Polymersomes with aggregation-induced emission based on amphiphilic block copolypeptoids

Article abstract of DOI:10.1039/c9cc07501a

Biocompatible polymersomes are prepared from amphiphilic block copolypeptoids with aggregation-induced emission, where the hydrophobic block P(TPE-NAG) is a tetraphenylethylene (TPE)-modified poly(N-Allylglycine) and the hydrophilic block is polysarcosine

Full text of DOI:10.1039/c9cc07501a

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Polymersomes with aggregation-induced emission
based on amphiphilic block copolypeptoids†
Cite this: Chem. Commun., 2019,
55, 13530
ab
b
d
Xinfeng Tao,
Min-Hui Li
Hui Chen,
Sylvain Trepout,c Jiayu Cen,d Jun Ling
and
´
be
Received 24th September 2019,
Accepted 14th October 2019
*
DOI: 10.1039/c9cc07501a
rsc.li/chemcomm
Biocompatible polymersomes are prepared from amphiphilic block Therefore, polypeptoids show better solubility, thermal processability,
copolypeptoids with aggregation-induced emission, where the hydro- cell permeability and proteolytic stability compared to polypeptides.4
phobic block P(TPE-NAG) is a tetraphenylethylene (TPE)-modified Polypeptoids are usually synthesized by ring-opening polymerization
poly(N-allylglycine) and the hydrophilic block is polysarcosine. These (ROP) of N-substituted glycine N-carboxyanhydride (NNCA).4 Recently,
nanoparticles are non-cytotoxic and show strong fluorescence emis- we developed an efficient alternative way to synthesize well-defined
sion in aqueous solution.
polypeptoids using a more stable monomer N-substituted glycine
N-thiocarboxyanhydride (NNTA).5
Nanoparticles composed of amphiphilic copolymers have
Nanoparticles based on polypeptoids have been widely
attracted extensive attention in the past decades due to their investigated.6 Luxenhofer and co-workers prepared worm-like
ordered nanostructures with various morphologies, including micelles and polymersomes by using amphiphilic block copoly-
spherical micelles, cylindrical micelles and vesicles, etc., which peptoids containing a hydrophilic polysarcosine (PSar) block and
can be potentially used in drug delivery and bioimaging systems.1 a hydrophobic polypeptoid block with butyl, pentyl, benzyl or
Among them, polymer vesicles, also known as polymersomes, are phenethyl side groups.7 We reported previously that PSar-block-
cell-mimicking hollow spheres with a polymer bilayer membrane poly(e-caprolactone)-block-PSar (PSar-b-PCL-b-PSar) formed unila-
enclosing an aqueous volume in its interior. They are more stable mellar sheets, worm-like cylinders, and multilamellar polymer-
and robust than lipid vesicles (liposomes) and have high encapsu- somes under different conditions.8 Spherical micelles formed in
lation capacity for both hydrophilic and hydrophobic molecules.2,3 water by poly(N-ethylglycine)-block-poly[(N-propargylglycine)-r-
Polypeptoids are a class of poly(amino acid)s suitable for the (N-decylglycine)] (PNEG-b-P(NPgG-r-NDG)) were studied by Zhang
preparation of polymersomes with the advantages of excellent et al.9 They used the hydrophobic core of micelles to encapsulate
biocompatibility, degradability, and low cytotoxicity.4 Polypep- doxorubicin (DOX) for further applications of controlled release
toids have a protein-mimicking structure with N-substituted of DOX.9 Sun et al. synthesized different PEG-block-polypeptoids
glycine as repeating units but lack intramolecular and inter- which self-assembled into two-dimensional nanosheets.10,11
molecular hydrogen bonding along the amino acid backbone.
All the above reported nanoparticles are based on polypeptoids
with normal aliphatic or aromatic side chains. Nevertheless, func-
tional side chains are often required in various applications, for
example, for the preparation of stimuli-responsive nanoparticles in
controlled release or fluorescent nanoparticles in bioimaging. In a
recent paper, we developed oxidation-responsive polymersomes
based on copolypeptoids, poly(N-3-(methylthio)propyl glycine)-
block-PSar (PMeSPG-b-PSar), containing thioether side-chains on
PMeSPG.12 However, this method has some limitations, i.e., we
need to develop a specific synthesis route for each functional
monomer, which is tedious and time-consuming. In this study,
we propose to use a functionalizable poly(N-allylglycine) (PNAG) as
a hydrophobic block, which is an excellent platform for modifica-
tion through a thiol–ene reaction to get functional polypeptoids.13
PNAG-b-PSar was synthesized using benzylamine-initiated ROP of
N-allylglycine NTA (NAG-NTA) and sarcosine NTA (Sar-NTA). To
a Shanghai Key Laboratory of Advanced Polymeric Materials, School of Materials
Science and Engineering, East China University of Science and Technology,
Shanghai 200237, China
b
´
Chimie ParisTech, PSL Universite Paris, CNRS, Institut de Recherche de Chimie
Paris, UMR8247, 11 rue Pierre et Marie Curie, 75005 Paris, France.
E-mail: min-hui.li@chimieparistech.psl.eu
c
´
Institut Curie, PSL Universite Paris, INSERM U1196 and CNRS UMR9187, 91405
Orsay Cedex, France
d MOE Key Laboratory of Macromolecular Synthesis and Functionalization,
Department of Polymer Science and Engineering, Zhejiang University, Hangzhou
310027, China
e Beijing Advanced Innovation Center for Soft Matter Science and Engineering,
Beijing University of Chemical Technology, 15 North Third Ring Road, Chaoyang
District, 100029 Beijing, China
† Electronic supplementary information (ESI) available. See DOI: 10.1039/
c9cc07501a
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mercaptoacetic acid (CA-SH) to get carboxylic acid-functionalized
P(CA-NAG)-b-PSar (Fig. 1C), which was then reacted with 3-(4-
(1,2,2-triphenylvinyl)phenoxy)propan-1-ol (TPE-C3OH) (Scheme
S4 and Fig. S7, ESI†) to get the AIE block copolymers P(TPE-
NAG)-b-PSar. The final structures of P(TPE-NAG)-b-PSar were
1
confirmed by H NMR spectra (Fig. 1D and Fig. S12, ESI†) and
SEC traces (Fig. S13, ESI†). Their molecular weights (MW) and
distributions (Ð) are summarized in Table S2 (ESI†). The hydro-
philic weight ratios ( fPSar) of P(TPE-NAG)-b-PSar are 18%, 30%
and 38%, respectively.
P(TPE-NAG)25-b-PSar46 was chosen as an example to study
the AIE properties of copolypeptoids. We found that it exhibited
obvious AIE fluorescence when dispersed in water owing to the
restriction of intramolecular rotation (RIM) of TPE moieties in
the aggregated state (Fig. 2A and discussion in the ESI†).18
Moreover, we were also interested in the morphology of aggre-
gates formed in this experiment. The dispersion in a DMF/H2O
mixture with fw = 99% was selected for DLS and cryo-EM
analysis. Fig. 2B shows the size distribution of aggregates
weighed by scattering intensity and by number. There are two
populations of nanoparticles, where smaller ones with an
average hydrodynamic diameter of about 56 nm is predomi-
nant and a few big particles with diameters 41000 nm co-exist.
Fig. 2C and D show the nanostructures observed by cryo-EM,
Fig. 1 (A) Synthesis of PNAG-b-PSar through block copolymerization of
NAG-NTA and Sar-NTA and its postpolymerization modification by reaction
with mercaptoacetic acid and TPE-C3OH successively to produce P(TPE-NAG)-
b-PSar. 1H NMR spectra of PNAG15-b-PSar54 (B), P(CA-NAG)15-b-PSar54 (C) and
P(TPE-NAG)15-b-PSar54 (D) in DMSO-d6 (*: diethyl ether; **: water).
highlight their ability to be functionalized at their side-chains and which confirm the results of DLS. Many nanoparticles with
to self-assemble into functional polymersomes, we attached tetra- diameters o50 nm were observed. Note that hydrophilic chains
phenylethylene (TPE),14–18 the emblematic luminogen with located on the membrane coronas (exterior and interior sides)
aggregation-induced emission (AIE), to PNAG side chains through of the polymersomes are flexible and swollen in water, which
postpolymerization modification (Fig. 1A). Polymersomes and do not constitute enough density contrast, relative to vitreous
micelles with AIE properties were obtained by using a nanopreci- ice at the sample grid, to be visible in cryo-EM.19 Therefore, it is
pitation method. Cytotoxicity tests showed good biocompatibility
of these nanoparticles. We believe that these AIE polymersomes
based on polypeptoids will have potential application in bio-imaging
and drug delivery.
We first synthesized Sar-NTA and the novel monomer NAG-NTA
through a phosgene-free method (Fig. S1–S6, ESI†).5 Homo-
polymerization of NAG-NTA initiated by benzylamine in acetoni-
trile at 60 1C was then studied (Table S1, ESI†). The well-controlled
characteristics of NAG-NTA polymerization based on single-site
initiation by benzylamine were supported by MALDI-ToF mass
spectra, 1H NMR spectra and SEC traces (see ESI† and Fig. S8–S10
for details). PNAG can be modified easily through a thiol–ene
radical addition reaction with mercaptan, e.g. mercaptoacetic
acid, mercaptoethanol, and benzyl mercaptan (Scheme S5, ESI†).
The 1H NMR spectra (Fig. S9, ESI†) indicated the quantitative
transformation of allyl groups to the selected functional groups.
PNAG-b-PSar with three different molecular weights were
then synthesized by sequential feeding of different amounts of
NAG-NTA and Sar-NTA (Fig. 1A). The successful synthesis of
PNAG-b-PSar was proved by 1H NMR spectra and SEC traces
Fig. 2 (A) Photo-luminescence (PL) spectra of P(TPE-NAG)25-b-PSar46 in DMF/
(see Fig. 1B and Fig. S11 and discussion in the ESI†). The H2O mixtures with different water fractions (concentration: 2.2 Â 10À5 M,
0.4 mg mLÀ1; excitation wavelength: 370 nm). The inserted photographs in
compositions of the three block copolymers were PNAG10-b-
PSar53, PNAG15-b-PSar54 and PNAG25-b-PSar46 as calculated
from NMR data.
(A) show samples with different water fractions under UV light at 365 nm.
(B) DLS profiles (Z-average with intensity and number average) of aggregates of
P(TPE-NAG)25-b-PSar46 in a DMF/H2O mixture with a water fraction of 99%.
AIE luminogens were attached to the block copolymers by
two-step reactions (Fig. 1A). PNAG-b-PSar was first reacted with DMF/H2O mixture with a water fraction of 99%.
(C and D) cryo-EM images of aggregates of P(TPE-NAG)25-b-PSar46 in a
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normal that the sizes measured by cryo-EM are smaller than the DLS curves are given here (Fig. 3A and D). The smaller one
hydrodynamic diameters. Meanwhile, a few vesicles (Fig. 2D corresponds to the micelles, while the larger one (very few in
and Fig. S15, ESI†) are also observed, and some of them have number as shown in the number profile of DLS) may be attributed
very big size, which corresponds effectively to the second to a few aggregates (clusters) of micelles (Fig. 3G and Fig. S16,
population of particles in DLS curves. These observations ESI†). When the fPsar decreases to 30% (P(TPE-NAG)15-b-PSar54),
showed that the copolypeptoid P(TPE-NAG)25-b-PSar46 had the both micelles and polymersomes were observed by cryo-EM
tendency to form vesicular structures. However, well defined (Fig. 3H and Fig. S18, ESI†) and TEM (Fig. S19, ESI†), whose
polymersomes were not obtained probably because of the quick diameters are 26 nm and 357 nm, respectively. The DLS profiles of
addition of a large quantity of water.
P(TPE-NAG)15-b-PSar54 nanoparticles show double peaks with
Then, we used a nanoprecipitation method with DMF/water as hydrodynamic diameters of 38 nm and 203 nm (Fig. 3B), which
the co-solvent and a slow water addition process to self-assemble is consistent with the TEM results. Note that the polymersomes
P(TPE-NAG)-b-PSar in a more controlled way. Typically, P(TPE- were a minor population as shown in the DLS number profile and
NAG)-b-PSar was first dissolved in 1 mL DMF at a concentration connected or fused polymersomes were observed in some cases
of 2 mg mLÀ1, and deionized water was then added very slowly (Fig. S18, ESI†). In the case of PNAG25-b-PSar46 ( fPsar = 18%), only
(2.5 mL minÀ1) with slight shaking. The slow rate of water addition polymersomes are formed according to cryo-EM images (Fig. 3I
could avoid the early freezing of the nanostructure and keep the and Fig. S20, ESI†), with two distributions of sizes: one around a
self-assembly dynamic as long as possible. When the water content Z-average diameter of 804 nm, and another around a Z-average
was increased to around 70% by volume, the dispersions were diameter of 136 nm as measured by DLS (Fig. 3C and F). As for the
dialyzed against deionized water to remove DMF and get P(TPE- sizes and size distributions, the results obtained from cryo-EM and
NAG)-b-PSar self-assemblies in pure water. Table S2 (ESI†) sum- DLS (Table S2, ESI†) are coherent to some extent.
marizes the hydrodynamic diameters of all nanoparticles and
With the increase of the hydrophobic P(TPE-NAG) chain
membrane thicknesses of the polymersomes. For P(TPE-NAG)10- length and the decrease of the hydrophilic ratio fPsar (here the
b-PSar53 ( fPsar = 38%), micelles with a diameter of around 26 nm length of the hydrophilic part PSar is similar for three copoly-
are mainly observed by cryo-EM (Fig. 3G and Fig. S16, ESI†) and peptoids), the nanostructures transform from micelles to vesi-
TEM (Fig. S17, ESI†). The DLS intensity profile shows a bimodal cles and their average sizes become larger. These tendencies
distribution (Fig. 3A) with hydrodynamic diameters of 29 nm and can be empirically explained by the increase of the packing
312 nm, respectively. When there are several populations of parameter p = v/al (v is the hydrophobic volume, a is the
nanoparticles, it is difficult to quantify the statistics with only optimal interfacial area and l is the length of the hydrophobic
one kind of size profile in DLS because the intensity profile favours block normal to the interface) when increasing the hydropho-
the big particles and the number profile favours the small bic block MW, as already reported previously.15,20,21
particles. Therefore, both intensity-weighed and number-weighed
According to the cryo-EM images, the thickness of the hydro-
phobic part of the membrane is measured to be 8 Æ 1 nm and
10 Æ 1 nm for P(TPE-NAG)15-b-PSar54 and P(TPE-NAG)25-b-PSar46
polymersomes, respectively. By using the MM2 energy minimi-
zation with the Chem3D software, the contour lengths of the
hydrophobic part P(TPE-NAG)15 and P(TPE-NAG)25 are about
4.4 nm and 7.3 nm, respectively. Consequently, a perfectly-
ordered tail-to-tail bilayer should have a membrane thickness
of 8.8 nm and 14.6 nm, respectively. The membrane thickness
measured by cryo-EM is lower than the double lengths of two
extended P(TPE-NAG) chains. Therefore, the obtained polymer-
somes should be unilamellar vesicles, in which the P(TPE-NAG)
chains might not be totally extended or might be interdigitated
between the two leaflets.
Then, we investigated the fluorescence of these well-defined
nanostructures formed by P(TPE-NAG)-b-PSar. The DMF solution
of P(TPE-NAG)-b-PSar is non-emissive while the aqueous solution
of P(TPE-NAG)-b-PSar micelles and vesicles shows strong emission
under a 365 nm UV lamp (Fig. 4A) due to the RIM of TPE moieties
in the core of polymer micelles and the polymersome membrane
as expected. The emission spectra of the P(TPE-NAG)10-b-PSar53,
P(TPE-NAG)15-b-PSar54 and P(TPE-NAG)25-b-PSar46 nanoparticles
are shown in Fig. 4B with maximal emission wavelengths of
478 nm, 474 nm and 471 nm, respectively, the excitation wave-
length being set as 370 nm which corresponded to the maximal
wavelength in the excitation spectrum (Fig. S21, ESI†). Rather high
Fig. 3 DLS profiles (Z-average with intensity (A–C) and number average
(D–F)) and cryo-EM images of nanoparticles in water formed by P(TPE-
NAG)10-b-PSar53 (fPsar = 38%) (A, D and G), P(TPE-NAG)15-b-PSar54 (fPsar
=
30%) (B, E and H) and P(TPE-NAG)25-b-PSar46 (fPsar = 18%) (C, F and I). The
dark densities present at the center of the polymersomes are caused by
the scattering of electrons due to the inherent thickness of the polymer-
some spheres.
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P(TPE-NAG)15-b-PSar54 and P(TPE-NAG)25-b-PSar46, respectively.
All the nanoparticles show strong AIE fluorescence in aqueous
solution. These AIE polymer micelles and AIE polymersomes
made of block copolypeptoids are non-cytotoxic, which guaran-
tees their safety in potential applications in bio-related fields.
Financial support from French National Research Agency
(ANR-16-CE29-0028) and the National Natural Science Founda-
tion of China (21674091) is acknowledged. We thank Prof. Ben
Zhong Tang (Hong Kong University of Science and Technology)
for fruitful discussions. We thank Prof. Zhengwei Mao (Zhejiang
University) for cytotoxicity analysis, and Dr Chao Deng (Zhejiang
University) for quantum yield measurement. The authors thank
the PICT-Ibisa for providing access to the cryo-EM facility at
Institut Curie in Orsay.
Fig. 4 (A) Schematic diagram of the self-assembly of P(TPE-NAG)-b-PSar
in aqueous solution and the AIE phenomenon of the polymersome
dispersion. (B) PL spectra of different nanoparticles (concentration:
0.4 mg mLÀ1; excitation wavelength: 370 nm). (C) Relative cell viability of
P(TPE-NAG)25-b-PSar46 polymersomes for human vein endothelial cells
Conflicts of interest
after 24 h incubation at a concentration of 23.7 mg mLÀ1, 47.5 mg mLÀ1
,
There are no conflicts to declare.
95.0 mg mLÀ1, 190 mg mLÀ1, 380 mg mLÀ1 and 760 mg mLÀ1, respectively.
Notes and references
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