Carbohydrate-Based Nanocarriers Exhibiting Specific Cell Targeting with Minimum Influence from the Protein Corona

Article abstract of DOI:10.1002/anie.201502398

Whenever nanoparticles encounter biological fluids like blood, proteins adsorb on their surface and form a so-called protein corona. Although its importance is widely accepted, information on the influence of surface functionalization of nanocarriers on the protein corona is still sparse, especially concerning how the functionalization of PEGylated nanocarriers with targeting agents will affect protein corona formation and how the protein corona may in turn influence the targeting effect. Herein, hydroxyethyl starch nanocarriers (HES-NCs) were prepared, PEGylated, and modified on the outer PEG layer with mannose to target dendritic cells (DCs). Their interaction with human plasma was then studied. Low overall protein adsorption with a distinct protein pattern and high specific affinity for DC binding were observed, thus indicating an efficient combination of "stealth" and targeting behavior.

Full text of DOI:10.1002/anie.201502398

Angewandte
Chemie
International Edition: DOI: 10.1002/anie.201502398
German Edition: DOI: 10.1002/ange.201502398
Nanocarriers
Carbohydrate-Based Nanocarriers Exhibiting Specific Cell Targeting
with Minimum Influence from the Protein Corona**
Biao Kang, Patricia Okwieka, Susanne Schçttler, Svenja Winzen, Jens Langhanki, Kristin Mohr,
Till Opatz, Volker Mailꢀnder, Katharina Landfester,* and Frederik R. Wurm*
Abstract: Whenever nanoparticles encounter biological fluids
like blood, proteins adsorb on their surface and form a so-
called protein corona. Although its importance is widely
accepted, information on the influence of surface functional-
ization of nanocarriers on the protein corona is still sparse,
especially concerning how the functionalization of PEGylated
nanocarriers with targeting agents will affect protein corona
formation and how the protein corona may in turn influence
the targeting effect. Herein, hydroxyethyl starch nanocarriers
(HES-NCs) were prepared, PEGylated, and modified on the
outer PEG layer with mannose to target dendritic cells (DCs).
Their interaction with human plasma was then studied. Low
overall protein adsorption with a distinct protein pattern and
high specific affinity for DC binding were observed, thus
indicating an efficient combination of “stealth” and targeting
behavior.
ethylene glycol) is the state-of-the-art approach to reducing
nonspecific interactions with plasma proteins; this effect is
often termed the “stealth effect”.[3] However, the stealth
effect alone is not enough: specific targeting agents have to be
attached additionally in order to reach the binding target.[1a]
But how does this additional moiety interact with plasma
proteins? A new corona could be generated, thereby altering
the in vivo performance by covering and deactivating the
targeting group.
The formation of a protein corona around single-compo-
nent nanoparticles (NPs), such as polystyrene,[2c,g,4] zinc
oxide,[5] silica,[2g,5,6] gold,[2o,7] silver,[2o] and titanium dioxi-
de[2n,5] nanoparticles, has been extensively studied, while the
surface modification of nanoparticles with PEG[8] and zwit-
terionic agents[9] has been proven to effectively reduce the
protein absorption. However, the combination of “stealth”
behavior with “on top” attachment of specific targeting
groups has not been studied. The protein interactions of
a PEGylated nanocarrier before and after the attachment of
an additional targeting group are of central importance for
the generation of efficient specific cellular uptake after blood
contact. For the first time, we compare the blood plasma
interaction of PEGylated nanocarriers with that of nano-
carriers that carry mannose groups attached to the PEG
chains. Isothermal titration calorimetry (ITC), dynamic light
scattering (DLS), and cellular uptake studies before and after
incubation with human plasma were performed and the
influence on the targeting of dendritic cells was evaluated. In
addition, proteomic mass spectrometry revealed a distinct
pattern of plasma proteins still present on all of the nano-
carriers that does not hamper the specific lectin binding of
mannose.
We have been studying hydroxyethyl starch nanocapsules
(HES-NCs) as biodegradable nanocarriers intensively.[10]
They are prepared through an inverse miniemulsion
method, can be loaded with hydrophilic cargo, and their
diameters can be adjusted precisely. More recently, well-
controlled PEGylation of HES-NCs was accomplished
through a number of different methods.[11] Herein, we
extend these methods to generate nanocarriers that can be
further functionalized at the outer layer with specific target-
ing groups. PEG diisocyanate (OCN-PEG110-NCO, Mn =
5000 gmolÀ1) reacts with the surface hydroxy groups of the
polysaccharide and subsequently with the targeting groups.
After one of the isocyanate groups has reacted with the
surface, the reactivity of the other one is drastically decreased
owing to steric hindrance and loss of mobility. If an excess of
PEG diisocyanate is used, there is a further reduction in the
formation of cyclic species and the second isocyanate group
N
anomedicine is a key technology for the 21st century.
Besides the initial development of various nanocarrier
systems and the development of specific targeting, the
development of protein-repellent surfaces is of high impor-
tance. When synthetic nanocarriers enter biological fluids, it is
known that, due to high surface energy and hydrophobic
interactions, they strongly adsorb plasma proteins.[1] Many
researchers have proposed that the in vivo fate of any
nanocarrier is determined by this protein “corona”, formed
post injection, instead of the intrinsic properties of the
(mostly polymeric) nanocarrier.[2] PEGylation (PEG = poly-
[*] B. Kang, P. Okwieka, S. Schçttler, S. Winzen, Dr. K. Mohr,
Dr. V. Mailꢀnder, Prof. Dr. K. Landfester, Dr. F. R. Wurm
Max Planck Institute for Polymer Research
Ackermannweg 10, 55128 Mainz (Germany)
E-mail: landfester@mpip-mainz.mpg.de
wurm@mpip-mainz.mpg.de
P. Okwieka, Dr. V. Mailꢀnder
Department of Hematology, Medical Oncology, and Pneumology
University Medical Center Mainz
Langenbeckstr. 1, 55131 Mainz (Germany)
Dipl.-Chem. J. Langhanki, Prof. Dr. T. Opatz
Institute of Organic Chemistry, University of Mainz
Duesbergweg 10–14, 55128 Mainz (Germany)
[**] Financial support by the BMBF (Cluster CI3) and the DFG
(Colaborative Research Projct SFB1066) is highly appreciated. We
would like to thank Christine Rosenauer for the DLS measurement,
and Katja Klein for the synthesis of polystyrene nanoparticles used
in this study. F.R.W. thanks the Max Planck Graduate Center for
support.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201502398.
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remains available for further reactions. Methoxy polyethylene
glycol isocyanate (MeO-PEG110-NCO, Mn = 5000 gmolÀ1)
was used to prepare PEGylated NCs as control samples for
further studies. Quantification of the degree of PEGylation of
the NCs was conducted by NMR spectroscopy[11] and
approximately 5 ꢀ 105 PEG chains were coupled to the surface
for both mono- and difunctional PEGs (Table S1 in the
Supporting Information), which is a first indication that PEG
diisocyanate reacts selectively at one chain end without the
formation of cyclic species. To attach additional targeting
groups, different synthetic routes were investigated. Various
small biomolecules, such as folic acid,[10,12] glucose,[13] man-
nose,[14] galactose,[14a] and disaccharides,[15] are known to bind
selectively to receptors on the cell surface. The coupling of
these targeting agents to NCs often involves several reaction
steps and quantification is often challenging. Herein, a robust
method for the coupling of such small biomolecules to the
surface of nanocarriers (Scheme 1) is presented that allows
precise quantification of the number of coupled biomolecules.
ized HES-NCs are named HES-PEG5000-C2-Man and HES-
PEG5000-C1-Man, accordingly). With this simple and fast
method, approximately 2 ꢀ 105 mannose units (Table S1 in the
Supporting Information) can be attached to each NC. Note
that copper-free click chemistry was also studied and led to
a 200 times lower mannose density than the isocyanate route
(see the Supporting Information). This general method can be
used for any type of small biomolecule, as long as it carries an
amine group.
The binding affinity of the mannose-functionalized HES-
NCs for immature dendritic cells (iDCs) was investigated by
fluorescence-activated cell sorting (FACS; Figure S1 in the
Supporting Information). In a recent study, our group
demonstrated that unmodified HES-NCs show a low degree
of unspecific cell uptake.[10] Besides the low uptake of HES-
NCs “as prepared”, subsequent PEGylation of the NCs
further reduces the uptake into iDCs (Figure S1). Further-
more, the NCs modified through the click method (HES-
PEG5000-DBCO-Man) exhibited a similar low uptake into
dendritic cells as the HES-PEG5000. This is attributed to the
rather low density of targeting units per capsule (0.0028
mannose units per nm2). The interaction between the receptor
and a single carbohydrate molecule is inherently weak and
has a binding constant in the range of 103–104 LmolÀ1. Upon
increasing the number of mannose molecules that interact
with the acceptor, the binding constant increases drastically to
106–107 LmolÀ1.[18] The NCs modified by NCO coupling
(HES-PEG5000-C2-Man) exhibit a 200 times higher mannose
density compared to HES-PEG5000-DBCO-Man and thus
exposes approximately 0.55 mannose units per nm2. Interest-
ingly, the binding affinity with iDCs is still low (Figure S1B).
The reason for this could be that a free hydroxy group at the
C-2 position of the mannose molecule is essential for
interaction with the acceptor protein.[19] Only, when 2-amino-
ethyl a-d-mannopyranoside (with the amine linked to the C-
1 position) was used, a much stronger binding of HES-
PEG5000-C1-Man to iDCs compared to unmodified and
PEGylated NCs was detected (Figure S1C). These results
clearly demonstrate that when mannose is attached via a PEG
linker to HES-NCs with sufficient density, a multivalency
effect is achieved, which enables cellular uptake into iDCs.
After the successful targeting of iDCs with mannose-func-
tionalized HES-NCs under nonphysiological conditions, the
influence of human plasma on the protein interaction and
targeting efficiency was studied. First, ITC was used to
determine the protein interactions from human plasma with
the prepared HES-NCs. The HES-NCs were titrated with
diluted human plasma to measure the heat released from the
protein adsorption at the surfaces after each titration step.
The corresponding values were corrected for the heat of
dilution. The same measurement was performed with stan-
dard polystyrene (PS) nanoparticles, which are known to
exhibit a high protein binding affinity,[20] as a reference
(Figure 1).
Scheme 1. General method for the coupling of mannose to HES NCs
via isocyanate chemistry. Reactants: A: Diisocyanate-PEG (OCN-
PEG110-NCO, Mn =5000 gmolÀ1) in acetone; B: d-mannosamine or 2-
aminoethyl a-d-mannopyranoside in water, followed by evaporation of
cyclohexane and dialysis.
Owing to the fast kinetics of the reaction between
isocyanates and amines,[16] amino-functionalized mannose
derivatives are directly coupled to the available NCO
groups during the redispersion procedure without the need
for an additional protective group (Scheme 1). The amino-
functionalized carbohydrates will preferably react with the
nanocarriers before hydrolysis of the isocyanates occurs. The
amount of amine-functionalized mannose in water (before
the addition of NCs) can be quantified by the fluorescamine
assay (see the Supporting Information).[17] After the coupling
reaction, the NCs are precipitated by centrifugation and the
remaining mannose concentration in the solution is quantified
by the same method to give the coupling efficiency.
The interactions between plasma proteins and the nano-
carriers were exothermic in all cases, thus indicating protein
interaction with all of the investigated NCs. However, there is
a significant difference between hydrophobic PS nanoparti-
cles and the HES-NCs: the binding affinity of the plasma
Two different mannose derivatives were used: d-mannos-
amine and 2-aminoethyl a-d-mannopyranoside, with the
amine groups at the C-2 or the C-1 position (the functional-
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Figure 1. ITC binding isotherms for the adsorption of plasma proteins
&
to different nanocarriers systems: unmodified HES-NCs (black ),
~
^
HES-PEG5000 NCs (red ), and HES-PEG5000-C1-Man NCs (blue
)
*
compared to polystyrene nanoparticles (green ) as a reference.
proteins, which is represented by the initial slope of the
binding isotherm, is strongly reduced for HES-NCs. This
matches the results published previously by our group and
also those reported in the literature regarding the low protein
affinity of HES[21] or other carbohydrates.[22] More interest-
ingly, the three different HES-NCs exhibit similar protein
adsorption: the ITC results show very little difference in the
affinity as a result of PEGylation or additional mannose
functionalization. This is an important finding since ITC gives
insight into the protein corona of the NC and it indicates that
the attachment of mannose does not cause significantly
increased protein adsorption. Furthermore, DLS was per-
formed in undiluted plasma to detect any aggregation of the
NCs.[23] The autocorrelation functions for the NCs/plasma
mixtures can be perfectly described by the so-called force fit
(Figure S2). This means that the sum of the autocorrelation
functions of both individual components is kept constant with
only the intensity fractions of plasma and nanocarriers as free-
fit parameters.[23] The results indicate that no structures larger
than the largest size of the NCs or plasma components are
formed in the mixture. However, it has to be mentioned that
size distribution changes caused by a monolayer of adsorbed
proteins on the NC surface cannot be detected by this
method.[24] The DLS results thus prove that no larger
aggregates of NCs caused by interaction with proteins are
observed. In combination with the ITC results, the most
probable scenario would be a minor amount of protein
adsorbed to the NC surface to partially cover the surface of
the NCs. This is further supported by protein quantification
and SDS-PAGE (Figure S3). Both methods indicate low
overall protein adsorption on all of the HES-NCs in
comparison to reference PS-NPs.
Figure 2. Heatmap of the most abundant proteins in the protein
corona of HES-NC, HES-PEG5000 NCs, HES-PEG5000-C1-Man NCs
and a reference PS-NP as determined by quantitative liquid chroma-
tography coupled with mass spectrometry. Only those proteins which
constitute at least 1% of the protein corona on one of the nanocarriers
are shown. Values were calculated from the molar masses of each
protein.
protein adsorption (Figure 2 and Figure S3). Importantly, the
protein adsorption cannot be suppressed completely in any of
the cases. In order to demonstrate the accessibility of the
mannose units, specific binding to C-type lectin was analyzed.
The assay was performed before and after incubation with
human plasma (Figure 3).
Neither unmodified HES-NCs nor PEGylated HES-NCs
show any specific binding to C-type lectin before or after
incubation in human plasma (the negative control without
NCs has the same readout). The mannose-functionalized
HES-NCs exhibit binding to the enzyme, however, the
protein corona influenced the binding of C-type lectin: with
adsorbed plasma proteins, the binding of C-type lectin is
reduced but clear binding can still be detected (Figure 3).
Combined with the data from MS, this strongly indicates that
the stealth properties of the PEG layer with mannose
attached at the outer layer allow control of the biological
identity of the nanocarrier to specifically target DCs. Owing
to their role in the immune system, the targeting of immature
dendritic cells (iDCs) is biologically significant. However, as
phagocytis cells, iDCs are known to also take up NCs
unspecifically. In order to verify that after adsorption of
plasma proteins, the uptake of mannose-functionalized HES-
NCs into DCs is a real receptor-mediated phenomenon,
mature dendritic cells (mDCs) were also investigated. mDCs
More importantly, proteomic mass spectrometry data
show that the protein pattern identified is rather similar for
the different NCs but different to the PS-control sample,
which is a promising prerequisite for specific targeting in
plasma (Figure 2). A comparable composition of the protein
corona was identified on all of the HES-NCs. Only the
PEGylated NCs show a slight difference with an overall lower
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adsorption to polysaccharides including HES,[25] chitosan,[26]
hyaluronic acid.[27]
In conclusion, hydroxyethyl starch nanocarriers were
efficiently PEGylated and functionalized at the outer-layer
with amino derivatives of mannose by a convenient two-step
method that relies on sequential nucleophilic additions to
isocyanates. The influence of mannose attached to the
PEGylated NCs on the pattern of the protein corona after
plasma incubation was shown to be minimal by ITC, DLS,
SDS-PAGE, and mass spectrometry. Furthermore, cellular
uptake of the NCs by dendritic cells and a binding assay with
C-type lectin showed that the targeting moieties are acces-
sible to the biological receptors after incubation with plasma.
Taking into consideration that rather simple carbohydrates
like the disaccharide in bleomycin[15] have been proven to be
able to selectively target cancer cells, the use of a combination
of stealth nanocarriers with saccharide-based targeting moi-
eties could overcome the problem of losing the stealth effect
after the coupling of other targeting agents.
Figure 3. Binding of unmodified (HES), PEGylated (HES-PEG5000),
and mannose-functionalized (HES-PEG5000-C1-Man) HES-NCs to C-
type lectin before or after incubation with human plasma (the negative
control was measured without the addition of HES-NCs).
have a much lower ability to function as phagocytic cells and
receptor-mediated uptake was clearly demonstrated.
Figure 4 shows a similar uptake of HES-PEG5000-C1-
Man into mDCs before (Figure 4A) and after (Figure 4B)
incubation with human plasma (Figure S5 shows the corre-
sponding microscopy images). These results demonstrate that
PEGylated nanocarriers can be further functionalized with
Keywords: calorimetry · carbohydrates · drug delivery ·
nanoparticles · proteins
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Received: March 14, 2015
Published online: && &&, &&&&
Angew. Chem. Int. Ed. 2015, 54, 1 – 6
ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
5
These are not the final page numbers!

.
Angewandte
Communications
Communications
Nanocarriers
Stealth nanocariers: The blood plasma
interactions and targeting properties of
PEGylated and mannose-functionalized
hydroxyethyl starch (HES) nanocarriers
were investigated. They exhibit colloidal
stability in human plasma, low protein
adsorption, a distinct protein pattern, and
highly specific cellular uptake into den-
dritic cells both before and after contact
with human plasma.
B. Kang, P. Okwieka, S. Schçttler,
S. Winzen, J. Langhanki, K. Mohr,
T. Opatz, V. Mailꢁnder, K. Landfester,*
F. R. Wurm*
&&&& — &&&&
Carbohydrate-Based Nanocarriers
Exhibiting Specific Cell Targeting with
Minimum Influence from the Protein
Corona
6
www.angewandte.org
ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2015, 54, 1 – 6
These are not the final page numbers!