Article

Cite This: ACS Nano 2018, 12, 6429−6442 www.acsnano.org

Multivalent Flexible Nanogels Exhibit Broad-

Spectrum Antiviral Activity by Blocking Virus
Entry
Pradip Dey,†,§,⊥ Tobias Bergmann,‡,⊥ Jose Luis Cuellar-Camacho,† Svenja Ehrmann,†
Mohammad Suman Chowdhury,† Minze Zhang,‡ Ismail Dahmani,∥ Rainer Haag,*,† and Walid Azab*,‡
†
Institut für Chemie und Biochemie, Freie Universität Berlin, Takustrasse 3, 14195 Berlin, Germany
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‡
Institut für Virologie, Robert von Ostertag-Haus, Zentrum für Infektionsmedizin, Freie Universität Berlin, Robert-von-Ostertag-Str.
7-13, 14163 Berlin, Germany
Downloaded via HUMBOLDT UNIV ZU BERLIN on May 14, 2019 at 14:00:35 (UTC).

§
Polymer Science Unit, Indian Association for the Cultivation of Science, 2A and 2B Raja S.C. Mullick Road, Kolkata 700032, India
∥
Institute of Biochemistry and Biology, University of Potsdam, Karl-Liebknecht-Str. 24-25, 14476 Potsdam, Germany
*
S Supporting Information

ABSTRACT: The entry process of viruses into host cells is

complex and involves stable but transient multivalent
interactions with different cell surface receptors. The initial
contact of several viruses begins with attachment to
heparan sulfate (HS) proteoglycans on the cell surface,
which results in a cascade of events that end up with virus
entry. The development of antiviral agents based on
multivalent interactions to shield virus particles and
block initial interactions with cellular receptors has
attracted attention in antiviral research. Here, we designed
nanogels with different degrees of flexibility based on
dendritic polyglycerol sulfate to mimic cellular HS. The designed nanogels are nontoxic and broad-spectrum, can
multivalently interact with viral glycoproteins, shield virus surfaces, and efficiently block infection. We also visualized
virus−nanogel interactions as well as the uptake of nanogels by the cells through clathrin-mediated endocytosis using
confocal microscopy. As many human viruses attach to the cells through HS moieties, we introduce our flexible nanogels
as robust inhibitors for these viruses.
KEYWORDS: multivalent, herpes simplex virus, heparan sulfate, nanoparticles, click chemistry, polyglycerol

T he parasitic nature and high diversity of viruses pose a

great hurdle in designing antiviral compounds that
could inhibit viral replication across virus families.
Antiviral drugs available to date mainly target specific viral
proteins in order to interrupt the replication cycle, which
results in reduced replication, giving the immune system time
to control or even clear the infection.1 Targeting early steps of
infection like entry of viral particles into host cells could
provide a broader inhibition approach. In general, viruses
attach to and enter host cells using multivalent interactions of
viral ligands with receptors and co-receptors present on the cell
membrane.2 The use of monovalent drugs to inhibit viral entry
and consequent spread is not very effective due to the strong
multivalent virus−cell interactions.3,4 Consequently, there is a
need for the development of antiviral agents based on
multivalent interactions to efficiently shield virus particles
and inhibit virus−cell interactions and subsequent entry and
infection. It is challenging, however, to maintain host cell
viability while targeting the virus.
Heparan sulfate (HS) moieties are cell surface molecules
that serve as primary receptors or co-receptors for several
microbial pathogens, including bacteria, viruses, and parasites.
It has been shown that HS is involved in the infection of many
viruses, including herpesviruses, arteriviruses, papillomaviruses,
and flaviviruses, through facilitating their internalization or
interaction with secondary receptors.5,6 Herpesviruses con-
stitute a large family of viruses that can infect a wide variety of
host species, including humans. Herpes simplex virus type 1
(HSV-1) is a human alphaherpesvirus with a high seropreva-
lence of 70−80% in adults.7 It primarily infects epithelial cells
and spreads to neurons, where it establishes life-long latent
infections to evade immune detection. Symptomatic reac-
tivations can present as orolabial and ocular lesions, herpes
Received: March 1, 2018
Accepted: June 12, 2018
Published: June 12, 2018

© 2018 American Chemical Society 6429 DOI: 10.1021/acsnano.8b01616

ACS Nano 2018, 12, 6429−6442
ACS Nano
Figure 1. Schematic depiction of flexible and rigid dPGS-based nanogels. Preparation was done using linPG and dPG as cross-linkers,
respectively, by strain-promoted azide alkyne cycloaddition via an inverse nanoprecipitation technique. The scheme shows the structure of
dPG and models of rigid and flexible nanogels. A denotes “upregulation in concentration, leading to cross-linking in the droplets”. B
indicates “addition of water, removal of acetone, and dialysis”. C denotes “nanogels dispersed in water”.
gladiatorum, and eczema herpeticum as well as central nervous
complications such as neonatal herpes and herpetic encepha-
litis.8,9 Several glycoproteins spike the viral lipid envelope and
are responsible for attachment, specific receptor binding, and/
or immune modulation. Herpesvirus particles initially attach to
the cell surface through a charge-based interaction of
glycoprotein (gC) and/or gB to heparan sulfate moieties of
the extracellular matrix.10 Although relatively nonspecific and
reversible, this first interaction is important to set the stage for
all subsequent processes including stable binding, fusion, and
penetration.11 Absence of HS,12−19 blocking with hepa-
rin,15,18,20−22 or deletion of the gC encoding genes results in
virions that are impaired in their ability to attach to the
cell.16,23,24 Currently, most of the herpesvirus therapeutics
include nucleoside, nucleotide, and pyrophosphate analogues
that target DNA replication. Acyclovir, Valacyclovir, Famcy-
clovir, and other relative drugs have been shown to reduce the
morbidity and mortality associated with encephalitis. However,
the development of resistance is a major obstacle.11 Previous
trials to block virus entry through targeting HS−virus
interaction showed promising results.25,26 However, the
developed compounds stuck with their weak antiviral activity
in vivo.25 No drugs inhibiting viral entry are commercially
available so far.
Another HS-binding virus is the unrelated porcine
reproductive and respiratory syndrome virus (PRRSV) that
has a major impact on the swine industry. It is a single-
stranded RNA virus that is classified within the family
Arteriviridae.27 The major envelope proteins M and GP5
form a complex that has been shown to bind to heparin, which
decreases virus infectivity in a dose-dependent manner.28
Until now, mainly dendritic and linear polymers, silver
(Ag)29,30 and gold (Au)31 nanoparticles, and functionalized 2D
nanomaterials (such as thermally reduced graphene oxide,32,33
6430
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carbon nanotubes, carbon dots,34 or nanodiamonds33)
functionalized with negatively charged functional groups such
as sulfonate (SO3−),35 sulfate (−OSO3−),33 or hydroxyl
(−OH) groups have been used as multivalent inhibitors of
viral entry.31,36,37 To effectively shield and block virus entry,
we have recently shown that sulfated nanoparticles of the same
size as virus particles significantly established multivalent
interactions and were capable of potently inhibiting virus
entry.33,38 However, using carbon nanomaterials in vivo is of
concern considering their toxicity due to reactive oxygen
species (ROS) generation in the target cells and oxidative
stress.39,40 Polymeric nanogels are cross-linked hydrogel
particles with a 3D network composed of water-soluble/
swellable polymers. They represent a better and promising
choice for the design of virus entry inhibitors41 due to the fact
that they can be degraded to smaller sized fragments, which
can be removed by renal clearance.42 In this context, polymeric
nanogels functionalized with bioactive ligands may play a
crucial role in terms of interfering with viral infection.
In this report, sulfated nanogels (NGs) based on dendritic
polyglycerol sulfate (dPGS) are prepared in the size range of
100−200 nm to match virus size.38 For nanogelation, we used
the inverse nanoprecipitation technique based on a bio-
orthogonal click reaction. Furthermore, the flexibility (defor-
mation) of these NGs is tuned by using two different types of
spacers, that is, dendritic and linear polyglycerols (Figure 1).
We hypothesize that flexible NGs (F-NGs) can efficiently
shield the virus particle and consequently are more potent
inhibitors for HS-binding viruses as compared to the rigid ones
(R-NGs). The rationale to use NGs with different deformation
potential is that flexible NGs can adapt to the virus surface
during the binding process, which leads to higher valent
interactions and hence reduces the probability of detachment
from the viral surface. To this end, we investigated the
DOI: 10.1021/acsnano.8b01616
ACS Nano 2018, 12, 6429−6442
ACS Nano Article

efficiency and the mechanism of inhibition of virus infection as

−34.3 ± 4.9

−16.5 ± 1.5

−5.1 ± 0.9
ζ-potential
well as the biological fate of NGs in cells.

(mV)
RESULTS AND DISCUSSION
Synthesis and Formation of Sulfated Nanogels Using

176 ± 47

174 ± 52

192 ± 71
Inverse Nanoprecipitation. Dendritic polyglycerol sulfate

(d, nm)
size by
NTA
azide (dPGS-N3), dPG-cyclooctyne (dPG-Oct), and linear
polyglycerol−cyclooctyne (linPG-Oct) were used as macro-
monomers for cross-linking by strain-promoted azide alkyne

0.043

0.107

0.079
from
DLS
cycloaddition reaction to form dPGS nanogels. The synthesis

PDI
of the macromonomers is discussed in the Supporting
Information. Inverse nanoprecipitation was introduced by

202.8 ± 4.1

211.5 ± 2.8

203.4 ± 2.3
our group for the preparation of hydrophilic nanogels as it has

intensity
(d, nm)
size by
several advantages: it is a surfactant-free technique, no
hydrophobic organic solvents are required, and size can be
controlled very easily by varying the good solvent to
nonsolvent ratio.43,44 The R-NGs (sulfated rigid NGs; dPGS-

Z average size

191.3 ± 3.9

191.4 ± 1.4

186.3 ± 0.8
dPG), F-NGs (sulfated flexible NGs; dPGS-linPG), and C-

in water
(d, nm)
NGs (nonsulfated control NGs, dPG-dPG) were formed by
the inverse nanoprecipitation technique,43 where the polymers
were dissolved in water and precipitated in acetone (Figure 1).
Here, R-NGs and F-NGs were the nanogels containing dPGS,

Z average size

not measured
156.2 ± 1.3

98.8 ± 0.06
in acetone
(d, nm)
where R-NG is the rigid particle composed of dendritic
polyglycerol (dPG), and F-NG is the flexible particle
composed of linPG. The dPGS content (Table 1, column 6)
in the polysulfated nanogels was determined from the
elemental analysis of freeze-dried nanogels measuring sulfur
(from CHNS, wt %)
dPGS content

not applicable
(S) content. They were found to be in the range of 40 to 28%
for R-NGs and F-NGs, respectively. Neutral C-NGs (control 40.4

28.4
nanogels) are solely composed of dendritic polyglycerol (dPG-
OH) and prepared by cross-linking of dPG-N3 and dPG-Oct.
Characterization of Nanogels. The particles were
characterized by dynamic light scattering (DLS), ζ-potential,
nanosight tracking analysis (NTA), 1H NMR spectroscopy,
volume of
acetone

200.0

200.0

320.0
(mL)

cryogenic transmission electron microscopy (cryo-TEM), and

atomic force microscopy (AFM). After nanoprecipitation, the
sizes were measured in acetone using DLS. The particle sizes
volume of

are shown in Table 1 (column 7). Afterward, the nanogels

(mL)
water

5.0

5.0

1.0

were transferred into water to quench them by adding excess

water and removing the acetone in vacuo (Figure 1). R-NGs
and F-NGs had different sizes (around 156 and 98 nm,
cyclooctyne/linPG-
cyclooctyne (mg)

respectively) in acetone. However, the hydrodynamic diame-

15.0 (16.0 μmol of

15.0 (6.64 μmol of

wt of dPG-

ters of both nanogels were found to be 191 nm in water with a

cyclooctyne)

cyclooctyne)

cyclooctyne)
2.0 (2.3 μmol

monomodal distribution (Table 1 and Figure 2a). As expected,

the nanogels swelled more in water compared to acetone. The
F-NGs swelled more (from 98 to 191 nm) compared to R-
NGs (from 156 to 191 nm). This can be explained due to the
inherent difference in cross-linkers used in the preparation of
10.0 (5.2 μmol)

10.0 (5.2 μmol)

3.0 (3.52 μmol)

wt of dPGS-N3

R-NGs and F-NGs. The presence of dPGS (in R-NGs and F-

NGs) and linPG (in F-NGs) was confirmed by 1H NMR of the
(mg)

dried nanogels (Supplementary Figures 1 and 2) and elemental

Table 1. Description of Nanogels

analysis. In addition, the size of the particles was measured by

NTA (Figure 2b). They were similar in size as those obtained
from DLS with a monomodal distribution (Figure 2a and
(dPGS-N3−linPG-cyclooctyne)
(dPGS-N3−dPG-cyclooctyne)

(dPG-N3−dPG-cyclooctyne)

Table 1, column 11). As expected, R-NGs and F-NGs both

have shown negative ζ-potential in the range from −15 to −35
mV compared to the control C-NGs (ζ-potential −5 mV;
nanogels

Table 1, column 12). Furthermore, the morphology of

nanogels was visualized by cryo-TEM (Supplementary Figure
3a,b). The diameters of individual nanogels were estimated by
analyzing the images taken by cryo-TEM using ImageJ
C-NGs
R-NGs

F-NGs

software, and histograms were plotted (Supplementary Figure

3c,d). The average sizes of dry R-NGs and F-NGs were found
6431 DOI: 10.1021/acsnano.8b01616
ACS Nano 2018, 12, 6429−6442
ACS Nano
Figure 2. Characterization of nanogels by DLS, NTA, and QNM
imaging. (a) Size distribution by intensity of all nanogels using
DLS. (b) Comparative size distributions obtained using NTA.
Morphology map (3D construction) of (c) rigid nanogels (R-NGs)
and (d) flexible nanogels (F-NGs). Cross sections for single NGs
as shown in the blue dashed lines (Supplementary Figure 4b,c) for
(e) R-NGs and (f) F-NGs. Arrows indicate the edges of NGs after
spreading on the substrate. (g) Simplified schematic representa-
tion of AFM photo detection system and tip applying repetitive
Nanoindentations across the scanned region. Here, the external
load applied by the AFM tip induces deformation onto the soft
nanogels, which can be determined as the difference in separation
distance from the tip initial contact point with the nanogels and its
separation distance at maximal loading force. The nanogels height
is obtained from the difference in separation distance from the
initial contact point on the nanogels surface and the lower region
corresponding in this case to the substrate. (h) Average
deformation obtained by applying same force on the nanogels.
Deformations were calculated by measuring 72 NGs, and the bars
indicate the mean with standard deviation of 72 NGs counted;
***p < 0.0001 (Kolmogorov−Smirnov normality test followed by
two-tailed Mann−Whitney test).
to be 109 ± 17 and 97 ± 18 nm, respectively. On the contrary,
the diameters obtained from DLS and NTA, which were
measured in solution (water), were ∼200 nm. These
differences can be attributed to the fact that DLS and NTA
measure the hydrodynamic diameter, whereas the hydration
shell is not visualized in cryo-TEM experiments.
Morphology and Flexibility of Nanogels Using
Atomic Force Microscopy. Sulfated nanogels (R-NGs and
F-NGs) were attached and imaged on poly-L-lysine-coated
mica substrates using AFM (3D reconstruction, Figure 2c,d;
2D image, Supplementary Figure 4b,c). An advantage of this
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technique is that changes in material properties can be
followed while imaging in liquid or buffer conditions even at
desired temperatures. The cationic nature of the lysine layer
allowed the nanogels to adhere on the surface, where spreading
of nanogels was different according to their flexibility, as seen
in the morphology maps (Supplementary Figure 4b,c) and
cross section profiles (Figure 2e,f) when imaged in water. For
R-NGs, the height image shows particles with a quasi-spherical
shape with a clear bumpy morphology. R-NGs had a mean
height of h = 65 ± 18 nm and a mean width of wm = 173 ± 56
nm (n = 74) (Supplementary Figure 4h). Surface topography
and cross-sectional height profiles of F-NGs are shown in
Figure 2d,f. A significant difference in morphology compared
to that of R-NGs (Supplementary Figure 4b,c) is observed for
F-NGs as they clearly spread out more on the substrate, and
their surface presented a more fibrous texture with clear distal
extensions from the center. A zoomed region for these with a
3D reconstruction is given in Supplementary Figure 5, where a
thread-like texture is resolved on their surface, likely generated
by a mesh of linear connecting polymers. Because of this more
enhanced spreading, the height of F-NGs was drastically
reduced to a mean value of h = 35 ± 12 nm with an increase in
measured width of wm = 228 ± 47 nm (Supplementary Figure
4h, n = 74). The pronounced spreading of these flexible
multivalent F-NGs driven by electrostatic interactions
resembles the adherence of cells on flat substrates upon first
contact with a substrate, a process that is mainly controlled by
their elasticity.45,46 This obvious difference in NG spreading
reflects their different degree of flexibility. We showed that the
size of rigid (R-NGs) and flexible (F-NGs) nanogels in
solution determined by DLS and NTA corroborated with each
other, whereas their height attained after spreading on the
substrate was very different and dependent on their flexibility.
The extent of spreading of a homogeneous soft particle on a
particular substrate will depend on its surface interactions,
interfacial surface energy, and elastic contributions from the
deformed particle. The atomic force microscopy results
revealed that NGs’ elasticity has a distinctive influence on
how spreading of NGs occurred on similar positively charged
surfaces. To quantify the degree of NG spreading, we
considered the ratio A = h/w for the determined values of
height h and width w. From the mean values for the height h
and width w for rigid and flexible NGs, ratios of w = 2.6h for
rigid (R-NGs) and w = 6.2h for flexible NGs (F-NGs) are
obtained. Imaging of soft nanoparticles with PeakForce QNM
mode (Supplementary Figure 4a)45−47 allows controlling and
minimizing the maximal applied normal force on the NGs’
surface by the AFM tip. This contributes to obtaining accurate
determination of the NGs’ height but still introduces an
overestimation of their width induced by the finite size of the
AFM tip. This contribution to the “true” NGs width (w) is
discussed below in the Methods section.
The process of adhesion to substrates induces deformation
in soft nanoparticles. Flexibility of different nanoparticles can
be compared through their degree of deformation induced by
the AFM tip (Figure 2g). Maps of deformation for R-NGs and
F-NGs are provided (Supplementary Figure 4d,e). The maps
show regions where the AFM tip penetrated the NGs to
different depths. In the image, brighter regions represent areas
where the tip penetrated deeper while indenting on the NGs
(Supplementary Figure 4d,e). On the other hand, the dark
backgrounds are regions scanned on the substrate where the
tip could not easily penetrate because the substrate is harder.
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Figure 3. Cytotoxicity and inhibition capacity of nanogels in Vero E6 cells. (a) Viability assay (WST-1) in Vero E6 cells for mock and
hydrogen peroxide (as negative controls) as well as C-NGs, F-NGs, and R-NGs (each 1 mg/mL). (b) Inhibition of HSV-1 infection at NGs
concentrations of 50, 100, and 200 μg/mL determined by flow cytometry. (c) Inhibition of HSV-1 infection with NGs at 200 μg/mL
determined by flow cytometry (black bars, postinfection; white bars, preinfection) or (d) plaque reduction assay. Postinfection refers to NGs
added to cells that had been infected with HSV-1 1 h before, and preinfection refers to NGs mixed with virus before being applied to the
cells. Virus was used at a multiplicity of infection (MOI) of 1. Normalized mean and standard deviation of three independent experiments
each in duplicates for (a−c). Circles represent individual plaque numbers from three independent experiments normalized to the mean
(with standard deviation) of the control (HSV) (d); *p < 0.01, **p < 0.001, ***p < 0.0001 (Kolmogorov−Smirnov normality test followed
by OneWay ANOVA with Bonferroni post-test for (a−c). OneWay ANOVA with Bonferroni post-test for (d). In (b), stars indicate a
significant difference of F-NGs and R-NGs to the contol C-NGs and between each other).

The profile of penetration on a single particle is given for R-
NGs and F-NGs (Supplementary Figure 4f,g, respectively).
Because both samples were imaged with the same type of AFM
tips and using the same maximal loading force (500 pN), it was
possible to directly compare their differences in deformability.
An average penetration depth of 6.3 ± 3.6 nm was obtained for
R-NGs, whereas a 22 ± 7.8 nm was found for F-NGs (Figure
2h). This represents a 10 and 62% deformation of their height
for R-NGs and F-NGs, respectively. This is a significant
difference in NGs’ deformation, which validates our chemical
approach for the preparation of rigid and flexible NGs with
controllable material properties.
Nanogels Are Potent Inhibitors of HS-Dependent
Viruses. At first, cytotoxicity of the prepared NGs was
assessed on Vero E6 and MARC-145 cells using the WST-1
viability assay.48 At a concentration of 1 mg/mL and 200 μg/
mL, respectively, none of the NGs significantly reduced cell
viability (Figure 3a and Supplementary Figure 7a). Next, the
efficiency of the NGs to inhibit infection of Vero E6 cells with
HSV-1 was evaluated. Nanogels in different concentrations
(ranging from 50 to 200 μg/mL) were incubated with HSV-1
[at a multiplicity of infection (MOI) of 1] on a monolayer of
Vero E6 cells for 24 h. Infected cells producing virus-encoded
green fluorescent protein (GFP)33 were quantified using flow
cytometry (Figure 3b). Although the control nanogels (C-
NGs) did not show any inhibition, the two sulfated NGs (F-
NGs and R-NGs: at a concentration of 200 μg/mL) were
active against HSV-1 and have inhibitory effect up to 90 and
60% in the case of F-NGs and R-NGs, respectively. The
effective concentrations (EC) with 50% inhibition were
calculated to be 90 and 164 μg/mL for F-NGs and R-NGs,
respectively. Here, the F-NGs were more potent compared to
the R-NGs though the concentration of dPGS present in both
nanogel preparations was in a similar range (Table 1, column
6433
6). To investigate the effect of the nanogels on virus entry, cells
were either treated with a mixture of NGs at 200 μg/mL and
virus at an MOI of 1 (preinfection) or first infected with HSV-
1 at an MOI of 1 for 1 h, then washed before nanogels were
added (postinfection). In the preinfection setup, the infection
was reduced to less than 5% and around 30% for F-NGs and R-
NGs, respectively. However, when the cells were infected first,
NGs were less or not effective in reducing the infection (Figure
3c and Supplementary Figure 6). After 1 h of infection, HSV-1
is expected to have shuttled its genome into the nucleus of the
infected cell and started gene expression.49 Therefore, it is
likely that the NGs exhibit their inhibiting effects at the level of
virus attachment, receptor binding, and/or penetration rather
than at downstream events. We clearly showed that both
functionalized NGs inhibited infection of cells with HSV-1
with high efficiency at the stage of viral attachment. Flexibility
of the nanogels has a significant impact on inhibition as only
around half of the concentration of F-NGs as compared to R-
NGs is needed to achieve 50% inhibition. The fact that the
infection was strongly reduced when NGs and virus particles
can freely interact (preinfection), but less inhibition is seen
when the cells are already infected (postinfection), shows that
inhibition occurs early in the infection cycle. The 25%
reduction in infection observed in the postinfection experi-
ments (Figure 3c) is likely due to the inhibition of egressed
virus particles that are newly synthesized in the infected cells.
Infection of cells with virus at an MOI of 1 statistically yields
an infection rate of around 70%. In a 20 h time frame, HSV-1
can well undergo an entire replication cycle, producing new
virions that can infect previously uninfected cells.50 The
presence of NGs in the supernatant inhibits this second round
of infection, leading to a smaller reduction in infection
compared to the controls. Accordingly, when the infection was
allowed to proceed for only 6 h, which is an enough time to
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produce GFP but not infectious progenies, no inhibition was
detected because no newly formed virus particles had been
released yet (Supplementary Figure 6a). Furthermore, in a
multicycle infection (MOI 0.01) over the course of 48 h, both
NGs reduced infection to 40−60% as at least two or three
additional rounds of replication had been inhibited (Supple-
mentary Figure 6b). Preincubation of NGs with cells for 24 h
and subsequent infection with HSV-1 (MOI of 1) for 6 h
confirmed that NGs are active for a long time as they still
inhibited the infection to the same extent (Supplementary
Figure 6c).
Similarly, the inhibitory potential of the NGs was confirmed
by plaque reduction assay. Here, NGs at 200 μg/mL were
either mixed with virus at 100 PFU (plaque-forming units)
incubated with the cells for 1 h, or the cells were infected first
before the NGs were added. To restrict virus cell-to-cell
spread, the cell monolayer was overlaid with DMEM
containing 0.5% methylcellulose. In accordance with the flow
cytometry experiments, F-NGs and R-NGs were able to reduce
plaque numbers by 50% in the preinfection setting, whereas in
the postinfection setup, no significant difference in plaque
numbers was observed for the NGs preparations (Figure 3d)
because we used an overlay medium, which abolishes a
subsequent second round of infection by newly made virus
particles.
To confirm the broad-range antiviral activity of our NGs, we
conducted preinfection experiments with the unrelated RNA
virus, PRRSV, which uses HS as an attachment receptor.28,51 F-
NGs were able to reduce infectivity of the input virus to
around 10%. The titer of the virus stock used was determined
to be 3.5 × 104 TCID50/mL, and mixing with 200 μg/mL F-
NGs reduced the infectivity to around 4 × 103 TCID50/mL
(Supplementary Figure 7b). Thus, our sulfated NGs were able
not only to inhibit entry of HSV-1 into cells but also to
strongly reduce infection with another unrelated, HS-depend-
ent virus. This finding strengthens the concept of sulfated NGs
being mimicking and competing with cellular HS in virus
binding.
The contact area (CA) of inhibitors is a very important
parameter while studying the interaction of soft multivalent
architectures with pathogens. A study on the dependence of
multivalency and steric shielding effects with size for hard
multivalent globular inhibitors has been reported recently from
our group.38 Hereby, a robust first approximation to estimate a
value for the NGs contact area CA can be made by considering
NGs’ attachment to be the area of a circle with diameter w,
thus CA = π(w/2)2, which leads to 22686 and 38687 nm2 for
R-NGs and F-NGs, respectively (Supplementary Table 1).
Now if we consider the size of HSV-1 particles in solution to
be around 200 nm and using the calculated values for the
average contact area CA of NGs, an estimation of the number
(NB) of NGs required to sterically shield the virus can be
made. A spherical virus with a diameter of around 200 nm
would have a surface area of about 125 600 nm2. This
estimated contact areas of nanogels (F-NGs and R-NGs)
would imply that to sterically shield a virus particle, around 5−
6 R-NGs are needed, whereas only 3 F-NGs would be required
(Figure 6 and Supplementary Table 1). This theoretical
observation, based on mathematical calculations, matches with
our experimental findings as discussed above.
To further determine the affinity of sulfated NGs to interact
with the virus, binding of sulfated nanogels (R-NGs and F-
NGs) with virus-mimicking positively charged nanogels
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(positive-NGs; 214 nm in size) was tested by surface plasmon
resonance (SPR). The sensorgrams are divided into three
distinct phases; (i) phase A, interaction of positive-NGs with
poly-L-lysine (PLY)-coated gold surface; (ii) phase B,
interaction of sulfated NGs with positive-NGs, and (iii)
phase C, dissociation of sulfated NGs (Supplementary Figure
8). Positive NGs were first injected, and the corresponding
change in SPR response units (RU; 500−700 RU) indicates
the immobilization and saturation of the SPR chip with
positive NGs. Sulfated and control NGs were then injected,
and significant interaction with positive NGs was seen for R-
NGs (Req = 7000 ± 1000 RU) and F-NGs (Req = 2000 ±
500 RU). As expected, C-NGs did not bind significantly to
positive NGs (Req = 775 RU). The increased signal obtained
for R-NGs compared to that for F-NGs is in line with our
steric shielding and flexibility hypothesis. For the same
concentration of NGs, the results suggest that less F-NGs
are required to cover the chip surface and to reach saturation.
This is consistent with our AFM results, which showed that F-
NGs spread out more (with a larger contact area) once they
are bound. Furthermore, due to the lower aspect ratio h/w of
the adhered F-NGs, they form a thinner layer onto the surface.
This explains the lower signal obtained with F-NGs compared
to R-NGs that form a thicker layer. The sensorgrams further
show the dissociation of C-NGs and, to lesser extent, of R-NGs
from the positive NGs; however, this does not stand for F-NGs
where the height of the signal remains constant. In conclusion,
sulfated NGs with a similar high multivalent capacity bind
significantly to positive surfaces; however, those with enhanced
flexibility resulted in stronger and stable binding.
While this work was in progress to be submitted, Cagno and
colleagues published results describing the use of gold
nanoparticles (NPs; core size 2−5 nm) decorated with long
flexible linkers mimicking HS as potent inhibitors of virus
infection at the entry level.52 They have shown that the
introduction of flexible linkers in the hard gold nanoparticle
core leads to better entry inhibition compared to the use of a
rigid linker, whereas in this report, our aim was to develop
anionic nanoparticles with inherent flexibility and degrad-
ability. Furthermore, we have correlated how deformation of
nanogels can lead to better steric shielding of virus particles. It
is worth noting that the use of hard-core nanoparticles may
lead to a long time accumulation in the body. In line with our
results, the synthesized gold NPs, mainly, inhibited infection
when first incubated with the virus before infection. The claim
that the sulfonated gold NPs can inhibit viruses after infection
needs further investigation as the effect was not assessed in the
real-time assay. The infected materials (infected cells/super-
natants and nanoparticles) were collected, and virus titration
was further conducted in another assay.52 It is expected that
the collected nanoparticles in the infected materials can still
inhibit the initially added viruses in the solution (not
necessarily inside the cells).
Nanogels Interact with HSV-1 and Inhibit Virus
Attachment to Cells. To better understand the mechanism
of inhibition as well as the fate of free NGs, we have
introduced a fluorophore into the nanogels by conjugating
cyanine dye in the dPGS azide and dPG azide (Supplementary
Figure 9). Similarly, three nanogels were prepared containing
cyanine dye by inverse nanoprecipitation as discussed above.
Experiments using confocal microscopy clearly show the
uptake of the functionalized (sulfated) NGs after 30 min
incubation at 37 °C into Vero E6 cells (Figure 4e,f). This
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Figure 4. Nanogels block virus attachment to cell membranes. Representative images of C-NGs as well F-NGs and R-NGs (green) (each at
10 μg/mL concentration). NGs were incubated with Vero E6 cells at 4 °C (0 min; a) or for 30 min at 37 °C (30 min; e) alone (top row) or
with HSV-1 (red) as well as virus alone (bottom row). Scale bars indicate 5 μm. (b−d) Median number of (b) nanogels or (c) virus particles
per cell in total, at the plasma membrane or intracellular, or (d) virus particles colocalizing with NGs for time point 0 min and (e−h) time
point 30 min. Virus was used at a multiplicity of infection (MOI) of 5. NGs and virus signals in 52 cells from three independent experiments
were counted; *p < 0.05, **p < 0.01, ***p < 0.001 (Kruskal−Wallis with Dunns post-test).

uptake can be completely blocked in cells kept continuously at
4 °C (Figure 4a,b and Supplementary Figure 10). Addition of
virus particles leads to significantly reduced numbers of both
virus and NGs at the plasma membrane (Figure 4a,c) and
inside the cells (Figure 4e,g). For F-NGs, the effect was more
prominent than for R-NGs, whereas C-NGs were not
detectable on or inside the cells. The number of virus particles
and NGs in the supernatant was significantly reduced when
added to cells alone, whereas co-presence of both virus and
NGs recovers concentrations in the supernatant to almost
preinfection levels (Supplementary Figure 11). The low
number of virus particles present at the membrane colocalizes
with almost 90% of F-NGs and with around 40% of R-NGs
(Figure 4d,h). Because HSV-1 uses its positively charged
glycoproteins to adhere to negatively charged heparan sulfate
on cell surfaces, shielding these glycoproteins by binding to
polyglycerol nanogels, providing high densities of negatively
charged sulfate groups, could neutralize the ability of the virus
and NGs to bind to cells, explaining the significantly reduced
amount of both on and inside the cells. Consequently, the
added input material (virus−nanogel complex) was found in
the supernatant, whereas viruses or NGs alone adsorb to and
enter the cells, which decreases the amount of virus−nanogel
6435
complexes in the supernatant. These effects are highly
significant for F-NGs, whereas tendencies with limited
statistical significance were detected for R-NGs. Interestingly,
NGs were rarely taken up in the presence of virus but
remained at the plasma membrane, and once viral particles had
entered a cell even in the presence of the nanogels, they did
not colocalize with NGs anymore. Both observations can be
explained with the interaction of NGs with viral glycoproteins
that the virus leaves behind at the plasma membrane when
entering Vero E6 cells via direct fusion.53
Nanogels Enter Cells through Clathrin-Mediated
Endocytosis and Route through the Classical Endo-
somal Pathway. To tackle the question of intracellular fate of
the NGs, subcellular structures were investigated for
colocalization with NGs. Internalization of extracellular cargos
into the cells occurs mainly through the endocytic pathway,
which is dependent on scaffolding proteins like clathrin and
caveolin.54 Macropinocytosis is also adopted by the cells to
uptake larger volumes of extracellular fluids.55 No colocaliza-
tion was detected with caveolin-coated pits or macro-
pinosomes (Supplementary Figure 12); however, 20−30% of
F-NGs colocalized with clathrin after 10 min incubation at 37
°C with gradually decreasing numbers over the course of 2 h
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Figure 5. Subcellular localization of F-NGs and R-NGs. Representative images of immunostained cells to detect colocalization of F-NGs
(green; 10 μg/mL) after 10 min (top row), 30 min (middle row), and 120 min (bottom row) at 37 °C with (a) clathrin, (b) EEA-1, (c)
RAB7−7, and (d) LAMP-1 (red). Scale bars indicate 5 μm and are 1 μm in the zoomed details. Median portion of F-NGs colocalizing and
noncolocalizing with (e) clathrin, (f) EEA-1, (g) RAB-7, and (h) LAMP-1. Median portion of R-NGs colocalizing and noncolocalizing with
(i) clathrin, (j) EEA-1, (k) RAB-7, and (l) LAMP-1 normalized to the total amount after 10, 30, and 120 min. NGs signals were counted in
52 cells from three independent experiments; ***p < 0.001 (Kruskal−Wallis with Dunns post-test).

(Figure 5a,e). Around the same percent of colocalization was
detected with an early endosome marker (early endosome
antigen 1; EEA-1) (Figure 5b,f) and to a lesser extent with a
late endosomal marker (Ras-related protein; RAB-7) (Figure
5c,g). Detection of lysosomes with the lysosome-associated
membrane protein (LAMP)-1 revealed increasing percent of
colocalization with up to 50% after 2 h (Figure 5d,h). R-NGs
behaved much the same way as F-NGs albeit with lower levels
of colocalization (Figure 5i−l). We showed that sulfated NGs
attach to the cell surface and are internalized at least in part by
clathrin-mediated endocytosis. They follow the classical
endosomal pathway accumulating in lysosomal structures as
seen for doxorubicin-loaded polyglycerol-based nanogels.56
The nonsulfated control C-NGs, however, failed to bind to
cells and were only found in the supernatant. Over a course of
6436
2 h, 70% of internalized F-NGs colocalized with markers
defining the endosomal−lysosomal pathway, although clearly
other, not yet identified, means of uptake are involved. It is
worth mentioning that not all sulfated NGs are uptaken by the
cells, and they remain active for a long time (24 h) outside the
cells (Supplementary Figure 6c). The proposed mechanism of
action is summarized in Figure 6.
To further elucidate the mechanism of uptake, we used
pharmacological inhibitors to study various cellular functions.
None of the inhibitors showed significant cytotoxicity at the
concentrations used (Supplementary Figure 13a). Vero E6
cells were preincubated with chlorpromazine, which inhibits
assembly of the clathrin adaptor protein 2, thereby preventing
formation of clathrin-coated pits,57,58 dynasore, an inhibitor of
a cellular GTPase (dynamin) involved in membrane fission
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Figure 6. Mechanism of inhibition. (i) HSV-1 initially attaches to heparan sulfate (HS) proteoglycan on the cell surface through
glycoproteins B (gB) and gC. Specific binding is next mediated by gD, and subsequent fusion releases the nucleocapsid into the host cell. (ii)
Sulfated nanogels (mimicking HS) shield viral particles and inhibit virus binding to cells. (iii) Only around three flexible as opposed to five
rigid nanogels are needed to completely cover HSV-1 particles. (iv) In the absence of virus particles, NGs are taken up by the cell via
clathrin-mediated endocytosis. Passed on to early endosomes, they are shuttled through late endosomes to lysosomes where they
accumulate. In the presence of virus particles, however, NG−virus complexes remain mainly in the supernatant and cannot internalize into
the cell.

and release of vesicles, as well as ethyl isopropyl amiloride
(EIPA), which inhibits macropinocytosis by targeting a
sodium-proton exchanger.59 Chlorpromazine was the most
potent inhibitor by reducing NG uptake to around 50%,
whereas 25% reduction was seen in cells treated with dynasore
or EIPA. Drugs (filipin or mβCD) targeting cholesterol-
dependent pathways, such as caveolin-mediated endocytosis,60
as well as the tyrosine kinase inhibitor (genistein) had no
significant effect on NG uptake (Supplementary Figure 13b).
The fact that NG uptake is sensitive to chlorpromazine and
dynasore treatment supports the role of clathrin-dependent
endocytosis as the primary means of internalization. It remains
unclear whether the low reduction of uptake caused by EIPA is
due to the inhibition of macropinocytosis, which would be
contradicted by the fact that no colocalization with the
macropinosome marker dextran was detected. However,
inhibition of the sodium-proton exchanger 3 (NHE3), which
is the primary target of EIPA, has been shown to block apical
receptor-mediated endocytosis in kidney cells and to prevent
cytoskeleton rearrangement;61,62 both can affect clathrin-
mediated uptake of NGs.
CONCLUSION
In summary, polyglycerol-based sulfated nanogels with differ-
ent flexibility were developed with defined sizes (100−200
nm) using a bio-orthogonal cross-linking chemistry, which
allows for an efficient gelation. The different flexibilities of the
nanogels were successfully proven by comparing the spreading
of nanogels to a positively charged mica surface. As expected,
the flexible particles have attained a smaller height and
increased width compared to those of the rigid ones.
Furthermore, the deformation data confirm that the
application of similar force (500 pN) to the NGs leads to
deformation in flexible NGs higher than that of the rigid ones.
Our experimental results showed that globular multivalent
inhibitors with different flexibility have a considerable impact
on pathogen inhibition through their increase in contact area.
Higher flexibility of these nanogels increases their antiviral
activity by better shielding the interactions of virus particles
with cellular surfaces, thereby inhibiting virus attachment to
cells and subsequent uptake of viral particles. Free NGs,
however, are endocytosed and routed through the classical
endosomal−lysosomal pathway. As many human viruses attach
to the cells through HS interactions, these flexible multivalent
dPGS nanogels have a great potential as robust inhibitors for
many virus species.
METHODS
Chemical Synthesis. All chemicals were reagent grade obtained
from Acros Organics (Geel, Belgium) or Sigma-Aldrich (Munich,
Germany) and used as received without further purification. The
SO3−pyridine complex was used as received from Fluka Production
GmbH (Buchs, Switzerland). Reactions sensitive to air or moisture

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were performed under anhydrous conditions in flame-dried glassware.
Detailed procedures for the synthesis of all compounds and their
characterization are provided in the Supporting Information (for 1H
spectra of all compounds, Supplementary Methods and Supplemen-
tary Figures 14−20).
Preparation of dPGS Nanogels. For R-NGs, dPGS-N3 (10 mg,
0.65 μmol) and dPG-cyclooctyne (15 mg, 2.0 μmol) were dissolved
separately in Milli-Q water (2.5 mL). The solutions were cooled to 4
°C, mixed, and added quickly to magnetically stirred acetone at 900
rpm (200 mL). Precipitated polyglycerol sulfate nanoparticles were
obtained as blue shining dispersions, and the particle size was
determined by DLS (Table 1). The reaction was quenched by the
addition of excess water after keeping the mixture overnight. Acetone
was evaporated to obtain blue shining nanogel dispersions in water.
The nanogels were dialyzed in water for 2 days and concentrated by
evaporation of water. Concentration was determined by lyophilizing
the solution.
The other nanogels, F-NGs and C-NGs, were prepared using the
same procedure, and the macromers used are described in Table 1.
Similarly, dye-functionalized nanogels were prepared using the dye-
functionalized dPGS azide and dPG azide following the procedure
described above.
Nanosight Tracking Analysis and Dynamic Light Scattering.
The particle size distribution and ζ-potential of the nanogels were
measured at a concentration of 1 mg/mL in Milli-Q water or
phosphate-buffered saline (PBS) at 25 °C using a Zetasizer (Malvern
Zetasizer-Nano ZS, Malvern Instruments Limited, Worcestershire,
UK) and temperature equilibration for 60 s. The intensity by size
distribution and the PDI values were recorded. In addition, the
hydrodynamic diameter of the nanogels was measured by NTA with a
Nanosight NS500. The concentration used for NTA was 1 μg/mL.
Cryo-Transmission Electron Microscopy. Five microliters of
nanogel solution (0.5 mg/mL) was drop-casted on hydrophilized
carbon film grids (Quantifoil R1/4) at room temperature. Then the
excess fluid was discarded using filter paper to generate an ultrathin
layer of the solution covering the holes in the carbon film. The grids
were vitrified in liquid ethane using a standard plunging device and
stored in liquid nitrogen prior to the measurement. The vitrified
samples were transferred under liquid nitrogen into a Tecnai F20
transmission electron microscope (FEI Company, Oregon, USA)
using a Gatan (Gatan Inc., California, USA) cryo-holder and stage
(model 626). Micrographs were recorded at a sample temperature of
around 100 K using the microscope’s low-dose protocol at a primary
magnification of 29000× and an acceleration voltage of 160 kV. Image
recording was done using an EAGLE 2k-CCD device (FEI Company,
Oregon, USA) at full 2048 × 2048 pixel size, and the defocus was
chosen to be ca. −10 μm in all cases to create enough phase contrast.
Graphical analysis of the images was performed using ImageJ v1.50b.
Atomic Force Microscopy. PeakForce mode was used for
imaging at high resolution with SNL-10 tips from Bruker (tip radius
2−12 nm and spring constant 0.3 N/m). Before each experiment,
cantilevers were first calibrated on cleaved mica surfaces to obtain the
cantilever sensitivity, and then the cantilever spring constant was
determined using the thermal noise method.63 Briefly, the negatively
charged nanogels were electrostatically attached to the surface of mica
coated with the cationic polymer poly-L-lysine. Round discs of
Muscovite mica of about 1 cm in diameter were cleaved with regular
adhesive tape, and 5 μL of poly-L-lysine (0.01 wt %, MW 70000
Sigma-Aldrich) was deposited and left to dry. Then the surface was
rinsed several times with Millipore water to remove any free unbound
polymer, and the discs were glued to round metal pucks. Ten
microliters of a 20-fold diluted solution (concentration ∼3 mg/mL)
was deposited on the mica-coated substrates and incubated for only
10 min. The samples were finally mounted on the AFM head, and a
fluid cell was assembled. Millipore water was introduced into the
sample chamber, and consequently, samples were never allowed to
dry. Scan Assyst was used during imaging, but maximal loading forces
were manually set to 500 pN in order to minimize damage on the
sample surface but still obtain reliable images. Imaging was taken at
512 points per line and a scan rate of 0.7−0.8 Hz.
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Approximations Applied to Correct Width and Height in
AFM. Determination of nanogel size using AFM imaging usually
considers the height of the measured particle as a more reliable value
than its width because the lateral resolution of the imaged object is
affected by the AFM tip size and induces an overestimation of the
particles’ “true” width w (Supplementary Figure 21). Geometrical
models to account for the induced convolution Δ requires knowledge
of the AFM tip radius, opening angle θ, and the size and shape of the
particle being scanned. In the case of R-NGs, an approximation to
obtain Δ and subsequently to estimate the “true” width w can be
made considering R-NGs as spherical particles of height h and h >
radius of tip (rtip). Here, we used the approach reported by Canet-
Ferrer and co-workers64 and obtained a value of Δ = 2.64 nm for the
overestimation of the width and a true value of w = 170.14 ± 56.29
nm (Supplementary Table 1). A schematic representation of this
approach and additional details to obtain Δ are provided in
Supplementary Figure 21. For the case of F-NGs, the AFM tip
could delineate a gradual increase in the NGs’ surface topography
from the very distal polymer extensions toward the center of the
particle, as shown in Figure 2d. Although small, the edge of these
distal extensions showed vertical deflections in the range of 1−2 nm,
which is still distinguishable from the substrate background. In this
particular situation, Δ was calculated using the approximation applied
by Winzer and co-workers for the case of h > rtip when the AFM tip
contacts the imaged object at its maximum height.65 Here, the vertical
height of the edge of F-NGs is about 1.5 nm. With this approximation,
we obtain a value of Δ = 5.52 nm for the tip convolution and of w =
222.04 ± 47.18 nm for a more accurate value of the F-NGs’ width.
It is worth mentioning that the geometrical approximations we
used to estimate the tip convolution will hold more accurate for rigid
objects and could have serious deviations for the case of very soft
particles as in the present case. The complex surface details of the
NGs’ shape or their deformation at the edges as a result from the
scanning process will obviously introduce uncertainties. Furthermore,
determination of “true” contact area for particles with a height larger
than the AFM tip could be well overestimated as determination of the
particle shape below its equator is not possible, and the NGs’ contact
angle could strongly vary depending on the surface interactions and
NGs’ elasticity. Moreover, our robust approximation of the contact
area CA takes into account an idealized circular region with diameter
w, which again is a simplification of the observed NG attachment.
Regardless of this, we consider that our approximation of the CA
provides a step forward in the characterization of the spreading of
multivalent inhibitors with different flexibility.
Surface Plasmon Resonance. Positively charged dPG nanogels
(positive-NGs; size determined by DLS was 214 nm, and the ζ-
potential was +24 mV)66 were immobilized on a PLY-coated SPR
gold sensor chip (XanTec bioanalytics GmbH). Prior to use, the
surface of the PLY-SPR chip was cleaned by injection of sodium
hydroxide (NaOH; 20 mM, 200 μL) and hydrochloric acid (HCl; 80
mM, 200 μL). Positive NGs (2.5 mg/mL) were first injected at low
flow rate (16 μL/min) to allow their immobilization on the PLY-SPR
chip. Association with the chip surface and saturation equilibrium
levels (Req) were monitored for 10 min. The corresponding change
in resonance units was monitored and recorded in the sensorgrams.
The dissociation phase was monitored for 5 min. C-NGs, R-NGs, and
F-NGs were injected at a concentration of 1 mg/mL at low flow rate
(16 μL/min). Regeneration of the surface of the SPR chip was
performed by short injection of 100 mM HCl then 50 mM NaOH.
SPR measurements were performed at 25 °C in 0.5× PBS (pH 7.5) in
a Biacore J SPR analyzer (Biacore AB, Uppsala, Sweden). SPR signals
were presented as a set of sensorgrams, which is a plot of resonance
units versus time. All solutions were freshly prepared, degassed, and
filtered through 0.22 μm pore filters.
Cells and Viruses. African green monkey kidney cells (Vero E6,
not listed as misidentified by ICLAC) were grown in MEM medium
(PAN Biotech P04-08050) supplemented with 10% fetal calf serum
(FCS, PAN Biotech P30-1402) and 100 U/mL penicillin (Roth
CN28.3) and 100 μg/mL streptomycin (Alfa Aesar J61299) in a 37
°C incubator with 5% CO2 atmosphere. Cells were grown to
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confluency, washed with PBS, and detached from the culture vessel
surface by the addition of 0.25% trypsin supplemented with 2.5 μM
EDTA. Cells were counted in a Neubauer counting chamber and
seeded at appropriate densities. MARC-145 (simian kidney epithelial
cells derived from MA-104, ATCC CRL-6489) were maintained as
adherent culture in Dulbecco’s modified Eagle’s medium (DMEM,
PAN Biotech P04-01548S1) supplemented with 10% FCS, 100 U/mL
penicillin, and 100 μg/mL streptomycin at 37 °C in an atmosphere
with 5% CO2.
HSV-1 BAC (pYEbac102)67 kindly provided by Dr. Yasushi
Kawaguchi, Institute of Medical Science, The University of Tokyo,
was reconstituted after transfecting BAC DNA into Vero E6 cells
using Lipofectamine 2000 (Invitrogen). HSV-1 encodes the gene for
enhanced green fluorescent protein to facilitate detection of infected
cells. PRRSV strain Lelystad virus (low pathogenic PRRSV-I
prototype strain,68 (GenBank accession number: M96262.2) was
maintained on MARC-145 cells.
Cell Viability Assay. Viability of Vero E6 and MARC-145 cells in
the presence of nanogels (1 mg/mL and 200 μg/mL, respectively) as
well as pharmacological inhibitors (see below) was determined using
the WST-1 assay after 20−24 h.48 Briefly, cells were seeded in 96-well
plates and incubated with NGs for 1 h on ice. Temperature was
shifted to 37 °C, and cells were incubated for 30 min or 20 h with
inhibitors or NGs, respectively. The WST-1 mixture was added
(Cayman Chemicals 10008883), and after 2 h, absorbance of each
sample was read using a microplate reader at 450 nm wavelength.
Controls used were untreated cells (mock) and cells treated with 0.1%
H2O2 (Sigma H1009) for 30 s.
Flow Cytometry. A mixture of HSV-1 at an MOI of 1 and
nanogels (ranging from 50 to 200 μg/mL for determination of EC50,
and at 200 μg/mL for inhibition experiments) were diluted in culture
medium and added to a confluent monolayer of Vero E6 cells and
incubated at 37 °C for 20 h (preinfection). Alternatively, cells were
infected with HSV-1 at an MOI of 1 for 1 h, and then cells were
washed with PBS. NGs (200 μg/mL) were added and incubated for 6
or 20 h at 37 °C. For multicycle experiments, cells were infected with
HSV-1 at an MOI of 0.01 for 1 h, washed, and incubated with NGs at
200 μg/mL for 48 h. For preincubation experiments, cells were
incubated with NGs at 200 μg/mL for 24 h before virus at an MOI of
1 was added for 6 h. Subsequently, cells were trypsinized and washed
twice with cold PBS. After centrifugation, cells were resuspended in
PBS, and 10 000 cells were analyzed with a CytoFLEX flow cytometer
(Beckman Coulter) to determine the percentage of infected cells by
fluorescence emission. Infection induced by parental viruses was set to
100%. Flow cytometry data were analyzed with FlowJo software with
equal gate settings according to the controls (Supplementary Figure
22).
Pharmacological Inhibitors. Cells were pretreated with different
drugs for 30 min at 37 °C before addition of fluorescent NGs (50 μg/
mL) in the presence of inhibitors. After 30 min incubation on ice,
cells were washed with prewarmed medium and reincubated at 37 °C
for 30 min in the presence of inhibitors. Cells were then trypsinized
and washed twice with PBS. After centrifugation, cells were
resuspended in PBS, and 10 000 cells were analyzed with a CytoFLEX
flow cytometer (Beckman Coulter) to determine the uptake of NGs.
The drug concentrations used were 50 μg/mL genistein (Sigma
G6649) dissolved in DMSO, 10 μg/mL chlorpromazine (Sigma
C8138) in PBS, 5 μg/mL filipin (Sigma 11078−21−0) in DMSO, 20
mM methyl-β-cyclodextrin (MβCD; Sigma 332615) in PBS, 50 μM
5-(N-ethyl-N-isopropyl)amiloride (EIPA; Sigma 1154−25−2) in
ethanol, and 150 μM dynasore (Sigma 1202867-00-2) in DMSO.
Mock samples were treated with 1% DMSO. Toxicity panels were
performed to ensure that the inhibitors did not cause an adverse effect
when used at the given concentrations.
Plaque Reduction Assay. Cells were grown in 24-well plates.
Virus suspensions (100 PFU per well) were mixed with the nanogels
(200 μg/mL), added to the cells, and incubated at 37 °C for 1 h.
Alternatively, cells were infected first with 100 PFU for 1 h, washed,
and treated with 200 μg/mL of the NGs for 1 h. Subsequently, cells
were washed with PBS, treated with citrate buffer pH 3 [40 mM citric
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acid (Roth 7624.1), 135 mM NaCl (VWR 27868.297), and 10 mM
KCl (Roth HNO2.3)] for 30 s, neutralized with medium, and washed
again with PBS before they were overlaid with MEM containing 0.5%
methylcellulose (Sigma M0262).69 At 2 days postinfection,
fluorescent plaques were counted using an inverted fluorescence
microscope (Zeiss Axiovert 100). As controls, mock-infected cells and
virus-infected cells in the absence of nanogels were included. All
plaques were counted, and the mean of three independent
experiments was calculated. Plaque numbers induced by parental
viruses were set to 100.
TCID50 of PRRSV. A 10-fold serial dilution of PRRSV stock was
prepared, mixed with NGs at 200 μg/mL, and added to MARC-145
cells in eight replicates. After 1.5 h, cells were washed with PBS, and
medium (DMEM supplemented with 2% FCS, 100 U/mL penicillin,
and 100 μg/mL streptomycin) with NGs at 200 μg/mL was supplied.
After 5 days, individual wells were analyzed for cytopathic effect. Viral
titers (TCID50/mL) were calculated by the Spearman-Kärber
algorithm.70,71
Confocal Microscopy. For binding assay, cells were seeded in an
8-well μ-slide (IBIDI) at low densities (1 × 104) and allowed to settle
for 2 h at 37 °C and 5% CO2. On ice, 1.5 μg (10 μg/mL) of
fluorescent NGs (excited at 560 nm) with or without HSV-1 (1 × 105
PFU) or virus alone was added to cells. Cells were rested at 4 °C for 1
h, washed with cold or prewarmed medium, and either kept at 4 °C (0
min time point) or shifted to 37 °C for 30 min (30 min time point).
Cells were washed twice with PBS and fixed with 4% paraformalde-
hyde (PFA) in PBS for 15 min at room temperature. Cells were
blocked with blocking buffer [BB; 4% FCS and 1% bovine serum
albumin (VWR 0332) in PBS]. BB containing 0.25% saponin
(AppliChem A2542) was used to permeabilize cell membranes for
30 min at 37 °C. Virus particles were detected using anti-gD (1:200 in
BB; Genetex GTX18160) or anti-VP5 (1:200 in BB; Abcam ab6508)
antibodies for the 0 and 30 min time points, respectively, and
incubated for 30 min at 37 °C. Cells were washed twice with PBS and
incubated with secondary goat anti-mouse IgG-Alexa Fluor 647
antibody (Abcam ab150115) or goat anti-rabbit IgG-Alexa Fluor 647
(Abcam ab150079) in a 1:1000 dilution in BB for 30 min at 37 °C.
For colocalization experiments, cells were stained with different
antibodies for 1 h at 37 °C to detect colocalization of NGs with
subcellular structures as described above. Antibodies used were
anticlathrin (Abcam ab21679), anticaveolin (Abcam ab2910), anti-
EEA-1 (Abcam ab2900), anti-RAB-7 (Abcam ab126712), and anti-
LAMP-1 (Abcam ab24170) in 1:200 dilutions. Dextran-FITC
conjugates (1 μg/mL; Sigma 46945) were used as markers of
macropinosomes. Finally, cells were washed three times with PBS, and
the nucleus was stained with 2 μg/mL 4′,6-diamidino-2-phenylindole
(DAPI, Life Technologies 62247).
To evaluate NGs and virus presence in the supernatants, samples
were prepared as described above. Twenty microliters of all samples
before addition to the cells (preinfection) and after 1 h incubation on
ice with the cells (postinfection) was dropped on prewet coverslips.
The samples were allowed to dry for 2 h at 37 °C and fixed with 4%
PFA for 15 min. Subsequently, cells were blocked and stained with
anti-gD antibody and secondary antibody as described above.
Coverslips were put on standard glass slides using Vectashield with
DAPI mounting medium (Vector Laboratories).
All samples were analyzed with a VisiScope spinning disc confocal
microscope (Visitron Systems GmbH). Acquisition settings (exposure
times, laser voltages) in each channel were kept constant within
experiments with small variations between experiments due to
technical reasons. Images were taken with the VisiView software
(Visitron Systems GmbH) and analyzed with ImageJ (ImageJ).
Brightness and contrast settings were adjusted according to the
controls and kept constant within experiments.
Statistical Analysis. All viability and flow-cytometry-based
experiments were blinded before analysis. All experiments involving
confocal microscopy were carried out in a blinded fashion.
Normalization was done in Microsoft Excel (Microsoft), and
statistical evaluation was performed using GraphPad Prism 5
DOI: 10.1021/acsnano.8b01616
ACS Nano 2018, 12, 6429−6442
ACS Nano
(GraphPad software). Values of p less than 0.05 are considered
significant.
ASSOCIATED CONTENT
*
S Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acsnano.8b01616.
Synthesis procedure and characterization data such as
1
H NMR, IR, and UV−vis spectra, additional results and
tables (PDF)
AUTHOR INFORMATION
Corresponding Authors
*E-mail: haag@zedat.fu-berlin.de.
*E-mail: walid.azab@fu-berlin.de.
ORCID
Pradip Dey: 0000-0002-1302-0874
Walid Azab: 0000-0003-2880-7255
Author Contributions
⊥ gIII with Heparinlike Moiety on the Cell Surface. Virology 1991, 181,
P.D. and T.B. contributed equally.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
We thank Florian Paulus for PG synthesis, Cathleen Schlesener
and Anja Stöshel for measuring gel permeation chromatog-
raphy and protected linear polyglycerol synthesis, Dr. Mathias
Dimde for providing positively charged dPG nanogels, and Dr.
Pamela Winchester for proofreading the manuscript. We
especially acknowledge Prof. Klaus Osterrieder, Dr. Sumati
Bhatia, and Dr. Dirk Steinhilber for the useful discussions. This
study was supported by the SFB 765 and the core-facility
biosupramol (www.biosupramol.de) to R.H. This study was
partly supported by grant from the DFG to W.A. (AZ 97/3-2).
P.D. thanks DRS-Honors Post-Doctoral Fellowship (2016-1),
Freie Universität Berlin (https://www.fu-berlin.de/en/sites/
drs/postdocs/drs_fellowships/index.html).
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