Preparation of membrane-mimicking lamellar structures by molecular confinement of hybrid nanocomposites

Article abstract of DOI:10.1039/c8cc09399g

Hybrid nanocomposites are multiphase systems with a wide range of applications. Some nanocomposites are water insoluble thereby preventing several applications. Thus, we prepared telechelic PEO with glucose molecules to form water-soluble lamellar nanostructures by co-assembly with metallacarborane. The lamellas formed by PEO/metallacarborane decorated by glucose molecules on the surface can serve as delivery agents for boron clusters and benzoxaboroles.

Full text of DOI:10.1039/c8cc09399g

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Preparation of membrane-mimicking lamellar
structures by molecular confinement of hybrid
Cite this:DOI: 10.1039/c8cc09399g
nanocomposites†
Received 27th November 2018,
Accepted 24th January 2019
a
a
a
b
b
David Vrbata,
Vladimır Dˇ or dˇ ovic,
Jani Seitsonen, Janne Ruokolainen,
´
ˇ
c
a
Olga Janouskova, Mariusz Uchman
and Pavel Matejıcek
ˇ´ˇ
*
ˇ
´
DOI: 10.1039/c8cc09399g
rsc.li/chemcomm
Hybrid nanocomposites are multiphase systems with a wide range Therefore, the shape and morphology of these nanostructures
of applications. Some nanocomposites are water insoluble thereby are determined by the relative size of the polymer blocks and by
4
preventing several applications. Thus, we prepared telechelic PEO the type of interaction between the building blocks.
with glucose molecules to form water-soluble lamellar nanostructures
5
Discovered by Hawthorne 50 years ago, the anion [cobalt
by co-assembly with metallacarborane. The lamellas formed by PEO/ bis(dicarbollide)](À1), also known as COSAN, is an intrinsically
metallacarborane decorated by glucose molecules on the surface can amphiphilic, surface-active, oil- and water-soluble metalla-
serve as delivery agents for boron clusters and benzoxaboroles.
carborane with unique chemical and physical properties (structure
6–8
in Fig. 1). Unsurprisingly, boron cluster compounds have been
Hybrid nanostructures based on weak interactions between extensively tested in medical applications, such as boron neutron
1
9
building blocks show unique features: (i) enhanced quality capture therapy, BNCT, and HIV protease and carbonic anhydrase
10
(synergistic effects); (ii) sensitivity to external stimuli and to inhibition. Furthermore, several metallacarboranes have been
boundary effects; (iii) ability to merge subunits with opposite proposed as functional building blocks of materials designed for
7c,11
polarity and dynamics (amphiphilic and amphidynamic nano- supramolecular chemistry and nanotechnology,
and for devel-
structures); (iv) quantum size effects (photonic crystals and oping devices suitable for the exchange of photons, ions or active
12
quantum dots); (v) partial or complete disassembly into the compounds.
original building blocks. The hybrid nanocomposite formed between the electro-
In particular, nanocomposites resulting from the co-assembly neutral hydrophilic poly(ethylene oxide) (PEO) and Na[COSAN]
of charged homopolymers (homopolyelectrolyte) and low molar- results from the spontaneous co-precipitation of both compo-
13
mass amphiphilic compounds, for example, surfactants with the nents in aqueous solution. The formation of this complex is
2
opposite charge have been extensively studied in recent decades
and show all the aforementioned features (i–v).
similar to the aforementioned co-assembly of homopolyelectro-
1c,e,f,2
However, these lytes with oppositely charged surfactants. Nevertheless, this
nanocomposites are usually insoluble in water, thus limiting their co-assembly process is driven not by electrostatic interactions
range of applications. Notwithstanding, they can be dispersed in
water by covalently attaching a soluble block to the homopolymer,
but this soluble block must not interact with the surfactant. The
resulting double hydrophilic block copolymers co-assemble with
3
surfactants into nanoparticles with a core–shell structure.
a
Department of Physical and Macromolecular Chemistry, Faculty of Science,
Charles University, Hlavova 2030, 128 43 Prague 2, Czech Republic.
E-mail: pavel.matejicek@natur.cuni.cz
b
Department of Applied Physics Nanotalo, Aalto University, Puumiehenkuja 2,
FI-02150 Espoo, Finland
c
Institute of Macromolecular Chemistry, v.v.i. Academy of Sciences of the Czech
Republic, Heyrovsk ´y Sq. 2, 16206 Prague 6, Czech Republic
†
Electronic supplementary information (ESI) available: Experimental details,
samples synthesis and characterization, additional NMR, confocal microscopy
and cell viability data. See DOI: 10.1039/c8cc09399g
Fig. 1 Structures of telechelic poly(ethylene oxides), PEOs, with attached
glucose, glc, molecules, and cobalt bis(1,2-dicarbollide) anion, COSAN.
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and counterion release but by dihydrogen bonding between the
8
PEO chain and COSAN clusters. The dihydrogen bond has
been predicted by quantum chemistry calculations and confirmed
14
experimentally. It is based on weak bonding between negatively
À
+
and positively charged hydrogen atoms: H(d )ÁÁÁH(d ) (example in
10a,13a
Fig. S24 in ESI†).
Consequently, the COSAN-to-polymer
segment ratio is not 1 : 1 but ca. 1 : 10–14.
1
3c
The PEO/
1
3a
13c
Na[COSAN] is stimuli responsive
and amphidynamic,
and it shows almost all the aforementioned features (except
point iv). Double hydrophilic block copolymers combining PEO
blocks with polyanionic or neutral blocks form spherical, com-
13a,15
pact, hybrid nanoparticles with a tunable inner structure.
Hence, to simultaneously simplify the polymer structure and
to broaden the range of applications, we prepared telechelic
PEO with glucose molecules (glc) attached to one or to both
1
6
ends of PEO via a standard CuAAC click reaction: PEO
m
-glc
and glc-PEO -glc (m and n are numbers of monomeric units;
n
the structures are shown in Fig. 1; synthesis, NMR and GPC
characterization, Scheme S2, and Fig. S1–S21 in ESI†). These
polymers are water soluble and biocompatible. They are a
Fig. 2 CryoTEM micrographs of PEO-glc/Na[COSAN] nanostructures in
17
0
.1 M NaCl oversaturated by COSAN; (A and B) PEO45-glc/COSAN, and
simpler version of the double hydrophilic block copolymer in (C and D) PEO114-glc/COSAN. Arrows within the micrographs indicate
which the stabilizing block shrinks into a single glucose molecule. multi-layer nanostructures in (A), single-wall nanotubes in (C and D), and
nanosheets in (B).
In addition to this stabilizing function, natural and artificial
glycopolymers are used as recognition or binding agents.
18
The polymers were designed to ensure a degree of PEO
polymerization (40–110) low enough to avoid canceling the
effect of the glc end-groups but not excessively low, which
would affect the polymeric features of PEO. The glucose mole-
cules, which do not interact with Na[COSAN] as proven by
NMR, act as molecular confinement agents that provide differ-
ent binding and solubility properties to the ends of PEO and to
the inner parts of glc-PEG
COSAN interaction with PEO are shown in ESI† (Fig. S22–S25).
Telechelic PEOs (PEO -glc and glc-PEO -glc) behave in a
n
-glc polymer chains. Details on
m
n
unique way. Though all PEO and glucose NMR signals dis-
appeared after adding Na[COSAN], which indicate the formation
of a compact structure (Fig. S23 in ESI†), the dispersions are
stable for at least several weeks or even months. The key to
prolonging dispersion stability is to add an excess of COSAN to
the medium, that is, a COSAN to PEO segment ratio Z1.0.
At ratios closer to ‘‘stoichiometry’’ (B0.1), the nanostructures
are prone to stick to the wall of the vial, forming an orange film.
The samples were analyzed by cryo-TEM imaging. The basic
motif of all nanostructures is a thin layer (lamella). PEO -glc
occurs in the form of nanosheets, multilamellar layers and
m
Fig. 3 CryoTEM micrographs of glc-PEO-glc/Na[COSAN] nanostructures
in 0.1 M NaCl oversaturated by COSAN; (A and B) glc-PEO45-glc/COSAN,
n
tubular structures (Fig. 2). The total confinement in glc-PEO -glc and (C and D) glc-PEO90-glc/COSAN. Arrows within the micrographs
indicate vesicular structures in (A and B), and flat mono- and multi-
reproducibly leads to vesicular structures (Fig. 3A and B). An
addition to the classical morphologies of microphase-separated
diblock copolymers in selective solvents (spheres, cylinders and
lamellae), atypical ribbon-like, tubular and helical structures have
layers in (C and D).
been observed in glycopolymers, in polypeptides and in similar ca. 5–10 nm, which is smaller than the PEO-linker contour lengths
1
8,19
amphiphiles.
bonding between amino acids or p–p stacking combined with molecular hydrogen bonds in polysaccharides (cellulose) and in
hydrophobic effects. related nanostructures, thereby stabilizing such nanostructures into
Their shape is typically determined by hydrogen (approximately 12 nm). Glucose has been shown to form inter-
20
Based on the cryoTEM micrographs of the vesicles, the thick- fairly compact polymers. However, the completely flat NMR spec-
ness of the glc-PEO45-glc/COSAN membrane was estimated at trum and too small changes in IR spectra shown in Fig. S23 and S24
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Fig. 4 Ideal structure of glc-PEO-glc/COSAN unilamellar membrane
+
(
Na counterions omitted for clarity). In the real system, such lamellas
are fused into layered nanostructures of lower degree of regularity (see
Fig. 2 and 3).
in ESI† prevents us from directly confirming this statement. Despite
the lack of a direct proof, all experimental data indicate that glucose
should be preferentially located on the surface. The driving force for
the membrane formation is the effect of the COSAN dihydrogen
bonding to PEO, restricted by glc moieties. The ideal structure of
the glc-PEO -glc/COSAN membrane is shown in Fig. 4. However,
the real nanostructures show lower degree of regularity (lack
of typical features of lamellas in SAXS/WAXS and XRD spectra,
Fig. S25 in ESI†). Fusion of several membranes into one undulating
layer due to the patchy surface of the membrane is expected as
observed in Fig. 3A.
4
5
Fig. 6 The confocal microscopy images of in vitro HeLa cell uptake of
(
A and B) glc-PEO45-glc/COSAN/GB179, and (B and C) glc-PEO45-glc/
COSAN/RhBB (composition in ESI†) after (A and C) 2 h and (B and D) 24 h
incubation.
To monitor the position of glucose molecules in the nano-
structures and to visualize the PEO-based lamellae, we prepared
(
in general, polymer/COSAN samples are more toxic than pure
COSAN). At given GB179 content, the inhibition activity of GB179
against HIV protease (IC50 B 100 nmol L
below the toxicity limit. Nevertheless, the toxicity excludes the
nanostructures from the use in BNCT.
2
1
22
two fluorescent probes: (1) GB179 and (2) RhBB (the struc-
tures in Fig. 5, and characterization in ESI†). GB179 is sparingly
soluble in water and has high affinity to PEO, which makes it
possible to selectively stain the inner parts of PEO/Na[COSAN]
À1 15a
)
is still slightly
Under fluorescence confocal microscopy, glc-PEO45-glc/COSAN/
GB179 and/RhBB nanostructures were observed outside cells
shortly after adding them to cell media to a final dilution lower
than the IC50 (Fig. 6A and C). After 24 h incubation, the HeLa cells
were evenly stained by both BG179 and RhBB (Fig. 6B and D),
whereas the probes were located exclusively in the remaining
compact nanostructures in the extracellular medium. Thus, we
assume the active transport of the nanoparticles into cells.
Complete cell staining indicates that the probes were released
from the polymer nanostructures (further comments on the
nanoparticle internalization in ESI†): GB179 should accumulate
2
1
lamellae. RhBB forms a dynamic covalent bond with glucose
2
3
in slightly alkaline buffers (pH 4 7).
Furthermore, GB179 exhibits inhibition activity against
1
5a
HIV protease (IC50 E 100 nM).
The advantage of using
benzoxaborole-containing fluorophores in sugar-containing
1
8c,22
nanostructures has also been previously demonstrated.
Although fluorescence microscopy has a lower resolution than
TEM, the structures observed in the fluorescence microscopy
study (solid films on glass plates; see sample preparation in
ESI† and Fig. S26) are similar to the micrographs in Fig. 2 and 3
(vesicular and tubular structures). Concurrently, the results
21
in proteins and in lipid membranes, whereas RhBB should be
preferentially bound to intracellular saccharides.
confirm that the probes are located only in nanostructures
and not evenly dispersed in the solution.
22
We prepared lamellar nanostructures by COSAN co-assembly
with telechelic PEO with glucose end groups, which act as
confinement agents, thereby forming artificial membranes with
embedded COSAN clusters. Their surface contains a high amount
of glucose molecules that can selectively attach to benzoxaboroles.
The lamellar nanostructures were visualized by cryoTEM, and
in vitro experiments in culture HeLa cell lines showed that the
nanostructures can be used for the delivery of both boron
cluster- and phenyl boronic acid-based conjugates thanks to
their dynamic bonding.
The cytotoxicity and uptake of the nanostructures were further
tested in vitro using HeLa cell lines. Although the pure polymer
showed no cell toxicity (details in Fig. S27 and S28 in ESI†), all
À1
COSAN-containing samples are toxic with IC50 B 10–30 mg mL
PM, VD and DV would like to acknowledge the financial
support of the Czech Science Foundation 17-00648S. OJ would
like to acknowledge the financial support by the Ministry of
Education, Youth and Sports of CR within the National
Fig. 5 Structure of GB179 (left) and RhBB (right) fluorescence probes.
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