(Oligo)mannose functionalized hydroxyethyl starch nanocapsules: En route to drug delivery systems with targeting properties

Article abstract of DOI:10.1039/c3tb20138d

Hydroxyethyl starch nanocapsules (NCs) are potentially interesting hydrophilic drug delivery carriers, since they do not show non-specific interactions with the living cells. Only the presence of a targeting agent on their surface allows them to target specifically the desired site of action. In this paper, we report the synthesis and cell uptake of crosslinked hydroxyethyl starch (HES) NCs decorated with (oligo)mannose, which is an effective targeting agent for macrophage and dendritic cells. The crosslinked HES NCs were prepared via the interfacial polyaddition of HES with 2,4-toluene diisocyanate (TDI) in inverse (water-in-oil) miniemulsion and then functionalized with (oligo)mannose following two different strategies. To compare the activity and availability of a targeting agent, different types of mannose molecules such as α-d-mannopyranosylphenyl isothiocyanate, 3-O-(α-d-mannopyranosyl)-d- mannose and α3,α6-mannotriose were used for the functionalization of NCs. The availability of the mannose was unambiguously assessed by interaction with a fluorescent lectin. Moreover, the accessibility of the pilot molecule was improved by the presence of a PEG linker at the surface of the NCs. To simulate in vivo conditions, where proteins interact with nanoparticles with a possible hindrance of the accessibility to the targeting agent, the mannosylated NCs were first incubated with human serum before interaction with the fluorescent lectin. Enhancement of uptake into dendritic cells demonstrates the targeting ability in in vitro studies. The Royal Society of Chemistry 2013.

Full text of DOI:10.1039/c3tb20138d

Journal of
Materials Chemistry B
View Article Online
View Journal | View Issue
PAPER
(Oligo)mannose functionalized hydroxyethyl starch
nanocapsules: en route to drug delivery systems with
targeting properties†
Cite this: J. Mater. Chem. B, 2013, 1,
4338
a
a
ab
Helene Freichels,‡ Manfred Wagner, Patricia Okwieka, Ralf Georg Meyer,b
´ `
ab
a
a
*
¨
Volker Mailander, Katharina Landfester and Anna Musyanovych
Hydroxyethyl starch nanocapsules (NCs) are potentially interesting hydrophilic drug delivery carriers, since
they do not show non-specific interactions with the living cells. Only the presence of a targeting agent on
their surface allows them to target specifically the desired site of action. In this paper, we report the
synthesis and cell uptake of crosslinked hydroxyethyl starch (HES) NCs decorated with (oligo)mannose,
which is an effective targeting agent for macrophage and dendritic cells. The crosslinked HES NCs
were prepared via the interfacial polyaddition of HES with 2,4-toluene diisocyanate (TDI) in inverse (water-
in-oil) miniemulsion and then functionalized with (oligo)mannose following two different strategies. To
compare the activity and availability of a targeting agent, different types of mannose molecules such as
a-D-mannopyranosylphenyl isothiocyanate, 3-O-(a-D-mannopyranosyl)-D-mannose and a3,a6-mannotriose
were used for the functionalization of NCs. The availability of the mannose was unambiguously assessed
by interaction with a fluorescent lectin. Moreover, the accessibility of the pilot molecule was improved by
the presence of a PEG linker at the surface of the NCs. To simulate in vivo conditions, where proteins
interact with nanoparticles with a possible hindrance of the accessibility to the targeting agent, the
mannosylated NCs were first incubated with human serum before interaction with the fluorescent lectin.
Enhancement of uptake into dendritic cells demonstrates the targeting ability in in vitro studies.
Received 29th January 2013
Accepted 24th June 2013
DOI: 10.1039/c3tb20138d
www.rsc.org/MaterialsB
Although many different kinds of carriers decorated with
mannose are described in the literature, until now there have
Introduction
Mannosylated nanocarriers that are able to transport hydro-
philic drugs like nucleic acids, peptides or proteins have
received considerable attention for medical purposes including
vaccination, and treatment of inammatory and bacterial
infectious diseases by targeting specically macrophages1 and
dendritic cells (DCs).2–4 In this area, several systems were
described: liposomes,5–8 polyester nanoparticles (NPs),9–12 poly-
anhydride NPs,13 gelatin NPs,14,15 pH-sensitive dextran nano-
particles,16 and chitosan based complexes.17–19
been no reports that describe the synthesis of nanocapsules
(NCs) having covalently attached mannose residues. NCs consist
of an aqueous core surrounded by a thin protective polymeric
shell and are very promising carriers, since they possess a high
loading efficiency. Additionally, the drug is protected inside the
core avoiding degradation, leakage and/or rapid release of the
drug. In order to obtain targeting nanocarriers, non-specic
interaction and uptake of the nanoparticles into non-target cells
have to be minimized; only then the specicity to the target tissue
or cell type can be achieved by the molecules (or ligands) that are
attached to the nanocarrier and they can interact specically with
the target structures in tissue.
Hydroxyethyl starch (HES) is a hydroxyethylated glucose
polymer. As a medical agent HES improves the transport of
oxygen to all organs of the body, maintains the normal blood
pressure/volume and is widely used for treatment of hypo-
volemia. The enzymatic degradation of HES is reduced with
increasing molar substitution (MS).
aMax Planck Institute for Polymer Research, Ackermannweg 10, 55128 Mainz,
Germany. E-mail: musyanov@mpip-mainz.mpg.de; Fax: +49 (0)6131-379100; Tel:
+49 (0)6131-379248
bDepartment of Hematology, Medical Oncology, and Pneumology, University Medical
Center Mainz, Langenbeckstr. 1, 55131 Mainz, Germany
† Electronic supplementary information (ESI) available: Conductometric titration
curves for HES and PU NCs, calibration curve for the determination of the amine
content by reaction with uorescamine, chemical structure of 13C-labeled
mannose at position C1 and C2 and its 13C-NMR spectrum, comparative
phenol/sulfuric acid assay of mannose and mannitol. See DOI:
10.1039/c3tb20138d
Until now only a few studies have been published describing
the formulation of HES capsules. For example, HES microcap-
sules with encapsulated bovine serum albumin (BSA) were
synthesized by crosslinking with terephthaloyl chloride at the
emulsion droplet interface.20 The release of BSA from the
‡ Present address: Interactive Materials Research, DWI an der RWTH Aachen e.V.,
Forckenbeckstrabe 50, 52056 Aachen, Germany.
4338 | J. Mater. Chem. B, 2013, 1, 4338–4348
This journal is ª The Royal Society of Chemistry 2013

View Article Online
Paper
Journal of Materials Chemistry B
toluene diisocyanate (TDI) in inverse miniemulsion (Scheme 1).
Aer the synthesis, some amount of non-reacted isocyanate
groups remain on the surface of capsules and are converted into
amine groups aer redispersion of the capsules in water, as was
shown for starch24,25 and polyurethane26 NCs. These amine
groups can be used for further functionalization of NCs, e.g.,
introduction of a PEG linker via a glutaraldehyde linkage,27,28 to
improve the accessibility of the targeting unit (Scheme 2). In
this paper, two different approaches for conjugation of HES NCs
are developed (Scheme 3). Amine groups on the surface of NCs
were reacted either directly with the isothiocyanate group (ITC-
Man) or through reductive amination (di-Man and tri-Man). The
access and specicity of mannose residues was studied by their
interaction with the uorescence-labeled lectin, Galanthus
nivalis agglutinin (GNA-FITC), which is known for its specic
interaction with mannose.29 The studies described herein
provide the evidence that mannose-functionalized HES NCs
possess promising future for targeted delivery to dendritic cells.
Scheme 1 A schematic representation of the procedure for the synthesis of
crosslinked HES NCs in an inverse miniemulsion and transfer of NCs from cyclo-
hexane into the water phase.
obtained microcapsules upon enzymatic degradation was
studied in vivo (aer intraperitoneal application) and in vitro
(using melanoma cells). The use of a miniemulsion approach
makes it possible to benet from the reduced size of the
obtained nanocapsules (below 300 nm).21,22 Furthermore, it was
shown that NCs composed of HES are very promising nano-
carriers as they are biocompatible23 and do not show any non-
specic cellular interactions.24
Experimental section
Materials
In the present work, two strategies for the synthesis of
crosslinked HES NCs functionalized with different mannose
molecules, namely a-D-mannopyranosylphenyl isothiocyanate
(ITC-Man), 3-O-(a-D-mannopyranosyl)-D-mannose (di-Man), and
a3,a6-mannotriose (tri-Man), were developed. The HES NCs
were prepared by interfacial crosslinking of HES with 2,4-
Hydroxyethyl starch, HES (10 wt% solution with 154 mM NaCl,
Mw ¼ 200 000 g molꢀ1, DS ¼ 0.40, MS ¼ 0.45), was purchased
from Fresenius Kabi. The synthesis of HES starts from the
hydrolysis of corn starch (consists of amylopectin), followed by
the hydroxyethylation reaction and ultraltration to get a fraction
with exact molecular weight. Tolylene 2,4-diisocyanate, TDI
Scheme 2 Introduction of a PEG linker onto the surface of NCs.
Scheme 3 Functionalization of nanocapsules with a-D-mannopyranosyl-phenyl isothiocyanate (ITC-Man), 3-O-(a-D-mannopyranosyl)-D-mannose (di-Man) and a3,a6-
mannotriose (tri-Man).
This journal is ª The Royal Society of Chemistry 2013
J. Mater. Chem. B, 2013, 1, 4338–4348 | 4339

View Article Online
Journal of Materials Chemistry B
Paper
(Sigma, > 98%), cyclohexane (Sigma-Aldrich, > 99%), uo- conductometric titration, the excess amount of 0.1 M HCl
resceinisothiocyanate (FITC) (Fluka, > 90%), a-D-mannopyr- aqueous solution was added to the aqueous dispersion of HES (or
anosylphenyl isothiocyanate (ITC-Man) (Sigma), DMSO (Sigma- PU) NCs with a solid content of 0.25 wt%. Aer 20 min of stirring,
Aldrich, 99.9%), GNA-FITC (Linaris), phenol (Acros Organics, the titration was started by slow addition of the standard 0.1 M
99+%), sulfuric acid (Sigma-Aldrich, 95–97%), 1,6-hexanediol NaOH solution to NC dispersion using a Titronic universal unit
(Sigma-Aldrich, 99%), 3-O-(a-D-mannopyranosyl)-D-mannose (di- (SCHOTT Instruments GmbH). The conductivity measurements
ꢁ
Man) (Carbosynth Limited), a3,a6-mannotriose (tri-Man) (Car- were performed at 22 C in the pH range from 2.5 to 12.5. The
bosynth Limited), 2-picoline borane (Sigma-Aldrich), citric acid amount of amine groups per gram of capsules was calculated
(Sigma-Aldrich), mannose (Fluka), PEG bis(amine) Mw ¼ 2000 g from the conductivity curve as a function of consumed NaOH
molꢀ1 (Aldrich), glutaraldehyde (25% solution, Serva GmbH), solution. The uorescence titration method was based on the
anthranilic acid (Acros Organic), and D-[1,2-13C2] mannose reaction of uorescamine with primary amine groups, yielding a
(Omicron Biochemicals, Inc) were used as received. Human highly uorescent product.27,32 The amount of amine groups was
serum (HS, 69 g molꢀ1) was collected from healthy blood donors. measured directly on the HES NCs. The calibration curve
The lipophilic block copolymer poly[(ethylene-co-butylene)-b- (Fig. S2†) was obtained by mixing known concentrations of hex-
(ethylene oxide)], P(E/B-b-EO), consisting of a poly(ethylene-co- ylamine and polystyrene particles (240 nm in diameter), stabilized
butylene) block (Mw ¼ 3700 g molꢀ1) and a poly(ethylene oxide) by the same amount of Lutensol AT50 as HES NCs. Polystyrene
block (Mw ¼ 3600 g molꢀ1), was synthesized starting from nanoparticles were used in order to have the same turbidity in the
u-hydroxypoly(ethylene-co-butylene), which was dissolved in calibration sample as it is in the measured samples of HES NCs,
toluene, by adding ethylene oxide under the typical conditions of thus avoiding the error in the obtained values due to the light
anionic polymerization.30,31 The non-ionic surfactant Lutensol scattering caused by the presence of NCs. The concentration
AT50, which is a poly(ethylene oxide)-hexadecyl ether with an (number) of polystyrene particles in the calibration samples was
ethylene oxide (EO) block length of about 50 units, was a gi from adjusted to the concentration (number) of NCs used in the ana-
BASF. MilliQ water was used throughout the work.
lysed sample. Briey, 0.1 mL of NC dispersion (solid content
0.15 wt%) was mixed with 2.9 mL of borate buffer (0.1 M, pH 9.2)
and then 1 mL of uorescamine solution (0.3 g Lꢀ1 in acetone)
was added. The excitation wavelength was 410 nm, and the
emission was measured at a wavelength of 470 nm.
Preparation of NCs
The HES and PU NCs were prepared as previously reported.24–26
Briey, for HES NCs: 1.4 g of HES solution (10 wt%) was mixed
with 7.5 g of cyclohexane containing 100 mg of P(E/B-b-EO). For
the synthesis of PU NCs the dispersed phase consisting of 0.07 g of
1,6-hexanediol, 0.015 g of sodium chloride and 1.2 g of MilliQ
Functionalization of NCs with a PEG bis(amine) linker
(Mw ¼ 2000 g molꢀ1) (Scheme 2)
water was mixed with 7.5 g of cyclohexane containing 100 mg of The dispersion of NCs was dialyzed for 2 h against PBS pH 7.4
P(E/B-b-EO). The obtained mixture (in both cases of NCs) was containing 0.1 wt% Lutensol AT50. Then 20 eq. of glutaraldehyde
stirred over 1 h at 25 ꢁC and then sonicated for 180 s at 70% (with respect to the amount of amine groups on NCs) was added
amplitude in a pulse regime (20 s sonication, 10 s pause) using a and the dispersion was stirred for 4 h at room temperature. The
Branson Sonier W450-Digital and a 1/200 tip. The sonication was capsules were puried from the non-reacted glutaraldehyde
performed under ice cooling in order to prevent evaporation of the through dialysis against Lutensol AT50 (0.1 wt%) aqueous solu-
solvent. A solution consisting of 5 g of cyclohexane, 30 mg of P(E/B- tion (2 days) and subsequently with PBS containing Lutensol
b-EO), and 100 mg of TDI was prepared and added dropwise over 5 AT50 (0.1 wt%) (2 h). The dispersion was diluted twice, then
min to the obtained miniemulsion at room temperature. The 50 eq. of PEG bis(amine) (with respect to the amount of amine
mixture was stirred overnight and aer that it was puried twice groups on NCs) and 50 eq. of 2-picoline borane, dissolved in
by centrifugation/redispersion in cyclohexane at 4000 rpm for 20 DMSO, were added and the dispersion was stirred for 16 h before
min. Then, 10ꢀ3 mol of TDI per g of NCs (solid content 0.6 wt%) purication by dialysis against Lutesol AT50 solution (0.1 wt%).
were added dropwise to the dispersion and stirred at room
temperature overnight. The NCs were puried twice by centrifu-
gation at 4000 rpm for 20 min and redispersed in cyclohexane. To
transfer the capsules to an aqueous medium, the following
procedure was used: 0.15 mL of NCs in cyclohexane (solid content
0.8 wt%) were slowly added to 2.5 mL of 0.1 wt% of Lutensol AT50
in an ultrasonication bath and kept for 3 min. Then the dispersion
was sonicated for 1 min at 10% amplitude in a pulse regime (5 s
pulse, 5 s pause). The NC dispersion was stirred in an open vial
overnight at 25 ꢁC in order to evaporate the cyclohexane.
Functionalization of NCs with uoresceinisothiocyanate (FITC)
and a-D-mannopyranosylphenyl isothiocyanate (ITC-Man)
The NCs were dialyzed for 13 h against 0.1 M carbonate buffer
(pH 9), containing 0.1 wt% of Lutensol AT50. Then, 10 eq. of
isothiocyanate molecules (with respect to the amount of amine
groups on NCs), dissolved in DMSO (maximum of 5% of DMSO in
the reaction mixture), were added. Aer 20 h of reaction, NCs were
puried by dialysis against Lutensol AT50 solution (0.1 wt%).
FITC-functionalized NCs were used for the calculation of the
functionalization yield. Aer synthesis, FITC-functionalized NCs
were centrifuged and the amount of non-reacted FITC was
Quantication of amine groups
The concentration of amine groups on the surface of NCs was determined by uorescence spectroscopy. The excitation wave-
determined by conductometric and uorescence titrations. For length was 490 nm, and the emission was measured at 525 nm.
4340 | J. Mater. Chem. B, 2013, 1, 4338–4348
This journal is ª The Royal Society of Chemistry 2013

View Article Online
Paper
Journal of Materials Chemistry B
Functionalization of NCs with 3-O-(a-D-mannopyranosyl)-D-
mannose (di-Man) and a3,a6-mannotriose (tri-Man)
Characterization
The size and size distribution of NCs were measured by
50 mg of citric acid per g of NC dispersion (0.15 wt%) was added dynamic light scattering (DLS) using a Nicomp particle sizer
to NC dispersion. Then, the pH was adjusted to pH 4 with 1 N (model 370, PSS Santa Barbara, CA, USA) at a xed scattering
ꢁ
NaOH and 2 eq. of carbohydrate dissolved in water and 20 eq. of angle of 90ꢁ at 23 C. The zeta potential was measured using a
2-picoline borane (dissolved in DMSO, with respect to the Zeta Nanosizer (Malvern Instruments) at 23 ꢁC. The samples
amount of amine groups on NCs) were added. The reaction were diluted in 10ꢀ3 M KCl. The morphology of the capsules was
mixture was stirred at 55 ꢁC for 3 days, and 20 eq. of 2-picoline studied by scanning electron microscopy (SEM) and trans-
borane were added every 12 h. The NCs were puried by dialysis mission electron microscopy (TEM). One droplet of the diluted
against Lutensol AT50 0.1 wt%.
sample (solid content about 0.01 wt%) was placed onto a silica
wafer (for SEM) or onto a 300 carbon-coated copper mesh (for
TEM) and dried under ambient conditions. SEM images were
recorded using a eld emission scanning electron microscope
LEO GEMINI 1530 at an acceleration voltage of 0.1 kV. TEM
studies were carried out with a Philips EM400 electron micro-
scope operating at an acceleration voltage of 80 kV. UV-visible
absorbance and uorescence measurements were performed
Quantication of mannose on the surface of NCs
The phenol–sulfuric acid colorimetric assay is known to be very
sensitive to quantify the amount of carbohydrate.33 However, in the
presence of phenol–sulfuric acid, both HES (shell of NCs) and
mannose will undergo similar reactions and lead to the formation
of furan derivatives. Therefore HES NCs, which were treated in the
same way as mannose functionalized HES NCs, but without addi-
tion of the mannose derivative, were used as a reference sample.
In addition, polyurethane (PU) NCs were synthesized and used
as the second reference NCs for the mannose quantication. The
amount of sugar was determined by measuring the absorbance at
486 nm and using the calibration curve obtained with a known
amount of mannose or 3-O-(a-D-mannopyranosyl)-D-mannose (di-
Man). In the case of HES NCs the value was calculated as a
difference between the values obtained for the mannosylated and
non-mannosylated HES NCs. The absorbance value of the
colorimetric assay performed on PU NCs, which were treated
under the same conditions, but without the addition of mannose
derivatives, was negative (ꢀ3.36 ꢂ 10ꢀ7 mol gꢀ1 NCs) thus indi-
cating that PU NCs do not contribute to any absorption under the
working coupling conditions.
using
a M1000 multimode Plate reader (Tecan GmbH)
controlled via i-Control soware (Tecan GmbH, version
1.6.19.2). 13C-NMR spectra of the chemicals dissolved in D2O
were obtained on an AVANCE 500 MHz Bruker. The 13C-NMR
experiments were performed with a 5 mm BBFO z-gradient
probe on the 500 MHz system. The quantitative 13C-NMR
studies were carried out with an external reference compound
(4,4-dimethyl-4-silapentane-1-sulfonic acid (DSS) in D2O) in a
special tube (diameter 1.3 mm in the centre of a 5 mm tube).
The temperature during the measurements (298.3 K or 353 K)
was dened with a standard 1H methanol or glycol NMR
sample. The control of the temperature was realized with a VTU
(variable temperature unit) and an accuracy of ꢃ 0.1 K, which
was checked with the standard Bruker Topspin 2.1 soware.
The spectra were obtained with p/2-pulse lengths of 13 ms (13C)
and a sweep width of 220 ppm for 13C. A relaxation delay of 3 s
was chosen for the 13C experiment and the used pulse program
for quantitative spectra was inverse gated decoupling. The
relaxation time (T1) of the labeled carbons that is equal to 1.01 s
in the mannose derivative was measured with the inversion
recovery method and the Carr–Purcell–Meiboom–Gill (CPMG)
method was used to determine the relaxation time (T2). Carbon
spectra were referenced using DSS as a standard at 0 ppm. The
13C-NMR study of the reductive amination between model
compounds was realized as follows: anthranilic acid (0.5 mg,
3.8 ꢂ 10ꢀ3 mmol) was added to a solution of citric acid (25 mg
in 500 mL of D2O adjusted to pH 4 with 1 N NaOH), followed by
10 mL of 140 mg mLꢀ1 D2O 13C labeled mannose (2 eq., 7.7 ꢂ
10ꢀ3 mmol, 1.4 mg) and 100 mL of 83 mg mLꢀ1 2-picoline
borane in DMSO (77.7 ꢂ 10ꢀ3 mmol, 8.3 mg, 20 eq.).
Briey, 0.5 mL of 5% aqueous phenol solution and 2.5 mL of
concentrated sulfuric acid were rapidly added to 0.5 mL of
redispersed NCs (0.15 wt%). The vial was allowed to stand for 10
min at room temperature, before it was vigorously shaken and
placed in a water bath at 23 ꢁC for 20 min before measuring the
absorbance at 486 nm.
Specic interaction of mannose with uorescence-labeled
Galanthus nivalis agglutinin (GNA-FITC)
The access and specicity of mannose on the surface of NCs was
studied with uorescence-labeled Galanthus nivalis agglutinin
(GNA-FITC). 30 mL of GNA-FITC in HEPES buffer (0.25 mg mLꢀ1
)
were added to 400 mL of HES NCs (0.1 wt%) in HEPES buffer
(0.1 M, pH 7.5). The dispersion was stirred at room temperature
in the dark for 3 h and then overnight at +4 ꢁC. The separation
Generation of immature dendritic cells (iDCs)
of non-reacted GNA-FITC was done by centrifugation at 4000
ꢁ
rpm for 20 min at 15 C. Aer centrifugation, the supernatant Peripheral blood mononuclear cells (PBMCs) were puried
was replaced by the HEPES buffer and the capsules were from healthy human donor buffy coats by standard Ficoll
redispersed using vortex. The uorescence intensity of the separation. Donor blood was obtained according to the decla-
supernatant was measured at 520 nm aer excitation at 494 nm. ration of Helsinki. Immature DCs (iDCs) were derived from
For the experiment in the presence of HS, 400 mL of HES NC monocytes selected by plastic adherence. 1.5 ꢂ 107 PBMCs were
dispersion were incubated with 50 mL of HS (69 g Lꢀ1) for 1 h at seeded in one well of a 6-well plate and allowed to adhere in a
ꢁ
37 C before interaction with GNA-FITC.
humidied environment at 37 ꢁC with 5% CO2 for 1 h in 3 mL of
This journal is ª The Royal Society of Chemistry 2013
J. Mater. Chem. B, 2013, 1, 4338–4348 | 4341

View Article Online
Journal of Materials Chemistry B
Paper
DC-medium (RMPI with 2% heat-inactivated pooled human with the oil-soluble block copolymer surfactant [P(B/E-b-EO)].
serum (HS), 100 U mLꢀ1 streptomycin (Sigma)). Non-adhering Miniemulsication by ultrasonication led to the formation of
cells were removed by washing three times with phosphate nanosized kinetically stable droplets. The addition of TDI
buffered saline (PBS, Invitrogen). Monocytes were cultured for allowed the reaction of the hydroxyl group from HES molecules
two days in DC-medium containing 800 U mLꢀ1 granulocyte/ with the isocyanate groups from TDI, and results in the
macrophage-colony stimulating factor (GM-CSF, Genzyme) and formation of a crosslinked polymeric shell. The obtained
500 U mLꢀ1 interleukin (IL)-4 (PromoKine) at 37 ꢁC/5% CO2. nanoparticulates have the core–shell structure: water sur-
The medium was replaced by DC-medium supplemented with rounded by the crosslinked HES. SEM and TEM studies per-
1600 U mLꢀ1 GM-CSF and 1000 U mLꢀ1 IL-4 on days 3 and 5. formed on the NC dispersion reveal the core–shell morphology
The cells were harvested on day 6 by incubation with PBS/EDTA (Fig. 1). The shell thickness was measured to be in the range
ꢁ
(2 mM) for 10 min at 37 C and subsequent washing with cold between 10 and 20 nm.
PBS. For treatment with nanocapsules, cryopreserved iDCs were
thawed in DC medium.
Aer the synthesis and thesecond addition of TDI (to
increase the content of the amine groups on the NC surface),
the capsules were redispersed in a nonionic surfactant solution
(Lutensol AT50 0.1 wt%). The hydrodynamic average sizes of the
NCs aer different reaction steps are shown in Fig. 2. As model
NCs for the coupling studies of different mannose molecules,
PU NCs were synthesized and characterized as well.
The amount of amine groups present on the surface of NCs
was determined by two different methods, i.e. conductometric
and uorescent titrations.
The conductometric titration was performed by adding an
excess of hydrochloric acid in order to protonate the entire
amine groups and follow the conductivity aer each addition of
sodium hydroxide. From the titration curves (Fig. S1†) and the
solid content of NCs, the amount of amine groups on the
surface of NCs was calculated for both types of NCs (Table 1).
Loading of iDCs with nanocapsules
1.5 ꢂ 105 iDCs were seeded per well of a 48-well plate containing
1 mL of DC-Medium with 1600 U mLꢀ1 GM-CSF and 500 U mLꢀ1
IL-4. Aer 1 h of incubation at 37 ꢁC, 300 mg mLꢀ1 of each
nanocapsule were added per well. Cells were incubated with
ꢁ
nanocapsules for 16 h at 37 C/5% CO2. Aerwards the nano-
capsule containing medium was discarded and the cells were
washed two times with pre-warmed DC-medium. Finally iDCs
were harvested by multiple washing with cold PBS.
Analysis of nanocapsule-uptake by ow cytometry
Flow cytometry was performed using a LSR-Fortessa FACS
analyzer equipped with FACSDivaTM soware (both Becton
Dickinson). The uorescence intensity of nanocapsules carrying
the dye SR101 was measured using a 586/15 nm bandpass lter
with excitation by a 100 mW/561 nm laser. iDCs were stained
with a FITC-conjugated monoclonal antibody against CD11c
(Miltenyi Biotec),
a DC specic expression marker and
measured with a 530/30 nm bandpass lter with excitation by a
100 mW/488 nm laser. NC uptake was assessed by using FlowJo
7.6.5 (Tree Star Inc.).
Confocal laser scanning microscopy (CLSM) of NC loaded
iDCs
Fig. 1 SEM (left) and TEM (right) images of crosslinked HES NCs from the
cyclohexane phase.
To conrm binding and intracellular localization of NCs in
iDCs, a Zeiss LSM 710-UV confocal laser scanning microscope
equipped with Zeiss LSM Image Examiner soware (Version
3.2.0.115, both Carl Zeiss) was used. iDCs were loaded and
incubated with NCs as described above. The cytoplasm of the
cells was stained using CellTrackerꢀ Green CMFDA (Life
Technologies) and the nucleus was stained with DRAQ-5 (New
England Biolabs). About 105 DCs were resuspended in PBS and
transferred onto a Lab-Tek 8-well chamber slide (Thermo Fisher
Scientic).
Results and discussion
Preparation and characterization of functionalized HES NCs
HES-based NCs were prepared via an interfacial polyaddition
reaction in inverse miniemulsion (Scheme 1) as previously
described.26 The aqueous dispersed phase consisted of HES
Fig. 2 Average hydrodynamic diameter of PU and HES NCs after different
solution and the continuous phase consisted of cyclohexane reaction steps.
4342 | J. Mater. Chem. B, 2013, 1, 4338–4348
This journal is ª The Royal Society of Chemistry 2013

View Article Online
Paper
Journal of Materials Chemistry B
Table 1 Amount of amine groups on the surface of PU and HES NCs determined
by conductometric and fluorescent titrations
glutaraldehyde. Subsequently, about 83% of the initial amount
of the amine groups were determined aer reaction with PEG
bis(amine). We assume that the residual 17% are not detectable
due to the possible reaction of PEG bis(amine) with both ends,
thus resulting in loop formation.
HES NCs
PU NCs
Conduct. Fluorescence Conduct. Fluorescence
titration
titration
1.14
titration
1.20
titration
1.08
Mol NH2 per g 1.59
NCs (10ꢀ5
)
Functionalization of NCs with uorescein isothiocyanate (FITC)
and a-D-mannopyranosylphenyl isothiocyanate (ITC-Man)
NH2 per NC
78 300
0.40
56 100
0.29
59 000
0.30
53 200
0.27
NH2 per nm2
As isothiocyanate reacts directly with amine groups, the amine
groups at the surface of NCs were used to react with commer-
cially available mannose isothiocyanate (ITC-Man) (Scheme 3).
For characterization and quantication, uorescein iso-
thiocyanate (FITC) was chosen as a model molecule for the
coupling on PU (used as a reference sample) and HES NCs, that
was realized in a 0.1 M carbonate buffer with 10 eq. of FITC in a
DMSO solution (5% volume with respect to the water volume).
Aer reaction the NC dispersion was centrifuged and the
amount of non-reacted FITC was determined in the supernatant
by uorescence spectroscopy. For both kinds of NCs, the
amount of coupled FITC is similar to the amount of amine
groups on the surface (Table 2), meaning that all amine groups
are accessible for the reaction with FITC. Moreover, the chosen
conditions are mild enough to avoid the degradation or aggre-
gation of NCs, as proved by DLS measurements (Fig. 1). In fact,
the size, i.e., 231 and 185 nm for PU and HES NCs respectively,
remains similar aer the reaction.
The uorescent titration also allows the determination of the
amine content. In this method, uorescamine reacts with the
amine group to form a highly uorescent compound.27,32
A comparison of the uorescence intensity of the NC sample
with a calibration curve permits the quantication of amine
groups on the NC surface (Table 1). From the obtained results it
could be seen that the amounts of amine groups determined by
conductometric titration are higher than those obtained from
the uorescent titration. On one hand, this might be due to the
difference in size of the reagents. The size of uorescamine
molecules is bigger as compared to the size of Na+ ions, and
therefore the ions can reach more amine groups than uo-
rescamine. On the other hand, it could be also due to quenching
of the uorescence (aer reaction with the uorescamine) when
the amine groups are placed very near to each other.
Next, a-D-mannopyranosylphenyl isothiocyanate (ITC-Man)
was coupled onto the surface of NCs. Aer the reaction under
the same conditions as for FITC, the excess of mannose was
Functionalization of NCs with a PEG-bis(amine) linker
The functionalization of the NC surface with a PEG linker was removed from the sample by repetitive centrifugation/redis-
realized in a two-step strategy (Scheme 2). In the rst step, the persion and the quantication of the mannose residues on both
amine group was reacted with glutaraldehyde to form a Schiff types of NCs was performed by the phenol–sulfuric acid assay.
base. In the second step, PEG-bis(amine) (Mw ¼ 2000 g molꢀ1
)
In the case of HES NCs, due to the overlap of the signals from
was reacted with the aldehyde group by reductive amination to mannose and HES, the value from HES NCs, which were treated
give NCs with a PEG linker possessing an amine group at the in the same way as mannose functionalized HES NCs, but
end of the molecule. As the chemicals used are bifunctional, without addition of the mannose derivative, was taken into
crosslinking between NCs was avoided by adding an excess of consideration during the data evaluation.
glutaraldehyde (20 times) and PEG-bis(amine) (50 times).
The amount of mannose measured on HES and PU NCs
Indeed, the DLS measurements did not indicate the presence of corresponds to 1.42 ꢂ 10ꢀ5 mol of mannose per g of NCs and
aggregates of NCs aer modication. Moreover, as expected, a 1.53 ꢂ 10ꢀ5 mol of mannose per g of NCs, respectively. The
slight increase of the size was observed by DLS aer coupling of values for ITC-Man functionalized NCs are in accordance with
PEG-bis(amine) (185 and 200 nm before and aer coupling, the amount of amine groups on the surface of NCs (Table 1).
respectively). The amount of surface amine groups remaining The amount of ITC-Man on NCs possessing a PEG linker is
aer each step was determined by uorescent titration. This test slightly lower, which might be due to the reaction of PEG with
revealed the disappearance of amine groups aer reaction with both ends, thus reducing the amount of available amine groups.
Table 2 Amount of FITC and ITC-Man molecules coupled to HES and PU NCs
HES NCs
FITC
PU NCs
FITC
ITC-Man
PEG + ITC-Man
ITC-Man
PEG + ITC-Man
Mol of molecules per g NCs (10ꢀ5
Molecules per NC
)
1.78
87 000
0.45
1.42
68 000
0.34
0.81
41 000
0.21
1.44
70 000
0.36
1.53
75 000
0.37
1.17
58 000
0.28
Molecules per nm2
This journal is ª The Royal Society of Chemistry 2013
J. Mater. Chem. B, 2013, 1, 4338–4348 | 4343

View Article Online
Journal of Materials Chemistry B
Paper
Functionalization of NCs with 3-O-(a-D-mannopyranosyl)-D-
mannose (di-Man) and a3, a6-mannotriose (tri-Man)
Functionalization of HES NCs with di- and tri-Man occurs
between the amine group on the NC surface and the aldehyde
group present on the acyclic form of the sugar (Scheme 3).34
This form is in equilibrium not only with the cyclic pyrannoses
and furanoses, but also with the hydrate form of the aldehyde.35
This strategy has the potential to be extended to every (oligo)-
sugar and it has been extensively used for the labeling of glycan,
for their identication and quantication36–38 as well as for the
immobilization of sugars on the surface plasmon resonance
(SPR) chips.39 The key factor for success of this reaction is the
presence of the acyclic form. The acetalization equilibrium
between the cyclic and the acyclic form is catalyzed by carboxylic
acid. Evangelista et al.40 have shown that malic, citric and
malonic acids are the most efficient carboxylic acids for this
function. In this work, citric acid was chosen as a catalyst.
Another key factor is the reduction of the Schiff base. Recent
publications proved the efficacy of 2-picoline borane for this
task.37,41,42 Moreover, this reducing agent has the advantage over
sodium cyanoborohydride of being non-toxic and therefore, it
was used in the present studies.
13C-NMR studies of reductive amination reaction
The reductive amination of mannose with amine was rst
studied with a model aromatic amine, anthranilic acid, which
possesses a structure similar to the amine from the non-reacted
and then hydrolyzed isocyanate group at the surface of the NCs.
The reaction was performed in water with citric acid (0.26 M) at
pH 4 and 55 ꢁC. To be able to quantify the reaction, 13C labeled
mannose was used for this study (Fig. S3†).
Fig. 3 Quantitative 13C-NMR (125 MHz, D2O, 353 K) spectra from the reaction
kinetics between 13C labeled mannose (2 eq. with respect to the amount of amine
groups that are present in the medium) and anthranilic acid in the aqueous citric
acid medium (pH 4) with 2-picoline borane as a reducing agent (20 eq.). The blue
and red asterisks show the characteristic carbon peaks of the labeled mannose (at
positions C1 and C2, respectively) and the orange and green – of the end-product.
The attribution of the other peaks is given in Fig. 4.
A
kinetic study was realized by quantitative 13C-NMR
measurements using the pulse sequence, where the Overhauser
effect is suppressed.43,44 The measurements revealed that aer 20
h of reaction the spectrum did not change anymore (Fig. 3).
Moreover, aer 5 h, in addition to the peaks from 13C labeled
mannose (71 and 94 ppm), from the citric acid (170–185, 75, 44
ppm), from DMSO (39 ppm), from DSS (external standard, 54, 19,
15 and ꢀ2 ppm), new peaks appeared at 70 and 49 ppm (orange addition this molecule does not possess any labeled carbon. This
and green asterisks in Fig. 3). These peaks are attributed to the experiment thus conrmed that the reaction is possible under
chemical shis of C1 and C2 of 13C labeled mannose aer reac- the chosen conditions. However, due to the hydrolysis of
tion with the amine group. The integration values of these peaks 2-picoline borane, the reducing agent was added in several steps
increase with time and nally stabilize (15 h of reaction). A to increase the efficiency of reductive amination.
comparison of the integration at 70 ppm with the integration of
Since the spin–spin relaxation time (T2) of mannose coupled
mannose at 94 ppm permits the determination of a reaction yield onto the surface of the NCs is too short, which leads to broad or
of around 66%. A closer look at the spectrum shows the vanished peaks, it was not possible to perform 13C quantitative
appearance of new peaks aer 10 h of reaction in the aromatic NMR on the HES NCs. Therefore, PU NCs were used to deter-
region (120–160 ppm). These peaks are attributed to 2-methyl- mine the coupling yield. 3-O-(a-D-Mannopyranosyl)-D-man-
pyridine, due to the hydrolysis of the reducing agent, 2-picoline nopyranose (di-Man) was coupled to the PU NCs. Aer 1, 2 and 3
borane. Indeed, these peaks were not visible aer 5 h of reaction. days of reaction, a sample was picked up, washed by repetitive
To support this hypothesis, the 13C-NMR spectrum (Fig. 4) of centrifugation/redispersion and freeze-dried. Quantication of
2-picoline borane (treated under the conditions of the reductive the mannose residue on the PU NCs was performed by the
amination) was recorded aer 12 h and revealed the presence of phenol–sulfuric acid assay as described in the experimental
the same peaks. It should be noted that the characteristic peaks part. In the present case, a disaccharide is graed onto NCs and
from the anthranilic acid are not visible in the 13C spectra, it remains in the form of monosaccharide aer the reaction
because the concentration of this molecule is too low and in (Scheme 3).
4344 | J. Mater. Chem. B, 2013, 1, 4338–4348
This journal is ª The Royal Society of Chemistry 2013

View Article Online
Paper
Journal of Materials Chemistry B
uorescence intensity became more signicant for mannose-
coated NCs with a PEG linker, due to the better accessibility of
the targeting agent on the surface of the NCs. Even if the
binding affinity for mannose is low (e.g. binding constꢀa1nt for:
methyl-a-mannoside is 41 Mꢀ1
;
45 di-Man is 3.3 ꢂ 103 M
;
46 tri-
Man is 3.3 ꢂ 103 Mꢀ1 (ref. 46)), the interaction was possible due
to the multivalence or cluster-glycoside effects such as the
higher binding affinity and specicity observed for a multiva-
lent ligand than for the sum of its parts.47,48
A comparison of different mannose molecules coupled onto
the surface shows a better interaction for 3-O-(a-D-mannopyr-
anosyl)-D-mannose (di-Man) and a3,a6-mannotriose (tri-Man)
in comparison to ITC-Man coated NCs in spite of a lower
density. This observation reects the signicantly higher
binding affinity of the lectin for these sugars, despite the fact
that in the present case one sugar is in the open form.
This might be due to the participation of some of the
hydroxyl groups in the hydrogen bonding with amino acid from
the lectin thus favoring the interaction.49 Similar uorescent
values for di- and tri-Man coated NCs are obtained for the NCs
with and without a PEG linker, because the binding constants
are quite similar.
Fig. 4 Quantitative 13C-NMR (125 MHz, D2O, 353 K) spectrum from 2-picoline
borane under the conditions of the reductive amination reaction. Peaks marked
with black, blue and red asterisks are attributed to the carbon from citric acid, DSS
(external standard) and DMSO, respectively.
Since the response of mannitol in comparison to mannose is
of the same order of magnitude as water in this colorimetric
assay (Fig. S4†), the values for different PU NC samples taken
aer each day of the reaction were compared with a calibration
curve obtained from mannose. The blank value, obtained from
PU NCs treated under the same conditions, but without addi-
tion of di-Man, was subtracted from the value of each sample.
The comparison with the initial amine content shows a
coupling yield that increases during the reaction: 8.5%, 25%,
39% aer 1, 2 and 3 days, respectively. This yield signied that
each PU NC possesses around 24 000 molecules of mannose on
its surface (0.12 molecules per nm2) aer 3 days of reaction. The
same procedure (3 days of reaction) was employed on HES and
PU NCs with and without a PEG linker. The obtained results (see
Table 3) show that the type of coupled sugar does not inuence
the coupling yield.
To mimic the in vivo conditions an additional set of experi-
ments was performed by incubating the NCs with human serum
before interaction with the uorescent lectin. Indeed, it is well
Recognition of surface-exposed mannose by interaction with a
uorescent lectin
The ability of mannose-functionalized HES NCs to interact with
the FITC labeled Galanthus nivalis agglutinin (GNA-FITC) was Fig.
investigated as a function of the mannose type that was coupled
onto the surface, the density of mannose and the presence of a
PEG linker. As seen in Fig. 5, the uorescence intensity is always
higher for mannose coated NCs as for the blank, proving the
5
Fluorescence of washed NC dispersion measured after interaction
between mannosylated HES NCs and GNA-FITC. The legend of the X-axis corre-
sponds to the following samples: (A) non-modified HES NCs; (B) HES NCs with ITC-
Man; (C) HES NCs with di-Man; (D) HES NCs with tri-Man; (E) HES NCs with a PEG
linker; (F) HES NCs with PEG/ITC-man; (G) HES NCs with PEG/di-Man; (H) HES NCs
with PEG/tri-Man. Di-Man, tri-Man, PEG correspond to 3-O-(a-D-mannopyr-
availability of sugar to bind with the lectin. Moreover, anosyl)-D-mannose, a3,a6-mannotriose and the PEG linker, respectively.
Table 3 Amount of 3-O-(a-D-mannopyranosyl)-D-mannose (di-Man) and a3,a6-mannotriose (tri-Man) coupled to HES and PU NCs
HES NCs without a
PEG linker
HES NCs with a PEG
linker
PU NCs without a
PEG linker
PU NCs with a PEG
linker
tri-Man
di-Man
tri-Man
di-Man
tri-Man
di-Man
tri-Man
di-Man
Mol of molecules per g NCs (10ꢀ6
Molecules per NC
)
2.83
13 000
0.07
3.24
15 000
0.08
3.26
16 000
0.08
3.67
17 000
0.09
4.45
22 000
0.11
5.24
26 000
0.13
4.79
23 000
0.12
5.13
25 000
0.13
Molecules per nm2
This journal is ª The Royal Society of Chemistry 2013
J. Mater. Chem. B, 2013, 1, 4338–4348 | 4345

View Article Online
Journal of Materials Chemistry B
Paper
Fig. 8 Confocal laser scanning microscopy of NCs loaded iDCs (maximum intensity
projection). The cytoplasm is stained in green, nuclear membrane in blue and NCs in
red. Non-modified HES NCs were only non-specifically attached to the cells (A),
whereas mannosylated HES/tri-Man NCs were taken up to a much greater extent (B).
Fig.
6
Fluorescence on washed NC dispersion measured after interaction
between mannosylated HES NCs and GNA-FITC without incubation with HS (dark
grey columns) and with incubation with HS (light grey columns).
Interaction of tri-Man functionalized HES NCs with dendritic
cells
known that proteins present in the biological medium interact
with the nanoparticles very fast.50 Therefore, this additional
step is important before testing these NCs in vivo. The uores-
cence intensities for NCs pre-incubated with the human serum
(HS) are lower than those for the NCs that were not incubated
with HS (Fig. 6).
In order to demonstrate the effectiveness of the mannosylated
NCs a cell culture experiment was performed. Dendritic cells are
one of the most interesting cell populations in immunological
applications and have been investigated widely e.g., for vacci-
nation trials and are also targets in other immunological
diseases.51,52 The uptake of NCs is highly enhanced by the
mannose on the surface of NCs as has been investigated by ow
cytometry and is demonstrated in Fig. 7. By using confocal laser
scanning microscopy (CLSM) we demonstrate that mannosy-
lated NCs are indeed taken up into the target cells (Fig. 8).
This difference was ascribed to the partial shielding of the
mannose molecules by the adsorption of the protein. Indeed,
the NCs are negatively charged (zeta potential around ꢀ15 mV)
and at physiological pH (pH 7.2), most of the proteins are above
their isoelectric point, leading to the electrostatic interaction
between the proteins and NCs. Nevertheless, the interaction
with the lectin was still possible, since a signicant difference in
uorescence is observed between the non-modied and the
mannosylated HES NCs. The observed tendency is similar to
that of the experiment without incubation with HS, i.e., a better
availability of the 3-O-(a-D-mannopyranosyl)-D-mannopyranose
(di-Man) was observed in the presence of a PEG linker. This
experiment proved unambiguously that despite the presence of
proteins, the targeting agent is still available for the interaction
with a receptor.
Conclusions
Taking advantage of the residual amine group from the cross-
linking coupling reaction for the formation of the NCs, two
strategies for the coupling of different (oligo)mannose mole-
cules onto the surface of the NCs were developed and charac-
terized. The rst relies on the reaction of the amine with the
isothiocyanate function present on mannose (ITC-Man), the
second with the aldehyde group of the open form of 3-O-(a-D-
mannopyranosyl)-D-mannose (di-Man) and a3,a6-mannotriose
(tri-Man). The rst strategy allowed a higher functionalization
of the amine present on the NCs by mannose (100% vs. ꢄ40%),
while the second is more versatile, since all saccharide mole-
cules possessing a reductive sugar can be coupled.
The interaction of these functionalized NCs with a uorescent
lectin shows the availability of the mannose coupled by both
strategies to interact with a model receptor. A better binding was
observed with di- and tri-Man coupled onto the NC surface, due
to the signicantly higher binding affinity of the lectin for these
sugars in comparison to mannose (one unit). Also the introduc-
tion of a PEG linker between the NCs and the saccharide leads to
the increased interaction with the receptor, which is mainly due
to the better accessibility of the sugar moiety.
The availability of the sugar at the surface to interact
specically with the mannose-receptor was evidenced by inter-
action with a uorescent lectin. The targeting function of
mannosylated HES NCs was shown in the example of dendritic
cells in in vitro studies.
Fig. 7 Flow cytometry analysis of NCs uptake by iDCs. Cells were gated for
expression of CD11c. Solid histogram indicates cells that were not treated with
NCs. Dashed histogram represents iDCs loaded with non-modified HES NCs, while
filled histogram shows cells treated with HES/tri-Man NCs.
4346 | J. Mater. Chem. B, 2013, 1, 4338–4348
This journal is ª The Royal Society of Chemistry 2013

View Article Online
Paper
Journal of Materials Chemistry B
20 J. Devy, E. Balasse, H. Kaplan, C. Madoulet and M. C. Andry,
Int. J. Pharm., 2006, 307, 194.
Acknowledgements
H.F. thanks the Max Planck Society for nancial support.
Gunnar Glasser (MPI-P) and Malte Paulsen (Institute of
Molecular Biology, Mainz) are acknowledged for their highly
skilled technical help with SEM and ow cytometry measure-
ments, respectively. Steffen Lorenz and Dennis Strand
(Department of Internal Medicine, University Medical Centre,
Johannes Gutenberg University, Mainz) are gratefully acknowl-
edged for their support in CLSM measurements.
21 K. Landfester, A. Musyanovych and V. Mailander, J. Polym.
Sci., Part A: Polym. Chem., 2010, 48, 493.
22 K. Landfester, Angew. Chem., Int. Ed., 2009, 48, 4488.
23 S. Mizrahy and D. Peer, Chem. Soc. Rev., 2012, 41,
2623.
24 G. Baier, D. Baumann, J.-M. Siebert, A. Musyanovych,
¨
V. Mailander and K. Landfester, Biomacromolecules, 2012,
13, 2704.
25 G. Baier, A. Musyanovych, M. Dass, S. Theisinger and
K. Landfester, Biomacromolecules, 2010, 11, 960;
K. Landfester, A. Musyanovych and V. Mailander, J. Polym.
Sci., Part A: Polym. Chem., 2010, 48, 493.
Notes and references
1 F. Chellat, Y. Merhi, A. Moreau and L. H. Yahia, Biomaterials,
2005, 26, 7260.
¨
26 U. Paiphansiri, J. Dausend, A. Musyanovych, V. Mailander
2 E. P. McGreal, J. L. Miller and S. Gordon, Curr. Opin.
Immunol., 2005, 17, 18.
and K. Landfester, Macromol. Biosci., 2009, 9, 575.
27 A. Musyanovych and H. J. P. Adler, Langmuir, 2005, 21, 2209.
3 P. Guermonprez, J. Valladeau, L. Zitvogel, C. Thery and 28 I. Migneault, C. Dartiguenave, M. J. Bertrand and
S. Amigorena, Annu. Rev. Immunol., 2002, 20, 621.
K. C. Waldron, BioTechniques, 2004, 37, 790.
4 S. J. van Vliet, J. J. Garcia-Vallejo and Y. van Kooyk, Immunol. 29 E. J. M. Van Damme, A. K. Allen and W. J. Peumans, FEBS
Cell Biol., 2008, 86, 580.
Lett., 1987, 215, 140.
5 G. Barratt, J. P. Tenu, A. Yapo and J. F. Petit, Biochim. Biophys. 30 H. Schlaad, H. Kukula, J. Rudloff and I. Below,
Acta, Biomembr., 1986, 862, 153. Macromolecules, 2001, 34, 4302.
6 S. Kawakami, J. Wong, A. Sato, Y. Hattori, F. Yamashita and 31 A. Thomas, H. Schlaad, B. Smarsly and M. Antonietti,
M. Hashida, Biochim. Biophys. Acta, 2000, 1524, 258. Langmuir, 2003, 19, 4455.
7 M. P. Leal, M. Assali, I. Fernandez and N. Khiar, Chem.–Eur. 32 F. Sauzedde, F. Ganachaud, A. Elaissari and C. Pichot,
´
J., 2011, 17, 1828.
J. Appl. Polym. Sci., 1997, 65, 2331.
8 J. Shao and J. K. H. Ma, Drug Delivery, 1997, 4, 43.
9 V. Fievez, L. Plapied, A. des Rieux, V. Pourcelle, H. Freichels,
33 M. Dubois, K. A. Gilles, J. K. Hamilton, P. A. Rebers and
F. Smith, Anal. Chem., 1956, 28, 350.
V. Wascotte, M. L. Vanderhaeghen, C. Jerome, 34 J. C. Gildersleeve, O. Oyelaran, J. T. Simpson and B. Allred,
A. Vanderplasschen, J. Marchand-Brynaert, Y. J. Schneider
and V. Preat, Eur. J. Pharm. Biopharm., 2009, 73, 16.
10 M. Nahar and N. Jain, Pharm. Res., 2009, 26, 2588.
Bioconjugate Chem., 2008, 19, 1485.
35 Y. Zhu, J. Zajicek and A. S. Serianni, J. Org. Chem., 2001, 66,
6244.
11 J. Rieger, H. Freichels, A. Imberty, J.-L. Putaux, T. Delair, 36 L. R. Ruhaak, G. Zauner, C. Huhn, C. Bruggink,
C. Jerome and R. Auzely-Velty, Biomacromolecules, 2009, 10,
651.
A. M. Deelder and M. Wuhrer, Anal. Bioanal. Chem., 2010,
397, 3457.
12 Z. Ghotbi, A. Haddadi, S. Hamdy, R. W. Hung, J. Samuel and 37 L. R. Ruhaak, E. Steenvoorden, C. A. M. Koeleman,
A. Lavasanifar, J. Drug Targeting, 2011, 19, 281.
A. M. Deelder and M. Wuhrer, Proteomics, 2010, 10,
13 B. Carrillo-Conde, E. H. Song, A. Chavez-Santoscoy,
2330.
Y.
Phanse,
A.
E.
Ramer-Tait,
N.
L.
Pohl, 38 L. R. Ruhaak, R. Hennig, C. Huhn, M. Borowiak, R. Dolhain,
M. J. Wannemuehler, B. H. Bellaire and B. Narasimhan,
Mol. Pharmaceutics, 2011, 8, 1877.
A. M. Deelder, E. Rapp and M. Wuhrer, J. Proteome Res.,
2010, 9, 6655.
14 S. K. Jain, Y. Gupta, A. Jain, A. R. Saxena, P. Khare and A. Jain, 39 Y. Suda, A. Arano, Y. Fukui, S. Koshida, M. Wakao,
Nanomedicine: Nanotechnology, Biology and Medicine, 2008, 4,
41.
T. Nishimura, S. Kusumoto and M. Sobel, Bioconjugate
Chem., 2006, 17, 1125.
15 M. Nahar, V. Dubey, D. Mishra, P. K. Mishra, A. Dube and 40 R. A. Evangelista, F. T. A. Chen and A. Guttman, J.
N. K. Jain, J. Drug Targeting, 2010, 18, 93. Chromatogr., A, 1996, 745, 273.
16 L. N. Cui, J. A. Cohen, K. E. Broaders, T. T. Beaudette and 41 V. A. Cosenza, D. A. Navarro and C. A. Stortz, ARKIVOC, 2011,
J. M. J. Frechet, Bioconjugate Chem., 2011, 22, 949. 182.
17 M. Hashimoto, M. Morimoto, H. Saimoto, Y. Shigemasa, 42 S. Sato, T. Sakamoto, E. Miyazawa and Y. Kikugawa,
H. Yanagie, M. Eriguchi and T. Sato, Biotechnol. Lett., 2006,
28, 815.
18 H.-L. Jiang, Y.-K. Kim, R. Arote, D. Jere, J.-S. Quan, J.-H. Yu,
Tetrahedron, 2004, 60, 7899.
43 R. Freeman, H. D. W. Hill and R. Kaptein, J. Magn. Reson.,
1972, 7, 327.
Y.-J. Choi, J.-W. Nah, M.-H. Cho and C.-S. Cho, Int. J. Pharm., 44 M. L. Martin, J. J. Delpuech and G. J. Martin, Practical NMR
2009, 375, 133. Spectroscopy, Heyden, London, 1980, pp. 231–235.
19 T. H. Kim, H. Jin, H. W. Kim, M. H. Cho and C. S. Cho, Mol. 45 H. Kaku and I. J. Goldstein, Carbohydr. Res., 1992, 229,
Cancer Ther., 2006, 5, 1723.
337.
This journal is ª The Royal Society of Chemistry 2013
J. Mater. Chem. B, 2013, 1, 4338–4348 | 4347

View Article Online
Journal of Materials Chemistry B
Paper
46 M. C. Chervenak and E. J. Toone, Biochemistry, 1995, 34, 50 G. Baier, C. Costa, A. Zeller, D. Baumann, C. Sayer,
¨
5685.
P. H. H. Araujo, V. Mailander, A. Musyanovych and
47 J. J. Lundquist and E. J. Toone, Chem. Rev., 2002, 102,
555.
48 Y. C. Lee and R. T. Lee, Acc. Chem. Res., 1995, 28, 321.
K. Landfester, Macromol. Biosci., 2011, 11, 628.
51 H. K. Kim, H. Wei, A. Kulkarni, R. M. Pogranichniy and
D. H. Thompson, Biomacromolecules, 2012, 13, 636.
49 W. I. Weis and K. Drickamer, Annu. Rev. Biochem., 1996, 65, 52 X. Sun, S. Chen, J. Han and Z. Zhang, Int. J. Nanomed., 2012,
441.
7, 2929.
4348 | J. Mater. Chem. B, 2013, 1, 4338–4348
This journal is ª The Royal Society of Chemistry 2013