Facile control of the self-assembled structures of polylysines having pendent mannose groups via pH and surfactant

Article abstract of DOI:10.1039/c002473b

A mannose-modified polylysine was synthesized, the self-assembly of which in aqueous solution led to the formation of spherical micelles, vesicles and rod-like micelles in a controlled manner by simply changing the solution pH and adding a surfactant to the solution.

Full text of DOI:10.1039/c002473b

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Facile control of the self-assembled structures of polylysines having
pendent mannose groups via pH and surfactantw
Rui Wang, Ning Xu, Fu-Sheng Du and Zi-Chen Li*
Received 4th February 2010, Accepted 30th March 2010
First published as an Advance Article on the web 21st April 2010
DOI: 10.1039/c002473b
A mannose-modified polylysine was synthesized, the self-assembly
of which in aqueous solution led to the formation of spherical
micelles, vesicles and rod-like micelles in a controlled manner by
simply changing the solution pH and adding a surfactant to the
solution.
polymerization (ROP) of e-benzyloxycarbonyl-L-lysine
N-carboxyanhydride (Z-Lys NCA), followed by removal of
the protective groups. 2,3,4,6-Tetra-O-acetyl-a-D-manno-
pyranosyl isothiocyanate (AcM-NCS) was then allowed to
react with the PLys in DMF to afford P(AcM/Lys-co-Lys).
In the 1H NMR spectrum of P(AcM/Lys-co-Lys), the methylene
protons adjacent to the amino groups (Hc, 2.98 ppm) shifted
to 3.35 ppm (Hc’) after formation of the thiourea bonds
(see ESI, Fig. S1).w Comparison of the relative integration of
peaks at 2.98 ppm to the methyl protons in the acetyl groups at
1.95–2.10 ppm allowed us to calculate the substitution degree
of mannose group, which could be controlled by the feed ratio of
AcM-NCS to polylysine. Deacetylation of P(AcM/Lys-co-Lys)
was then carried out in basic methanol aqueous solution. After
dialysis (water/cellulose tube, MW cutoff 1000 g/mol) and
freeze-drying, two copolymers, P(M/Lys-co-Lys), were
obtained, whose structures were confirmed by FTIR, 1H
NMR and 13C NMR spectra, and the substitution degree of
the mannose group is 16% for copolymer 1 and 23% for
copolymer 2 (see ESI, Fig. S2, S3 and S4).w
As a glycopolymer, P(M/Lys-co-Lys) is supposed to be
highly water-soluble in an acidic conditions because PLys
dissolves in water below pH 10 as a weak cationic poly-
electrolyte.22 However, when we dissolved P(M/Lys-co-Lys)
directly in water (0.1 mg/mL) at pH 4, we found the formation of
spherical micelles as evidenced by TEM and AFM (Fig. 1a and d),
the diameters of which were about 10–15 nm for copolymer 1.
Similar spherical micelles were also observed for copolymer 2,
with a slight increase of diameters. We measured the CD
spectra (see ESI, Fig. S5)w of copolymer 1 at pH 6.25 in water,
and detected one positive band at 217 nm, suggesting that
copolymer 1 existed as random coil as for unmodified PLys.
Therefore, the introduction of pendent mannose groups
(16% relative to the total lysine units) has very little effect
on the secondary structure of PLys (see ESI, Fig. S6).w
Schlaad and co-workers recently reported that a hydrophilic
Amphiphilic copolymers are known to form a variety of
molecular assemblies in solution, the morphology and
structure of which depend not only on the polymer structure,
composition and topology, but also on the nature of solvents
and various environmental factors. Micelles and vesicles
formed in aqueous media by amphiphilic copolymers are
among the most interesting and widely studied assembled
structures due to their applications in controlled and targeted
drug delivery.1–5 Considering the biodegradability, bio-
compatibility, tunable functionality and defined secondary
structures of polypeptide, self-assembly of polypeptide-based
block copolymers in aqueous solution has been the subject
of many recent researches.6–9 Among these copolymers,
polylysine-based block copolymers have been demonstrated
to be particularly interesting, mainly due to the controllable
manipulation of the secondary structure and amphiphilicity of
polylysine in response to an environmental stimulus (such as
pH, surfactants, and temperature).10–15 Since synthetic glyco-
polymers have shown enhanced interactions with cells, and
found potential applications in cell sensing, drug delivery, and
others,16–18 synthesis and self-assembly of glycopolypeptide
have recently attracted a great deal of attention.19–21 However,
these block copolymers are relatively difficult to synthesize,
and manipulation of morphological transformation of the
self-assembled structures from a single copolymer is hard.
We present here a simple approach for the synthesis of a
polylysine-based glycopolymer by simple modification of
polylysine with mannose. This simple polymer is capable of
self-assembling into a variety of structures by simply changing
the pH of the solution and adding surfactant to the solution
(see Scheme 1).
Copolymers 1 and 2 used for this study (see structure in
Scheme 1) were synthesized by post-polymerization modification
of poly(L-lysine) (PLys) (Scheme 2). Two PLys (DP = 32 or 61,
determined by NMR) were obtained by ring-opening
Beijing National Laboratory for Molecular Sciences (BNLMS),
Key Laboratory of Polymer Chemistry & Physics of Ministry of
Education, Department of Polymer Science and Engineering, College
of Chemistry and Molecular Engineering, Peking University, Beijing,
100871, China. E-mail: zcli@pku.edu.cn; Fax: +86-10-6275-1708;
Tel: +86-10-6275-5543
Scheme 1 Schematic diagram showing the morphological transition
among spherical micelles, vesicles and rod-like micelles formed by
P(M/Lys-co-Lys) [copolymer 1 (x = 5, y = 27) and copolymer 2
(x = 14, y = 47)] in aqueous solution by changing the pH and adding SDS.
w Electronic supplementary information (ESI) available: Synthesis,
characterization and spectroscopy studies. See DOI: 10.1039/c002473b
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copolymers. The wall thickness of the vesicle was 12 nm
for copolymer 1 and 20 nm for copolymer 2 (Fig. 1b and c).
The CD spectrum at this pH shows that the copolymer adopts
an a-helix conformation (see ESI, Fig. S5),w which is due to
the formation of intramolecular hydrogen bonding among
PLys backbones. Because the spherical micelles built upon
hydrogen bonds at lower pH are dynamic structures,28 the
stronger hydrogen bonding among PLys backbones drives the
morphology change from micelles to vesicles. Pendant
mannose units remain water-soluble, thus contributing to the
stabilization of the vesicles in solution. To confirm this
assumption, we measured the 1H NMR of the vesicular
solution in D2O (see ESI, Fig. S3);w it was found that the
signal appeared at 2.99 ppm (–CH2–NH3+) in neutral
D2O shifted to 2.72 ppm in basic conditions, and at the same
time the a-methine proton signals of PLys (COCHNH, at
4.30 ppm) almost completely disappeared. These results
indicated that the chain mobility of the PLys segments was
greatly reduced, the PLys helix should exist in the hydrophobic
vesicle wall, while the mannose groups are on the vesicle
surfaces.11
Scheme 2 Synthesis of P(M/Lys-co-Lys).
glycopolyamide homopolymer can self-assemble into spherical
vesicles and nanofibers through intermolecular hydrogen
bonding between amide and glucose units.23 In our case,
introduction of pendant mannose groups and thiourea groups
to PLys should also be responsible for the micellar structure
formation. Our copolymers contain thiourea groups, which
have proven useful in constructing hydrogen bonding
receptors.24–26 The relatively acidic thiourea NH protons have
a strong hydrogen-bond donor capability.27 In order to know
whether the thiourea is involved in hydrogen bonding
formation between polymers, we measured the 13C NMR spectrum
of copolymer 1 in the presence of urea (see ESI, Fig. S7),wand
found that the C-1 signal obviously shifted downfield, while
other carbon signals of the mannose unit changed only
slightly. C-1 is the carbon adjacent to the thiourea group,
therefore, the observed shift variation presumably indicates
that the hydrogen bonds contributed by thiourea groups
dissociate after adding urea. The micellar aggregation was
probably driven by the intermolecular hydrogen bonds from
the thiourea N–H to the amide CQO on the PLys backbones,
which mainly affect the chemical shift of the C-1 signal.25,26
The proposed drawing at pH 4 in Scheme 1 just shows that the
intermolecular hydrogen bonds are necessary for the
formation of the micellar structures, however, the detailed
structure of the micelles needs further investigation.
The formation of vesicles was further ascertained by
studying the container property with calcein as a probe.29
An aqueous solution of calcein-loaded vesicles was prepared
by mixing 5 mM calcein with 0.1 mg/mL copolymer 2 at pH 2,
then the pH was adjusted to higher than pH 10 with aqueous
NaOH, finally the solution was dialyzed against pH 10.83
carbonate buffer for 16 h. For these dye-loaded vesicles, the
calcein concentration was 55 mM, and the loading efficiency
was estimated to be 17%. The release of calcein was monitored
by measuring the absorbance at 497 nm. The vesicle solution
showed a much slower elution profile (see ESI, Fig. S8),w
indicating a significantly retarded release of the calcein at this
pH due to its entrapment within the vesicles. Moreover, if the
initial calcein concentration was increased to three times
higher, a more rapid release was observed. As expected, a
control experiment in the absence of any vesicles revealed a
more rapid dye elution.
When the pH of the solution was adjusted to higher than 10,
vesicles with diameters of 45–80 nm were observed for both
We further confirmed that mannose groups do locate on the
surface of the vesicles. It is well-known that mannose moieties
have strong and specific interactions with Concanavalin A
(Con A).30 Soon after a Con A solution was added to the above
vesicle solution at pH 10, the turbidity of the solution initially
increased quickly and reached a plateau within 10 min (see
ESI, Fig. S9),w a similar phenomenon was observed for other
sugar-covered aggregates.31
When the pH was continuously increased up to 12,
precipitation of the copolymer was observed for both samples.
These precipitates could hardly dissolve in any common
solvents, such as THF, DMF, DMSO. The FTIR spectrum
(see ESI, Fig. S10)w of the lyophilized precipitates shows a
strong band at 1628 cmÀ1 and a weaker band at 1686 cmÀ1
,
indicating the formation of antiparallel b-sheet conformation
of the peptide chains.32
Fig. 1 TEM images of copolymer 1 and 2 at different pHs. (a)
Copolymer 1, pH = 4, (b) copolymer 1, pH>10, (c) copolymer 2,
pH>10. (d) AFM images of copolymer 1 at pH = 4. Inset: Height
information of copolymer 1 spherical micelles. The initial copolymer
concentration is 0.1 mg/mL. Uranyl acetate was used as a staining
agent.
Besides pH, adding anionic surfactants could also induce the
change of self-assembled morphologies of P(M/Lys-co-Lys).
When adding SDS into the copolymer solution (0.2 mg/mL) at
pH 4, we found changes of both the secondary structure of the
copolymer main chain and the self-assembled structures; the
ꢀc
This journal is The Royal Society of Chemistry 2010
Chem. Commun., 2010, 46, 3902–3904 | 3903

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This work was supported by the National Natural Science
Foundation of China (NSFC, No.50573002). We thank
Hao Sun and Prof. Yan Li (Peking University) for their
general help in AFM measurements. We also thank
Prof. Xin-Hua Wan (Peking University) for accessing the
CD spectra measurement equipment.
Notes and references
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in Fig. 2, with increasing the SDS/lysine unit ratio, the
conformation of the copolymer changes gradually from
random coil to b-sheet, where the CD spectrum has one minimum
at 215 nm and a maximum at 195 nm. This conformation
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or tethered on surface, which was ascribed to the decreased
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rod-like micelles at a SDS/lysine unit ratio up to 0.75. We
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spherical micelles are transformed to rod-like micelles.
In summary, we demonstrated that a simple mannose-
modified polylysine can self-assemble into spherical micelles,
vesicles and rod-like micelles in aqueous solution via simply
changing pH or adding a surfactant. The transformation
between different morphologies was due to the formation of
hydrogen bonding, secondary structure transition, and the
change of hydrophobicity. Vesicle formation from such
a simple polymer may be very promising for constructing
cell-recognized drug deliver systems.
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Fig. 3 TEM image for aggregate prepared from 0.2 mg/mL copolymer
2 aggregate in pH = 4 aqueous solution by adding 1 mM SDS with
SDS/lysine unit ratio = 3/4. Uranyl acetate was used as the staining
agent.
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This journal is The Royal Society of Chemistry 2010
3904 | Chem. Commun., 2010, 46, 3902–3904