C. Striegler et al. / Polymer 80 (2015) 188e204
189
assumed that a preferential decoration of all primary and sec-
2.2. Characterisation
ondary amino groups with oligomeric/polymeric chains was
realized.
The NMR spectroscopy measurements were carried out on a
Bruker DRX 500 NMR spectrometer operating at 500.13 MHz for 1H
and at 125.75 MHz for 13C. Various NMR methods were used to
evaluate the structural composition of the coreeshell architectures.
Our own efforts are directed to establish sugar-decorated den-
dritic polyamines as neutral, cationic or anionic coreeshell archi-
tectures suitable for application as polymeric therapeutics and
diagnostics [29e33], but also addressable as polymeric drug [34].
More recently, anionic glycodendrimers were found to be suited
materials to support the degradation of various prion strains by
protease attacks [35]. Moreover, dense-shell glycodendrimers
randomly decorated with few phenyl groups in the sugar shell
resulted in the formation of aggregates with defined dimensions,
while their treatment with sonication promotes the disaggregation
process in smaller aggregates followed up by the time-dependent
formation of necklace- and donut-like supramolecular sugar
structures [36]. Furthermore, weakly cationic maltose-modified PEI
can be used as an artificial tubulating protein to tailor the
morphological transformation of anionic vesicles into tubular-like
entities [37].
Motivated by our own success in the formation of dendritic
supramolecular structures [36e38] and the fabrication of defined
polymeric micelles received by the self-assembly of poly(amino
acid) containing coreeshell architectures [21e25], we became
interested in the realization of under physiological condition pref-
erentially non-assembled anionic coreeshell architectures, also
additionally modified with sugar units. Such new kind of anionic
coreeshell (glyco-)architectures (Scheme 1) would be very inter-
esting for forthcoming application in the construction of poly-
electrolyte complex particles [39] or as delivery system for
retarding release of cationic drugs [40]. In the here presented
concept, the hyperbranched PEI is the core macromolecule, while
the shell component consists of poly(amino acid) chains equipped
with and without disaccharide units. This kind of anionic coree-
shell architecture is significantly distinguishable from other anionic
or cationic coreeshell architectures possessing binary shell [14,22]
and double shell [21,25] at which anionic poly(glutamic acid)
chains or cationic polylysine chains are integrated in such specific
coreeshell architectures [14,21,22,25]. To our knowledge there
exist no studies in which investigations of PEI equipped only with
unprotected poly(glutamic acid) or poly(aspartic acid) chains are
described.
The 1H NMR spectra of
g-BLG-NCA, b-BLA-NCA, PEI-PBLG-346 and
PEI-PBLA-346 were measured in DMSO-d6. The 1H and 13C NMR
spectra of the water-soluble polymers were measured in D2O. A
capillary tube filled with
a sodium 3-(trimethylsilyl)-3,3,2,2-
tetradeuteropropionate solution in D2O was used for external
calibration (
d
(1H) ¼ 0 ppm;
d(
13C) ¼ 0 ppm).
The FTIR spectroscopic investigations were carried out on a
Bruker IFS 28 spectrometer (Bruker Optics GmbH, Ettlingen, Ger-
many) equipped with globar source and MTC detector as well as
attenuated total reflection (ATR)-attachment (Optispec, Neeerach,
Switzerland) located in the original sample compartment of the
spectrometer. FTIR spectra were recorded by collecting 100 scans at
a spectral resolution of 2 cmꢀ1. The polymer films were prepared by
casting from polymer solutions (0.1e1 mg/mL) onto germanium
(Ge) internal reflexion elements (KOMLAS GmbH, Berlin, Germany).
SEC-MALLS (multi angle laser light scattering) measurements
were performed using PolarGel-M column (Polymer Labs, UK),
Agilent isocratic pump Series 1200 (Agilent Tech, USA), differential
refractive index (RI) detector K2301 (Knauer, DE) and a MiniDAWN
MALLS Detector (Wyatt Technologies, USA). The eluent was N,N-
dimethylacetamide (DMAc) with 3 g/L LiCl and a flow rate of
1.0 mL/min. The dn/dc of 0.09 mL/g was calculated from the mea-
surements for all samples by assuming full mass recovery between
injected and detected sample by neglecting the adsorptive effects.
AF4 (asymmetric field flow-field fractionation) measurements
were performed on an Eclipse DUALTEC system (Wyatt Technology
Europe) which was connected with an Agilent pump system series
1260. Poly(ether sulfone) membranes with a molecular weight cut
off of 3000 Da were used. The detection system consists of RI de-
tector Optilab T-rEX (Wyatt Technologies Corp., USA) and LS de-
tector DAWN HELEOS II (Wyatt Technologies Corp., USA). Nitrate
buffer (50 mM), containing 0.02% (w/v) sodium azide to prevent
bacteria growth, was used as eluent. A short channel with height of
350 mm was used. The channel flow rate was maintained at 0.8 mL/
min for all AF4 operations. The fractionation of PEI-PGlu-346 was
performed under isocratic cross flow of 4 mL/min over 20 min. PEI-
PGlu-346-Mal was fractionated over 20 min by a linear cross flow
gradient from 3 to 0 mL/min. The dn/dc was determined external by
RI detection.
The pH dependent titration was performed with a particle
charge detector (PCD-03, Mütek, Germany) in the pH region 3e10.
The corresponding surface charges were recorded by zeta potential
measurements at 25 ꢁC on a Zetasizer Nano ZS from Malvern
Instruments.
Here, we report the synthesis and characterisation of coreeshell
architectures where the terminal amino groups of anionic poly(L-
glutamic/aspartic acid) chains are partly decorated with maltose
or lactose units (Scheme 1). Solution properties of the anionic en-
tities in various aqueous solutions were determined by using DLS,
streaming potential pH titration, zeta potential measurements,
TEM, cryo-TEM, AFM, in-situ AFM and FTIR spectroscopy, including
its complexation properties against cationic dye by using UVeVis
spectroscopy.
UV/Vis spectra of acridin orange were recorded at room tem-
2. Experimental section
perature (22
2
ꢁC) on a Cary 6000i spectrophotometer (Varian).
Spectral bandwidth and increment were 1 nm.
2.1. Materials
Titration experiments for UV measurements were carried out at
room temperature (22
2
ꢁC) in a sealed cuvette (quartz glass
Hyperbranched poly(ethylene imine) (PEI, Lupasol G100
suprasil, HELLMA, Germany) by stepwise addition (gas-tight micro
litre syringe, through the serum cap) of appropriate amounts of the
titrant solution in water. At the end of the titration, the solutions
were significantly diluted. For each titrant concentration, the
spectra were corrected for the dilution by applying the appropriate
factor.
Mw ¼ 5000 g/mol) was obtained from BASF SE (Ludwigshafen, Ger-
many) and dried in vacuum overnight before use. g-Benzyl-L-gluta-
mate,
b-benzyl-L-aspartate,
bis(trichloromethyl) carbonate,
borane*pyridine complex (8 M BH3 in pyridine), D-(þ)-maltose-
monohydrate (Mal), D-(þ)-lactose-monohydrate (Lac), N,N-dime-
thylformamide, tetrahydrofuran and poly(glutamic acid sodium salt)
(PGlu 61 K, Mw ¼ 61 kDa) were used as purchased from Fluka and
DLS experiments were performed at 25 ꢁC at a fixed angle of
173ꢁ using the Zetasizer Nano ZS (Malvern Instruments) equipped
with a He-Ne-laser (wavelength 633 nm).
Aldrich. Poly(
purchased from Polyscience Inc. (Germany).
g
-benzyl-
L
-glutamate) (PBLG, Mw ¼ 150e300 kDa)was
The morphology of the accumulated macromolecules in dried