well as the protein corona developed

upon nanoparticle exposure to the biological media. [ 16–19 ]

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Scheme 1 . Disc-shaped zeolite L nanocrystals with different surface modifi cations. The abbreviations of nanoparticles types were given under each illustration.

Besides all the advantages that nanotech-

nology could bring to the biological world,

there are also other aspects like cytotox-

icity which need to be addressed. Several

groups have found that cytotoxicity effects

appear most of the time at high dosages,

depending on the duration of exposure. [ 20 ]

To study the issues mentioned above, we

have chosen as our nanoparticle model

disc-shaped zeolite L nanocrystals, which

have been discussed much less than other

Table 1. Size distributions of zeolite L nanocrystals before and after modifi cations characterized by DLS in relevant media.

D-Zeo C-D-Zeo PEG-D-Zeo N-D-Zeo PAH-D-Zeo

PBS Z-Average [nm] 112 119 127 123 130

Polydispersity 0.03 0.19 0.17 0.13 0.16

Serum-free Z-Average [nm] 179 152 131 206 201

Polydispersity 0.32 0.32 0.18 0.43 0.43

Serum Z-Average [nm] 197 144 138 124 147

Polydispersity 0.43 0.23 0.19 0.32 0.17

shapes such as spheres or rods. The unique property of such

nanocrystals is that they can form mono-dimensional nano-

channels consisting of pores with about 0.71 nm apertures,

leading to unit cells with 1.26 nm at the widest point and 0.75

nm in length, with a typical Si/Al ratio of 3.0. They have tune-

able size and shape from 30 nm to 10 000 nm, from cylindrical

to disc-shaped. The well-defi ned channels are ideal hosts for

guest molecules such as fl uorophores, contrast agents, and

potentially certain drug molecules. [ 21 , 22 ] Additionally, selec-

tive modifi cation of external surfaces can be realized by

stepwise procedures. [ 23 ] Among them, 200 nm disc-shaped

particles have the advantage that they have fewer tendencies

to form aggregates in suspension. Loaded with highly fl uores-

cent molecules, such nanoparticles have shown great poten-

tial in immunoassays. [ 24 ]

To understand how nanocontainers could infl uence cancer

cells during therapy and to obtain information about their

cytotoxic potential knowledge about the route of uptake and

the intracellular fate of the particles is most important. We

investigated the behaviours of disc-shaped zeolite L nanoc-

rystals with different surface coating properties in diverse

media as well as the cytotoxicity and the interactions between

Human Cervical Cancer cell line (HeLa) and these nanoparti-

cles. Fluorescent confocal microscopy was applied to observe

the internalization of the disc-shaped zeolite L nanocrystals

in HeLa cells at different time scales, temperatures and cul-

ture media. Whereby, endocytosis inhibitors such as dynasore,

chloropromazine and amilorid and doublestaining experi-

ments were applied to study nanoparticles internalization

pathways for further understandings.

2. Results and Discussion

2.1. Physicochemical Characterization of the Nanocrystals and their Dispersion in Different Media

Disc-shaped zeolite L nanocrystals were synthesized via a

hydrothermal process as reported in the literature. [ 24 ] The

X-ray diffraction pattern show (Figure S1) that the obtained

crystals have the complete structure of zeolite L nanocrystals.

The scanning electron microscopy (SEM) images (Figure S1)

indicate that such crystals have disc-shaped geometry with

diameters of about 150 to 200 nm and a thickness of about 50

to 70 nm. As already well-studied zeolite L nanocrystals are

known for their 1D channels, here we inserted neutral fl uo-

rescent DXP molecules to visualize the particles, since neutral

dyes have minimal leakage into the media due to insolubility.

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The procedure and spectra are shown in the Supporting

Information. According to different modifi cation methods

(Supporting Information), we coated various groups onto

the zeolite L nanoparticle surfaces: NH 2 groups (N-D-Zeo);

COOH functionalization (C-D-Zeo); PEG 500 groups (PEG-

D-Zeo); cationic polymer polyallyamine (PAH-D-Zeo),

and; bare-surfaced (D-Zeo) as illustrated in Scheme 1 .

These coatings introduce different physicochemical proper-

ties to the nanoparticles, which will play an important role

besides the particle geometry when they are in contact with

cells in the media. Instead of SEM, we have used dynamic

light scattering (DLS) to observe size differences before and

after modifi cation. Since our particles are not spherical, the

z-average diameters were used as references. Results of diam-

eter and polydispersity measurements of the nanoparticles

in phosphate buffered saline (PBS) are shown in Figure S3

and summarized in Table 1 . Z-Average diameter is between

the diameter and thickness of disc-shaped nanocrystals as

observed under SEM. The polymer PAH-modifi ed nanoc-

rystals showed the thickest coating layers. Although zeolites

with coated surfaces have a certain degree of heterogeneity,

the polydispersity before and after modifi cation is below 0.2,

which indicates the relatively good homogeneity of all par-

ticle types.

Since nanoparticles are always in contact fi rst with the

culture media before they are in contact with the cells, it is

important to check the nanoparticle stabilities in these media.

Thus, similar DLS studies were performed in serum-free

and serum media (Table 1 ) which are often applied in such

experiments. The sizes of all nanoparticles shifted to higher

values in cell culture media compared to those in PBS. There

were no obvious size variations of the different nanoparticles

between these two media. With only one exception of PAH-

modifi ed zeolites, it shows clearly much bigger agglomerates

in serum-free media than in serum media.

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Internalization of Zeolite L Nanocrystals in HeLa Cells

Figure 1 . Variation in the zeta potential of different zeolite L nanocrystals in PBS, serum-free, and serum containing media.

Another factor to determine the physical stability of a col-

loidal suspension is its zeta potential. The higher the absolute

value, the higher the electrostatic repulsion between the par-

ticles and the higher the physical stability. Differently coated

nanocrystals were suspended individually in PBS + + buffer,

serum-free media, and serum media ( Figure 1 ). There were

no proteins in the serum-free media, which was the opposite

of the serum-containing media. As a rule of thumb, suspen-

sions with an absolute zeta potential above 30 mV are physi-

cally stable and below 20 mV is the limit of stability. In PBS + +

and serum-free media, the zeta potential values were more

or less consistent with their intrinsic surface properties. Non-

modifi ed and carboxyl-functionalized disc-shaped zeolite L

showed negative zeta values of around –30 mV or even lower.

At these values, the colloidal systems were rather stable. For

PEGylated nanocrystals the zeta value was around –10 mV,

which is less stable than non-modifi ed ones. However, it does

not necessarily mean that the physical stability decreased.

Figure 2 . A) Linear range of standard calibration curve of BSA for the Bradford assays. The assay demonstrates a linear regression with increasing BSA concentrations. B) Protein adsorption on zeolite L surfaces.

Aside from electrostatic stabilisation, there

is also steric stabilisation introduced by the

PEG chain, which shifts the shear plane

further away from the particle surfaces. As

for amino- and PAH-modifi ed zeolite L,

although they both carried intrinsic posi-

tive charges, only amino-modifi ed ones

showed positive zeta potentials at about

10 mV in PBS + + buffer, with the other

being slightly negative (Table 1 ). This may

due to the presence of Ca 2 + , Mg 2 + in the

PBS + + media, which alters ionic strength, or

due to the presence of other small organic

molecules which could adsorb onto the

surfaces of the nanoparticles. However, in

serum (10% FBS)-containing media, zeta

values shifted to around –10 mV for all

particles regardless of their surface prop-

erties. This is clear evidence that serum

proteins were adsorbed onto zeolite L,

masking their surfaces. Adsorbed proteins

will then alter nanoparticles and cell inter-

actions subsequent to their internalization

pathways. Even though the zeta potential

data indicates that some of the colloidal

systems were not stable, we found only big

aggregates of samples of PAH-D-Zeo and

N-D-Zeo in serum-free media. This may be

© 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheimsmall 2013, 9, No. 9–10, 1809–1820

attributed to interactions of amino groups

on the surfaces of the nanoparticles with

components in the serum-free media.

To further understand the infl uences

of coatings in serum media, we quantifi ed

protein surface adsorption on different

nanoparticles from serum by Bradford

assay. Bovine serum albumin (BSA) was

chosen as a standard to determine the

unknown protein concentration from

nanocrystal samples, because it is one of

the most abundant proteins in serum. A

calibration curve was created using dif-

ferent concentrations of BSA in the presence of Brilliant

Blue G-250 dye molecules ( Figure 2 A). Some of the serum

proteins were adsorbed onto the surface of the zeolites, while

others stayed in solution. Total protein concentrations as well

as the concentrations in the supernatants were measured

using Bradford methods at an absorption wavelength of 595

nm. Subtracting the concentration of proteins in supernatant

from the total amount of proteins therefore gives the amount

of proteins adsorbed on the nanoparticles. With this prepara-

tion method we have removed loosely adhered proteins, and

determined the amount of relatively strongly adsorbed pro-

teins on different nanoparticles.

The results are summarized in Figure 2 B. For the different

surfaces of the same size of disc-shaped zeolite L nanocrystal,

positive surfaces adsorbed larger amounts of serum proteins.

PEG-functionalized and bare surfaces showed less adsorp-

tion, while carboxyl zeolite displayed the lowest adsorption.

This may be attributed to the surface charges, which introduce

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Figure 3 . Cytotoxicity and cell viability in presence of Zeolite L nanocrystals. The fl uorogenic GF-AFC Substrate can enter live cells, where it is cleaved by the live-cell protease to release AFC. The luminogenic AAF-Glo Substrate cannot enter live cells but can be cleaved by the protease activity released by dead cells to generate aminoluciferin, which is detected in a luminescent reaction. A,B) Cytotoxicity and viability of HeLa cells after exposed to zeolite L nanocrystals at different concentrations in serum-free media.

electrostatic interactions between the nanoparticles and pro-

teins. The effect of charge on protein adsorption has also been

observed on cerium oxide nanoparticles. Nanoceria samples

with positive zeta potentials were found to adsorb more BSA,

while the samples with negative zeta potentials showed little

or no protein adsorption. [ 25 ] However, different results have

been also observed by other groups with ‘soft’ polymeric nan-

oparticles with sizes of about 100 nm and 50 nm. [ 26 ]

This shows that protein adsorption is not only charge-

directed but also material-related. Indeed in a given media,

the important nanoparticle characteristics that determine the

interactions are the material’s chemical composition, surface

functionalization, shape and angle of curvature, porosity and

surface, crystallinity, roughness, and hydrophobicity. [ 27–29 ]

Although the different nanoparticles showed the same zeta

potential values in serum media, protein adsorption pro-

fi les could be quite diverse. [ 30 ] This makes different particles

unique to cells in serum media even if the zeta values are the

same. In other words, they would still behave differently in

the process of internalization.

2.2. Cytotoxicity and Cell Viability

Cytotoxicity of nanoparticles is another important issue. In this

report we employed the commercially available Multi Tox-Glo

assay. It sequentially measures two protease activities; one is

a marker of cell viability, and the other is a marker of cyto-

toxicity, which indirectly indicates the cell membrane integrity.

Digitonin was applied in the assay as a positive control to kill

cells. Because of high background noise induced by serum, this

assay was performed in serum-free media for all particles.

The results ( Figure 3 ) show that both measurements are

in good agreement with each other. Four concentrations of

the zeolites, 50, 100, 250 and 500 μ g/mL, were tested in the

assay. The cellular toxicities of different nanoparticles were

all dosage dependent. At the lowest concentration (50 μ g/mL)

no toxic effects were observed regardless of size or surface.

PEGylated disc-shaped zeolite L showed no toxic effect at

all even at the highest concentration. This is in agreement

with the literature, whereby PEGylation was applied to

decrease the cytotoxicity of dendrimes via attenuation of

oxidative stress. [ 31 ] The nanoparticle cytotoxicity depends on

several parameters, including the properties of the nanopar-

ticles and the type of cell analyzed. The predominant factor

which infl uences cytotoxicity of nanoparticles of similar

chemistry can also vary with particle size. Napierska et al.

reported that, with amorphous silica nanoparticles, the cyto-

toxicity was strongly related to particle size, independent of

morphology. Smaller particles showed signifi cantly higher

toxicity to endothelial cells than the bigger ones when dose

was expressed in mass concentration. [ 32 ] For particles of 500

nm, using RAW 264.7 cells increased toxicity in this order of

functional groups anchored to the particles: thiol > carboxy >

amine. This is in contrast to HEK 293 where the differences

were insignifi cant. [ 33 ] Additionally, in the same report it was

demonstrated that the toxicity was dosage-, time-, and cell

line type-dependent as well. In our case, in terms of surface

property with same morphology, non-functionalized disc-

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shaped particles showed the most dose-dependent behavior.

This may be attributed to the abundant surface acidic sites

on bare zeolites due to the alumina component, which can

catalyze some chemical reactions on the cells. However, two

positively charged zeolites were only toxic at the highest

concentrations. Usually the positively charged surface has a

higher affi nity for negatively charged cell membranes, thus

introducing higher cytotoxicity. This seems to be a cummu-

lative effect with our positive-coated zeolite L nanoparticles.

Carboxyl zeolite had unusual toxicity at the specifi c concen-

tration of 250 μ g/mL instead of at the highest concentration,

which requires further investigation.

As mentioned before, the Multitox Glo assay was not

applicable in our case to test the cytotoxicity of nanoparti-

cles in serum due to the high signal variations. Thus we have

chosen two different assays to evaluate the cytotoxicity in

serum/serum-free medium. Membrane integrity is the central

parameter to detect living cells in both assays. To evaluate

the cytotoxicity of zeolite L nanocrystals in serum media, we

applied a standard Trypan blue staining to test membrane

integrity and a Multitox Glo assay (Promega Corp, Germany).

The trypan blue cannot pass through the cell membrane when

they are alive, as Multitox Glo assay measures the activity of

proteases that are secreted by dead cells. According to the

manufacturer’s information, both methods produce compa-

rable results. In different experiments, Trypan blue was used

by them to verify the results of the Multitox Glo assay. The

Multitox Glo Assay provides additional information about

the cell viability and better handling. A possible disadvantage

of this assay could be that the proteases of the dying cells

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Internalization of Zeolite L Nanocrystals in HeLa Cells

attach to the surface of the nanoparticles, building a corona

itself and thus minimizing the cytotoxicity result. As there

are also other, nonproteolytic active proteins in the solution

which could attach to the nanoparticles, it could be generally

considered that cells dying during the assay alter the results.

But to our knowledge such an effect is not yet reported

for this assay. It is also not reported that, in the case of the

lumiogen-peptide complex attached to the surface of nano-

particles, a cleavage by protease is prevented.

These experiments were performed slightly differently

from the previous ones. Cells were seeded on cover slips in

a 12-well microplate. Different zeolite L nanocrystals at two

concentrations, 50 mg/mL and 250 μ g/mL,were added into

the wells and cells were stained at four time intervals 6 h,

12 h, 24 h, and 48 h. Images were taken immediately after

staining by light microscope and are shown in the Supporting

Information (Figure S4).

At low concentrations of 50 μ g/mL little toxicity was

observed for all nanoparticles in serum media over a pro-

longed time, which is in agreement with experiments

performed in serum-free media. However, at the higher con-

centration of 250 μ g/mL, except for PEG- and carboxyl-func-

tionalized disc-shaped zeolites, all particles demonstrated a

toxic effect even after the fi rst 6 h. This is not surprising for

bare disc-shaped zeolite L nanocrystals since we observed

low concentration toxicity in serum-free media. On the other

hand, for two positively charged zeolite coated samples with

higher protein adsorption analyzed by Bradford assay, the

toxicity also started at the concentration which was nontoxic

in the serum-free media. Different reports have demonstrated

that the presence of serum can mitigate the toxicity of nano-

particles. [ 34 , 35 ] For carbon nanoparticles, the extent of toxicity

attenuation increased with increasing amounts of serum pro-

teins adsorbed. [ 36 ] Another reason why we observe the early

toxic effect for these two coatings in serum media could

be that they showed smaller sizes in the presence of serum

media in the previous agglomeration experiments. Usually,

smaller nanoparticles result in higher cytotoxicity. Since two

different toxicity assays were applied in our experiments in

the absence and presence of serum, we cannot compare the

results directly due to the different assay sensitivities. How-

ever, it was also reported that zeolite L nanoparticles with

cylindrical shape display a clear dose-dependent toxicity by

other assays, as the viability of exposed HeLa cells decreased

signifi cantly with increasing nanozeolite LTL dosage (from

50 to 200 μ g/mL). [ 37 ] In our case, the cytotoxicity attenuation

of the serum was not effective for bare and positively coated

disc-shaped zeolites. Nevertheless, at the low concentration of

50 μ g/mL, coated and non-coated disc-shaped zeolites were

all nontoxic to the cells in both culture media. So this concen-

tration was chosen as a standard concentration for HeLa cell

zeolite L nanocrystal internalization experiments.

2.3. Uptake and Internalization of Zeolite L Nanocrystals by Cancer Cells

Nanoparticle cell uptake is one of the most important issues

in their application in nanomedicine, as it offers insight in

© 2013 Wiley-VCH Verlag Gmbsmall 2013, 9, No. 9–10, 1809–1820

the mechanisms of cytotoxicity. At the same time it is the

most complicated process. In order to reduce the number of

variables in the following experiments, we have focused only

on the chemical nature of the surface modifi cations of disc-

shaped zeolite L nanocrystals. We have tested at two different

temperatures the infl uence of the presence of serum protein

at two time intervals. Upon the addition of different nanoc-

rystals into the cells seeded on microplates, different uptake

conditions were applied immediately. Cells were afterwards

fi xed and stained with a green fl uorescent dye, DiOC 6 , to vis-

ualize the intercellular part for imaging. 1,4 diazabicylo[2.2.2]

octane (DABCO) was added in fl uoromount mounting media

to reduce the photobleaching of stained cells.

As shown in the Supporting Information, upon the addi-

tion of different zeolite L nanocrystals HeLa cells were kept

at 4 ° C in either serum-free or serum-containing media for

24 h. The uptake activities of nanoparticles by cells were not

observed after this time by a confocal laser scanning microscope

(Figure S5). A number of mechanisms, including phagocytosis

and endocytosis, could account for the uptake of nanoparticles

in a temperature-dependent active transport manner. Consid-

ering the size and the rigidity of our nanoparticles, we did not

expect that they would pass through the cell membrane in a pas-

sive manner. Thus, internalization of zeolite L nanocrystals into

HeLa cells were examined also at 37 ° C in serum-free and serum

media. We also performed these experiments at 2 h duration.

3D Z-stack images taken by confocal laser scanning micro-

scopy (CLSM) were applied to prove the internalization of

zeolite L nanocrystals by HeLa cells. If the intercellular parts

stained green overlapped with nanocrystals (red) in three

dimensions, we could then be certain that the nanocrystals

were inside of the cells ( Figure 4 ). All types of zeolite nano-

particles did not show clear internalization activities by cancer

cells within the fi rst 2 h in serum media. Zeolites with posi-

tively charged surfaces (N-D-Zeo and PAH-D-Zeo) attached

to a high amount to the cellular membrane, which might be

the reason for the higher toxicity of these samples. We could

also see the bare zeolites scattered around the cells in consid-

erably less numbers. There were few nanoparticles coated with

COOH or PEG found close to the cells. Neither nanoparticle

adhesions nor internalizations were observed for the HeLa

cells in serum media with neutral or negative surfaces.

The situation was different in the serum-free media after

the fi rst 2 h: all nanoparticles except for amino-functional-

ized ones demonstrated a certain degree of uptake activity

by cancer cells. Almost no amino-coated zeolites were found

inside the cells, but bigger agglomerations were clearly seen

at the cellular membrane, which was not the case in other

images. By prolonging the incubation time to 24 h, all nano-

particles were taken up by HeLa cells regardless of the pres-

ence of serum in media and nanoparticle coating.

Positively coated nanoparticles, like silica nanoparticled

coated with PAH, showed similar results when analyzed by fl ow

cytometry in serum-free media. In fact, it was reported that

there was no toxic effect of PAH on stem cells. [ 38 ] Jiang et al.

demonstrated that, even among all-cationic surfaces, slight

changes of surface functionality would result in differences in

nanoparticle cell internalization quantities and pathways. [ 39 ]

We have also observed this phenomenon in serum-free media

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Figure 4 . Confocal laser scanning microscopy images (z-stack) of zeolite L nanocrystals interactions with HeLa cells at 37 ° C for 2 and 24 h in serum and serum-free media. Intercellular parts were stained by DiOC6 represented in green colour. Nanoparticles were shown in red colour. Scale bar unless stated were 10 μ m.

between our differently coated positively charged surfaces

(amino and PAH). The uptake of nanoparticles by cells could

be viewed then as a two-step process: fi rst the nanoparticles

quickly accumulate on the cell membrane or in its proximity.

Then they are internalized by the cells, whereby the nanopar-

ticle clusters diminished considerably.

For negatively charged disc-shaped zeolite L, serum pro-

tein delayed this internalization process, as seen when com-

paring the images taken at 2 h from both media. The study

of protein adsorption and cellular uptake, of cerium oxide

nanoparticles as a function of zeta potential, demonstrated

that nanoceria samples with positive zeta values adsorb more

BSA, while the samples with negative charges showed little

or no protein adsorption. The cellular uptake studies show

preferential uptake of the negatively charged nanoparti-

cles. [ 25 ] The other study from Asati et al. which used the same

nanoparticles declared that positive or neutral nanoparticles

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entered most of the cell lines studied, while

negative ones were internalized mostly

in the cancer cell lines. [ 40 ] There was also

a recent study of the effect of the protein

corona on bare silica nanoparticle uptake

and the impact on the cells, where similar

results concluded that, in the absence of

serum, nanoparticles demonstrate a higher

internalization effi ciency. [ 41 ] Different

from the literature, in our images, we did

observe strong adsorptions of nanopar-

ticles on the cell membrane. It seemed

that, in our cases, the adsorption and

internalization occurred at the same time

in serum-free media. In the serum media

the albumin coating hampered the interac-

tion with cells probably because of steric

effects. On the contrary, it increased nano-

particle capture by macrophages. [ 42 ] Iron

oxide nanoparticles that are stabilized by

carboxyl-functionalized 3rd-generation

poly(amidoamine) dendrimers have also

shown internalization ability into human

epithelial carcinoma cells, presumably

either through pinocytosis or via direct

diffusion through the cell membrane. [ 43 ]

As described above, we have quantifi ed

serum protein adsorption, which proved

that positively charged surfaces had the

highest amount of proteins. If steric effects

played a role, we would observe no nano-

particle adsorption of N-D-Zeo and PAH-

D-Zeo on cell surfaces in serum containing

media, which was not the case. We assume

that, beside the effect of steric hindrance,

there must be other effects like protein

types or quantities which infl uence the

uptake activity. The surfaces of different

zeolites are not directly exposed to cell

membranes, but through diverse adsorbed

proteins. Thus, even in serum media where

the zeta potentials were the same for all

surfaces (around –10 mV), we still noticed different nanopar-

ticle adhesion or internalization behaviours. Actually corre-

lating, from serum media, the amount of protein adsorbed on

the surface of the crystals with the amount of nanoparticles

attached on the cells, we found that the nanoparticles with

the most proteins adhered to them adsorbed faster to the cell

surface. Similar trends have been noted for polystyrene nano-

particles with amino or carboxyl surfaces, although the pro-

tein adsorption behavior was the opposite of our results. [ 27 ]

Protein adsorption may not be the determining factor for

nanoparticle adhesion to cells. However, it has a great effect

on the kinetics of such processes and may even lead to dif-

ferent pathways of cellular uptake.

Since we have observed different nanoparticle internali-

zation behavior under different conditions, it is interesting

to compare their pathways during these processes. To

simplify the experimental conditions, serum-free media

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Internalization of Zeolite L Nanocrystals in HeLa Cells

Figure 5 . Confocal images (Z-stack) of zeolite L nanocrystals interactions with HeLa cells at 37 ° C for 4 h in serum-free media. Cell membrane was stained by DiD represented in red colour. Nanoparticles were shown in green colour.

Figure 6 . Quantifi cation of nanoparticle C-D-Zeo and PAH-D-Zeo uptake by HeLa cells. Mean values and standard error of three independent triplicate experiments were analyzed.

and 4 h duration of uptake were applied for these tests at

37 ° C. Otherwise similar parameters as before were applied

and the samples were analyzed by CLSM. In order to better

understand the mechanism, we also developed a method to

quantify the cells with internalized or attached nanoparticles

according to the nanoparticles’ fl uorescent intensities on the

microplates. In the inhibitor-absent experiments, we observed

similar phenomena as before. The PAH- and carboxyl-func-

tionalized nanoparticles demonstrated the highest uptake

activities ( Figure 5 A). Therefore, we focused on these two

types of particles, and the others are shown in the Supporting

Information (Figure S5 and S6). Dynasore is an inhibitor

which is specifi c for protein dynamin-involved clathrin- and

caveolin-mediated endocytosis. [ 45 ] Even though PAH-D-Zeo

particles were less internalized in the presence of dynasore

(shown in the images), the nanoparticle–cell membrane asso-

ciations were not affected. The reason could be that dynasore

only acts on the hydrolysis of guanidine triphosphate (GTP)

which controls the cleavage of formed membrane endocytosis

vesicles. For negatively charged C-D-Zeo, reduced internaliza-

tion was also observed without many nanoparticles attaching

to the membrane. To clarify the internalization pathways,

we also applied chlorpromazine to the cells, which specifi c

inhibits the formation of clathrin-coated pits at the plasma

membrane. [ 44 ] The addition of chlorpromazine signifi cantly

suppressed C-D-Zeo nanoparticle internalization (by about

30% more as compared to dynasore samples), which can

be clearly observed in the quantifi cation results ( Figure 6 ).

It is a good indication that carboxyl-functionalized disc-

shaped zeolite L nanoparticles are internalized mainly via a

clathrin-mediated endocytosis pathway. This is not so obvious

for PAH-modifi ed zeolite L samples, where chlorpromazine

only resulted in slightly fewer nanoparticle uptake activities

compared to dynasore inhibition (Figure 6 ). According to

the literature, clathrin-mediated endocytosis-formed vesicles

have an average size of about 120 nm. [ 45 , 46 ] Although this

is smaller than the size of our nanoparticles, our nanoparti-

© 2013 Wiley-VCH Verlag Gmbsmall 2013, 9, No. 9–10, 1809–1820

cles have a fl at shape with one dimension of about 50 nm.

It was also reported that the internalization of nanoparti-

cles smaller than 200 nm mainly involves clathrin-mediated

endocytosis. [ 47 ] From the images it is shown that the C-D-

Zeo nanoparticles were much better dispersed than PAH-

D-Zeo nanoparticles, where the latter attached signifi cantly

to the cell surfaces. This good dispersion offered the C-D

Zeo nanoparticles a chance to maintain their single crystal

1815www.small-journal.comH & Co. KGaA, Weinheim

Z. Li et al.

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shapes during the internalization process, so that clathrin

vesicles could be formed. With larger aggregates of PAH

samples, due to the bigger size in serum-free media shown in

agglomeration data, this is unlikely to happen. Therefore, we

chose another inhibitor, amiloride, which is used to block the

activity of macropinocytosis for bigger agglomerates. It is an

actin-driven process to generate vesicles with size of 0.5-10

μ m which serves as a non-specifi c pathway to internalize

large particles. The microscopy as well as quantifi cation of

results revealed that this endocytosis route was not the main

internalization pathway for either PAH-D-Zeo or C-D-Zeo

nanoparticles. The internalization pathway of PAH-coated

disc-shaped zeolites would require further investigation.

6 www.small-journal.com © 2013 Wiley-VCH Verlag Gm

Figure 7 . Intracellular distribution of zeolite L nanocrystals. A) Colocalizaclathrin coated vesicles. Both shown with the respective antibodies and was shown at three different scales.

After excluding macropinocytosis for the uptake pathway,

double staining experiments were performed with antibodies

against caveolin I, clathrin, and with fl uorescently marked

zeolite L nanoparticles, in order to characterize the intracel-

lular distribution of differently coated nanoparticles.

C-D-Zeolites were found in caveolae as well as in clathrin-

coated vesicles as shown in Figure 7 A,B. All other zeolites

used in these experiments are absent in the caveolae. Coating

with PEG, NH 2 or a lack of coating results in their absence

in clathrin-coated vesicles. PAH-D-Zeolites are at least asso-

ciated and partly also uptaken by clathrin-coated vesicles.

These data are in line with results obtained by uptake inhibi-

tion studies with chlorpromazine, as shown above. Regarding

bH & Co. KGaA, Weinheim

tion of the zeolites with caaveolae B) Colocalization of the zeolites with fl uorescence marked zeolites. Each co-staining of the respective coating

small 2013, 9, No. 9–10, 1809–1820

Internalization of Zeolite L Nanocrystals in HeLa Cells

the number of uptaken nanoparticles, it seems that COOH

coating results in the highest uptake by HeLa cells, followed

by the uptake rate of PAH-D-Zeo particles.

Quantifi cation of the uptake in the presence of inhibitors

was shown in Figure 6 . As the inhibitors used here reduced

also the surface association of zeolites, quantifi cation as

decribed here was only possible in experiments where inhibi-

tors were involved. Otherwise the attachment of particles

infl uenced the results of the uptake. The corresponding data

are shown in Figure S6 (Supporting Information).

In the experiments described here, clathrin- and caveolin-

independent uptake was found for all types of nanoparticles

used, though to a smaller extent than the uptake of COOH-

coated nanopartices. This effect was already observed in

lung epithelial cells (A549). [ 49 ] The mechanism of uptake

remains unclear and might be the focus of further studies.

Though these studies were performed under serum-free con-

ditions, which seem to be unrealistic for natural conditions,

the results might be important for in vivo conditions. Reports

that the corona can be intracellularly destroyed, for example

in lysosomes, hints that at least under some conditions the

nanoparticle surface (without a mask of proteins) is in direct

contact with cellular components. [ 50 ] Further studies are also

necessary here.

3. Conclusion

Disc-shaped zeolite L nanocrystals have been demonstrated

to be readily internalized by HeLa cancer cells in different

ways. They have shown good dispersion capability in PBS

buffer and media with the exception of PAH-coated nano-

particles, where we observed slightly bigger agglomerates.

In serum-containing media we detected a protein corona

on all nanoparticles investigated with the highest amount of

proteins on zeolites with positively charged surface modi-

fi cations. Zeolite nanocrystals exhibited little cytotoxicity

at low concentrations. At high concentrations, positively

charged particles demonstrated higher toxicity compared

to other nanoparticles used in the experiments described

here.

Independently of experimental conditions, disc-shaped

zeolite L nanocrystals were considerably internalized by

HeLa cells after 24 h. However, by reduction of the incuba-

tion time to 2 h we have shown that the initial uptake proc-

esses can be infl uenced by different surface modifi cations as

well as serum protein adsorption. In the presence of serum

protein on the nanoparticles surfaces, the uptake was delayed

compared to results obtained in serum-free media. Investiga-

tion of the uptake route in serum-free media by applying inhi-

bition and double labeling experiments clearly demonstrated

that a COOH coating results in an uptake via caveolae and

the clathrin-mediated pathway. The internalization pathway

of PAH-modifi ed zeolite L was neither dynamin-dependent

nor via macropinocytosis.

These in vitro experiments offer valuable information

about the heterogeneity of nanoparticle cancer cell interac-

tions driven by surface alteration of anisotropic zeolite L

nanocrystals.

© 2013 Wiley-VCH Verlag Gmbsmall 2013, 9, No. 9–10, 1809–1820

4. Experimental Section

4.1. Materials

Dulbecco’s modifi ed eagle medium (DMEM), penicillin,

streptomycin, as well as membrane staining dye DiD were

purchased from Invitrogen. The chemicals for inhibitor assays

(chlorpromazine hydrogen, dynasore and amiloride hydro-

chloride hydrate) were all obtained from Sigma Aldrich.

The MultiTox-Glo assay kit was purchased from Promega.

Nutrient Mixture F-12 HAM, paraformaldehyde, digtionin,

minimal essential medium eagle’S (EMEM), Bradford rea-

gent, Fluoromount and DABCO were purchased from Sigma

Aldrich, anti-clathrin (CatNo ABIN968006)- and anti-cave-

olin (CatNo. ABIN363230)-antibodies were purchased from

antibodies-online.com. For the secondary antibody staining

Alexa Fluor 633 coated rabbit anti- mouse-antibody pro-

vided by life technologies GmbH, Darmstadt, Germany.

Fetal bovine serum (FBS), L-glutamine and non-essential

amino acids (NEA), trypsin, Ethylenediaminetetraacetic

acid (EDTA) and phosphate buffer saline (PBS) were pur-

chased from Biochrom. Succinic anhydride was purchased

from ABCR. Dimethyl sulfoxide (DMSO) and n-butanol

were purchased from Merck. Accutase was purchased from

PromoCell.

4.2. Instrumentation

The morphology of the zeolite L nanocrystals was investi-

gated using a Zeiss 1540 EsB Dual Beam Focused Ion Beam/

Field Emission Scanning Electron Microscope (SEM) with

a working distance of 8 mm and an electronic high tension

(EHT) of 3 kV. Absoption spetra were recorded by Varian

Cary 100 scan UV-Visible spectrophotometer. The emission

spectra were recorded on a Horiba Jobin-Yvon IBH FL-322

Fluorolog spectrometer equipped with a 450 W xenon arc

lamp, double grating excitation and emission monochro-

mators (2.1 nm/mm dispersion; 1200 grooves/mm) and a

TBX-4-X single-photon-counting detector (emission). Zeta

potentials were measured by DelsaTMNano zeta potential

and submicron particle size analyzer (from BeckmanCoulter)

coupled with fl ow cell sampler. Flow cells were rinsed with

suspension media before sample addition.

Fluorescence images in Figure 4 were done by confocal

laser scanning microscope and those of Figure 5 and Figure 7

were obtained with a commercial 4Pi microscope (TCS 4Pi

microscope type A, Leica Microsystems) employing oil

immersion objective ( × 100, numerical aperture 1.46). The TCS

4Pi is a confocal laser scanning microscope of type TCS SP2

incorporating one- as well as two-photon excitation, photon-

counting by avalanche photodiodes, and a 4Pi attachment.

Because of these features the microscope could be employed

in the confocal mode with upright or inverted beam path and

single- or two-photon excitation, or as a two-photon excita-

tion 4Pi microscope. Since the nanoparticles are not excitable

in two-photon excitation the microscope was used in the con-

focal mode. The upright beam path was applied.

1817www.small-journal.comH & Co. KGaA, Weinheim

Z. Li et al.

1818

full papers

Single-photon excitation wavelengths used in the mem-

brane staining and anti-clathrin-/anti-caveolin-antibodies

staining were 488 nm for the nanoparticles and 633 nm for

the staining, yielding best case resolutions of 170 nm resp.

221 nm in both x and y direction and 390 nm resp. 506 nm

in z direction. The beam expander was set to 6. Fluorescence

originating from the sample was passed through a fi lter cube

(beam splitter 625 nm, band-pass 535-585 nm, and band-pass

647-703 nm), and its intensity was measured by photon-

counting avalanche photodiodes (PerkinElmer). The detec-

tion pinhole was set to 1 Airy unit. Raw images were linearly

brightened, rescaled, and linearly fi ltered by a subresolu-

tion mask employing the image processing program ImageJ

(Wayne Rasband, National Institutes of Health, USA, http://

rsb.info.nih.gov/ij) or the Leica TCS 4Pi software. 3D recon-

structions of cells or cell segments were derived from image

stacks using Leica software ditto.

4.3. Zeta Potential of Nanocrystals in Different Media and Quantitative Protein Adsorption in Serum Media

4.3.1. Zeta Potential Measurements

All the dried zeolite L nanocrystals were resuspended in

PBS + + (Ca 2 + , Mg 2 + ), serum-free and serum (10% FBS) media

individually at concentration of 0.5 mg/mL for 1 h. All the

samples were sonicated in water bath for 20 min before

measurement.

4.3.2. Quantitation of Protein Adsorption (Bradford Assay)

Zeolite L nanocrystals surfaces adsorbed protein were indi-

rectly determined by reduced protein amount from sus-

pended FBS media. Calculated amount of BSA was dissolved

in culture media without FBS to reach concentration at

10 mg/mL. They were further diluted to 50, 100, 150, 200, 250,

300 μ g/mL. 3 mL of Bradford reagent was added to each

dilution and kept at room temperature for 5 min with gentle

mixing. Absorbance was measured at 595 nm against blank

by UV/Vis spectrophotometer. Standard calibration curve

was then plotted for unknown protein concentration detec-

tion. 10% FBS media was diluted at 1 to 20 in media without

serum for adsorption measurements.

Zeolite L nanocrystals were resuspended in PBS + + buffer

as stock concentration at 4mg/ml. 100 μ L of such samples were

added into 100 μ L this serum containing media. The mixtures

were gently mixed at room temperature for 1.5 h. All the

samples were centrifuged at 12000 rpm by eppendorf centri-

fuge. Supernatants were aspirated into new tubes. 100 μ L of

each supernatant and diluted serum media were pipetted into

3 mL Bradford reagents and kept at room temperature for 5

min. Absorbance were measured at 595 nm. Relative protein

concentrations were calculated from BSA standard curve.

4.4. Cell Culture

HeLa cells were cultured in EMEM containing 10% FBS, 1%

l-glutamine and 1% NEA, in 75cm 2 culture fl ask (Greiner)

www.small-journal.com © 2013 Wiley-VCH Verlag Gm

and sub-cultured 2 till 3 times per week depending on prolif-

eration. Cells were maintained at 37 ° C in a humidifi ed atmos-

phere of 5% CO 2 . Sub-confl uent cell layers were washed with

PBS − − and incubated for approx 2 min with 0.02 mL/cm 2 of

trypsin/EDTA solution (0.05%/0.02% [w/v]. After detaching

of the cells, the activity of trypsin was inhibited by the addi-

tion of EMEM media.

4.5. Nanocrystals Cytotoxicity in Serum and Serum-free Media

The MultiTox-Glo assay (Promega GmbH, Mannheim, Ger-

many) was used to test the cytotoxicity of different zeolite L

nanocrystals to HeLa cells. Cells were seeded in 96-well glass

bottom culture plate with cell number of 2.5 × 10 4 per well and

incubated at 37 ° C and 5% CO2 overnight. In the second day

plates were washed twice with PBS and then were replaced

by nanocrystals containing serum-free media. Nanocrystals

samples were calculated to obtain different concentrations

at 50, 100, 250 and 500 μ g/mL. Plates were incubated in the

same condition as before for 24 h. In the third day MultiTox-

Glo assays were performed. Digitonin was applied as positive

control to kill cells. It was fi rst prepared as 1 mg/mL in water

as stack solution and then further diluted to an end concen-

tration of 100 μ g/mL in serum-free media. Reagents were

prepared as recommended in the protocol. 50 μ l of GF-AFC

reagent were added to all wells. Plates were mixed by orbital

shaking and kept at 37 ° C without CO2 in darkness for 1 h.

Samples signals were measured by fl uorescence at excitation

380 + /- 10nm and emission 510 + /- 20 nm by Optima Fluostar

well plate reader (BMG Labtech). 50 μ l AAF-Glo reagents

were added to all wells in the same plates. Subsequently

plates were mixed by orbital shaker and incubated at room

temperature for 15 min in the darkness. Dead cell lumines-

cence was measured by OPTIMA as well.

HeLa cells were seeded on cover slips in 12-well micro-

plate overnight. Two concentrations of different zeolite L

nanoparticles 50 μ g/mL and 250 μ g/mL in serum containing

media (10% FBS) were added to each well. At different

time interval 6 h, 12 h, 24 h and 48 h the media in the wells

were replaced by trypan blue solution. Cells were incubated

for further 5 min. Cover slips were then taken out imme-

diately for Leica light microscopy observations and images

record.

4.6. Uptake Activity by Confocal Laser Scanning Microscopy

For confocal laser scanning microscopy (CLSM) imaging, glass

cover slips were sterilised in ethanol and washed twice by with

PBS buffer. All zeolite L particles were autoclaved as well.

HeLa cells were incubated in serum media overnight at 37 ° C

5% CO 2 on these cover slips placed in 12-well plates with cell

concentration at 2.5 × 10 5 cells/mL. 1 mL per well. Different

zeolite particles were sonicated in PBS for 10 min and then

diluted in serum and serum-free (Ham f-12) media to reach

the concentration 50 μ g/ml. Cell culture media was replaced

by 1 mL of particle suspension. Particles cell mixtures were

incubated individually at 37 ° C and 4 ° C for 2 h and 24 h. After

bH & Co. KGaA, Weinheim small 2013, 9, No. 9–10, 1809–1820

Internalization of Zeolite L Nanocrystals in HeLa Cells

washed twice by PBS + + buffer cells were fl uorescent stained

with DiOC 6 solution in PBS + + which was diluted 100 times

from 1 mM DMSO stock solution. It took 10 min and then

cells were washed with two times before fi xation. 1 mL of 4%

paraformaldehyde was added to each cover slip containing

well and kept at room temperature for 15 min. Two times

of washing were performed again. DBACO was mixed into

fl uoromount at concentration 20 mg/mL to form mounting

media. One drop of this media was placed on top of glass

slide. Cover slips with cells were put on the drops and kept at

room temperature until everything became solid. Images were

taken with Leica software on a Fluoview 300 equipped with

an IX71 with two lasers, 488 and 543 nm, and a × 63 oil objec-

tive. To avoid crosstalk between the channels, emission signals

were collected independently in a serial mode.

4.7. Inhibition Tests with higher Resolution Confocal Images at a 4 Pi Microscope and Quantifi cation

For inhibition studies, HeLa cells in a density of 6.41 × 10 4

cells/cm 2 were seeded on glass cover slips, in 12 well plates

and incubated overnight at 37 ° C at 5% CO 2 . Three hours

before particles exposure, EMEM was exchanged to un-

supplemented HAM F-12 medium. After desired time, cells

were incubated with 0.5 mL of individual inhibitor solution

(dynasore 80 μ M, chlorpromazine hydrogen 50 μ M, amilo-

ride hydrogen 100 μ M) made up in un-supplemented HAM

f-12 medium for 30 min at 37 ° C and 5% CO 2 . Subsequently,

another 0.5 mL of un-supplemented HAM f-12 medium,

containing the same concentration of inhibitor and zeo-

lite nanoparticles at fi nal concentration of 50 μ g/mL, were

added to the cells and incubated for further 4 hours. After-

wards cells were washed twice with PBS + + and stained with

DiD (15 μ M) for 30 minutes at 37 ° C at 5% CO 2 . Cells were

washed again twice and fi xed for 15 min at 37 ° C at 5% CO 2 ,

using 4% paraformaldehyde. The cells were then imaged with

4 Pi microscope in the confocal mode as illustrated in the

instrumentation.

To quantify the uptake activity of PAH-D-Zeo nanoparti-

cles by cells in the presence of inhibitors, we have developed

a method as follow: For fl uorescence intensity measurement

three independent experiments with three replicates were

prepared to study the uptake of particles. The samples prepa-

rations were the same as for the 4 Pi microscopy experiments

except that the cells were not stained with DiD and cul-

tured directly in microplates. Cells were then detached from

the well plate using Accutase (0.5 mL/well). After stopping

Accutase by addition of 1 mL EMEM, cells were washed

once with PBS + + and then fi xed in suspension for 15 min at

RT using 4% paraformaldehyde and centrifuged for 5 min

at 1 g . After one more washing step using PBS + + , all the cells

were counted and fl uorescence intensities of nanoparticles

were measured by Optima Fluostar well plate reader using

an excitation of 485B 12 nm and an emission of 580nm.

Quantifi cation of C-D-Zeo nanoparticles uptake activity

was slightly different from above method. Cells were

detached from wells by using EDTA and were resuspended

in 1 mL EMEM. After centrifugation for 3 min at 0.8 g cells

© 2013 Wiley-VCH Verlag GmbHsmall 2013, 9, No. 9–10, 1809–1820

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4.8. Double Staining with Zeolites and Anti-Clathrin-/Anti-Caveolin-Antibodies

For antibody staining the cells were seeded and incubated

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Supporting Information

Supporting Information is available from the Wiley Online Library or from the author.

Acknowledgements

This work was supported by the German Federal Ministry of Education and Research foundation Grant FKZ 03X0015 and FKZ0315773A. Z. Li thanks the European Community’s Seventh Framework Programme for fi nancial support under grant agree-ment CP-FP 228622-2 MAGNIFYCO. LDC thanks the ERC Advanced for the grant award number 247365. We thank Mrs. Kathrin Hardes for perfect technical assistance and for great help from Mrs. Faria Sarbrin for living cell experiments. We also thank Harald Fuchs for proof reading of the article.

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Received: July 17, 2012 Revised: September 3, 2012Published online: January 18, 2013

mbH & Co. KGaA, Weinheim small 2013, 9, No. 9–10, 1809–1820