Unimolecular Polypeptide Micelles via Ultrafast Polymerization of N-Carboxyanhydrides

Article abstract of DOI:10.1021/jacs.0c01173

Polypeptide micelles are widely used as biocompatible nanoplatforms but often suffer from their poor structural stability. Unimolecular polypeptide micelles can effectively address the structure instability issue, but their synthesis with uniform structure and well-controlled and desired sizes remains challenging. Herein we report the convenient preparation of spherical unimolecular micelles through dendritic polyamine-initiated ultrafast ring-opening polymerization of N-carboxyanhydrides (NCAs). Synthetic polypeptides with exceptionally high molecular weights (up to 85 MDa) and low dispersity (? < 1.05) can be readily obtained, which are the biggest synthetic polypeptides ever reported. The degree of polymerization was controlled in a vast range (25-3200), giving access to nearly monodisperse unimolecular micelles with predictable sizes. Many NCA monomers can be polymerized using this ultrafast polymerization method, which enables the incorporation of various structural and functional moieties into the unimolecular micelles. Because of the simplicity of the synthesis and superior control over the structure, the unimolecular polypeptide micelles may find applications in nanomedicine, supermolecular chemistry, and bionanotechnology.

Full text of DOI:10.1021/jacs.0c01173

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Communication
Unimolecular Polypeptide Micelles via Ultra-
fast Polymerization of N-Carboxyanhydrides
Shixian Lv, Hojun Kim, Lin Feng, Ziyuan Song, Yingfeng Yang, Ryan Baumgartner,
Kuan-Ying Tseng, Shen J. Dillon, Cecilia Leal, Lichen Yin, and Jianjun Cheng
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.0c01173 • Publication Date (Web): 20 Mar 2020
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Unimolecular Polypeptide Micelles via Ultra-fast Polymerization of
N-Carboxyanhydrides
Shixian Lv,†,‡ Hojun Kim, †, § Ziyuan Song,† Lin Feng,† Yingfeng Yang,† Ryan Baumgartner,† Kuan-
Ying Tseng,† Shen J. Dillon,† Cecilia Leal,† Lichen Yin,*,‡ Jianjun Cheng*,†
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† Department of Materials Science and Engineering, University of Illinois at Urbana-Champaign, Urbana, Illinois, 61801,
United States
§ Center for Biomaterials, Biomedical Research Institute, Korea Institute of Science and Technology (KIST), Seoul 02792,
Republic of Korea
‡ Jiangsu Key Laboratory for Carbon-Based Functional Materials and Devices, Institute of Functional Nano and Soft
Materials (FUNSOM), Collaborative Innovation Center of Suzhou Nano Science and Technology, Soochow University,
Suzhou, 215123, China
Supporting Information Placeholder
success has only limited to a few specific systems, although
micellar drug delivery systems have been studied for 2-3 decades.
ABSTRACT: Polypeptide micelles are widely used as
biocompatible nano-platforms, but often suffer from their poor
structural stability. Unimolecular polypeptide micelles can
effectively address the structure instability issue, but their
synthesis with uniform structure, well controlled and desired sizes
remains challenging. Herein, we report the convenient preparation
of spherical unimolecular micelles through dendritic polyamine-
initiated ultrafast ring-opening polymerization of N-
carboxyanhydride (NCA). Synthetic polypeptides with
exceptionally high MWs (up to 85 MDa) and low dispersity (Ð <
1.05) can be readily obtained, which are the biggest synthetic
polypeptides ever reported. The degree of polymerization was
controlled in a vast range (25 - 3200), giving access to nearly
monodisperse unimolecular micelles with predictable sizes. Many
NCA monomers can be polymerized using this ultrafast
polymerization method, which enables the incorporation of
various structural and functional moieties to the unimolecular
micelles. Given the simplicity of the synthesis and superior
control over the structure, the unimolecular polypeptide micelles
may find applications in nanomedicine, supermolecular chemistry
and bio-nanotechnology.
To overcome the instability of self-assembled micelles, efforts
have been made to develop cross-linking strategies or design
unimolecular micelles.13-16 Unimolecular micelles are defined as a
type of macromolecules having three-dimensional/colloidal
structure analogous to conventional micelles but the polymer
chains are covalently bound together.17-19 Due to this unique
architecture, unimolecular micelles possess excellent stability
against extreme conditions.20-23 Unimolecular micelles including
dendrimers, hyperbranched polymers and cylindrical brushes are
usually prepared via step-by-step synthesis or polymerization.24-28
However, these methods still have limitations. For instance, step-
by-step synthesis is able to produce monodisperse polymers, but
is only economic for low molecule weights (MWs).
Polymerization methods (e.g., grafting-through/from) can give
high MW, while the control of polymers at high MWs (e.g., ˃ 106
Da) is still unsatisfactory for most polymerization systems.29-31
Therefore, the development of simple methods to prepare
unimolecular micelles with predictable size and low dispersity is
still in high demand.
Polymeric micelles have attracted enormous attention for
applications in drug/gene delivery.1-2 Conventional polymeric
micelles are spherical and prepared by self-assembly of
amphiphilic copolymers which is driven by various intermolecular
forces such as hydrophobic and electrostatic interaction.3-6
However, several key drawbacks of polymeric micelles have
limited their potential as delivery vehicles for clinical application.
Polymeric micelles are usually heterogeneous mixtures consisting
of not only micelles but also free polymers, even above the
critical micelle concentration (CMC),7-8 which make it very
challenging for chemistry, manufacturing and controls in micellar
drug development. Despite the ease of preparation via copolymer
self-assembling, polymeric micelles generally suffer from their
structural instability,9-10 and may easily dissemble to free
polymers under dilute conditions or exposed to environmental
variations (e.g., pH and ionic strength).11-12 These drawbacks
have hindered the applications of polymeric micelles. Clinical
Scheme 1. Spherical unimolecular micelles from surface NCA
polymerization.
Recently, a polypeptide-based cooperative polymerization was
reported in solvents with low dielectric constants such as
dichloromethane (DCM).32-33 The unique feature in this
polymerization is that the rate of a polymer’s chain growth is
catalyzed by its own macromolecular structure. The system
consists of a linear scaffold with a high density of initiating
groups, where amino acid N-carboxyanhydride (NCA) monomers
can grow into polypeptide brushes. After reaching a critical chain
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length, the polypeptides fold into α-helices, which dramatically
conversions (˃ 98%) even at extremely high DP, yielding
polymers of exceptionally high MW via a one-pot reaction (Table
S2). The G5-inititated polymerization at the [M]0/[I]0 of 3200
yielded the synthetic polypeptide with highest DP and MW ever
reported (Mn = 8.5 × 107 Da), almost 20-fold bigger than the
largest protein identified (titin, ~4 000 kDa).36
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enhance the polymerization rate through their cooperative
macrodipole interactions among neighboring α-helices.32 We
hypothesize that a cooperative NCA polymerization similar to that
observed with a brush macroinitiator may proceed on the
spherical surface of macro-initiators in proximity (Scheme 1),
allowing synthesis of uniform polypeptide unimolecular micelles
with the same spherical morphology adopted by most polymeric
micelles.
Such elegant control of polymerization in DCM indicated that
the PAMAM-initiated NCA polymerization may also have
cooperativity due to proximity of α-helices. To verify this
assumption, in situ Fourier transform infrared (FT-IR)
spectroscopy was utilized to monitor the detailed progress of the
polymerization (Figure 2a). It was noted that the surface NCA
polymerization occurred at a very high rate in DCM similar to the
previously published brush polymerization system.32 Particularly,
at the [M]0/[I]0 of 100, NCA conversion was > 99% within 8 min
(Figure 2b). And at higher [M]0/[I]0 ratio (400), the
polymerization was almost complete within 40 min (Figure 2c).
Due to the ultra-fast polymerization kinetics, we did not observe
the first, slow stage. However, the fast kinetics is consistent with
the previously reported cooperative polymerization systems.32, 37
Monitoring changes of circular dichroism (CD) at 222 nm during
the polymerization further confirmed the formation of right-
handed α-helical structures (Figure S5). The initial slow increase
of CD signal at 222 nm in the first 90 s was consistent with the
previously reported results, indicating a two-stage, cooperative
polymerization kinetics.32 In addition, compared to the typical
slow polymerization initiated by hexylamine (monomer
conversion of 37% within 24 h at the [M]0/[I]0 ratio of 100, Figure
S6), such fast polymerization further indicated the cooperative
NCA polymerization at the surface of the spherical macro-
initiators.
To validate this hypothesis, we selected polyamidoamine
(PAMAM) dendrimers as the macro-initiators (denoted as Gx
where x represents the generation), because they adopt nearly
spherical structure and the primary amines anchored on the
surface can serve to initiate NCA polymerization. γ-Benzyl-L-
glutamate NCA (BLG-NCA) was chosen as a model monomer
due to its convenient synthetic as well as purification process and
good solubility in various solvents. The resulting polymer, poly(γ-
benzyl-L-glutamate) (PBLG), is known to adopt α-helical
conformation under various conditions,34-35 thus allowing
acceleration of the polymerization through inter-helix cooperative
macrodipole interactions. The polymerization was first performed
in DCM with low dielectric constant, which minimized the
disruption of the macrodipole interaction between α-helices. The
obtained polymers with different degree of polymerizations (DPs)
were termed as Gx-g-PBLGn (n = designed DP), and gel
permeation chromatography (GPC) was utilized to characterize
the MWs of polymers. The DP values refer particularly to the
feeding ratio of monomer to the primary amines of PAMAM.
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Figure 1. (a) Chemical structures of PAMAM and BLG-NCA.
(b) GPC traces of polymers synthesized from different PAMAM
initiators. (C) GPC traces of polymers synthesized from G5 at
various designed DPs. (D) MWs and dispersity of G5-g-PBLG
polymerized in DCM at the [M]0/[I]0 ratio from 25 to 3200.
Figure 2. (a) FT-IR spectra showing the conversion of BLG-
NCA initiated by G5 in DCM. (b) Conversion of BLG-NCA
initiated by different PAMAM initiators at the [M]0/[I]0 ratio of
100. (c) Conversion of BLG-NCA initiated by G5 at
various[M]0/[I]0 ratios. (d) Conversion of BLG-NCA, BDG-NCA,
and BDLG-NCA initiated by G5 at the [M]0/[I]0 ratio of 100. All
the polymerizations were conducted in DCM.
In DCM, the polymerization of BLG-NCA conducted with
different PAMAM macroinitiators resulted in polypeptides with
predictable MWs and low dispersity (Figure 1b and Table S1).
GPC light scattering (LS) and differential refractive index (dRI)
responses demonstrated the monomodal distributions for all the
resulting polymers (Figure S1-2). In addition, such controlled
polymerization can proceed at a wide range of monomer/initiator
ratio ([M]0/[I]0, the initiator refers to total primary amines) (from
25 to 3200), yielding polymers with predictable MW (Figure 1c,
S3 and Table S2). The dispersity values remained low at
extremely high DP of 3200 (˂1.05, Figure 1d). Further addition
of monomers into the pre-existing polymer can successfully
extend the polymer chains with low dispersity and expected MW,
indicating the living feature of the polymerization (Figure S4).
Moreover, the polymerization featured almost complete monomer
To further confirm the necessity of α-helices to induce fast
polymerization rate, we polymerized the racemic monomer
(BDLG-NCA) instead of enantiopure monomers. It was observed
that NCA monomers of L or D enantiomers can be polymerized
with approximately the same rate (Figure 2d), and the PBDG
polymer also showed predictable MW and low dispersity values
(Figure S7 and Table S3). However, racemic BDLG-NCA
exhibited dramatically slower polymerization, and it took over
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100 min to reach > 98% monomer consumption at the [M]0/[I]0
DP (˂400), which was largely attributed to the low initiation
efficiency because the ultra-fast polymer rate.32 Cryogenic
transmission electron microscopy (cryo TEM) was further utilized
to observe the morphology of micelles. As expected, the micelles
are spheroidal and uniform (Figure 3d). The contrast of the cryo
TEM image was not very clear because the solvent was DMF, not
water. Due to the uniform structure, the initiating efficiency of the
polymerization can be estimated assuming that the polymers
grown from each surface amine groups had the similar chain
length. Based on the average length of an α-helix (0.15 nm per
amino acid residue) and the size of G5 PAMAM (5.4 nm), the
theoretical sizes of the unimolecular micelles can be obtained
assuming 100% initiating efficiencies (Figure 3C). Therefore, the
initiating efficiencies of the G5-PBLG50/100/200 polymers were
estimated to be 65%, 83% and 91%, respectively. The sizes of the
micelles with higher DP were nearly identical with the theoretical
values, revealing that the initiating efficiencies were very close to
100%. After removal of the benzyl groups, uniform water-soluble
poly(L-glutamic acid) unimolecular micelles can be readily
obtained (Figure S16-20).
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ratio of 100, suggesting that the formation of α-helices was
essential for the polymerization rate enhancement (Figure S8-10).
Additionally, the polymerization cannot reach sufficient NCA
consumption using the racemic BDLG-NCA at higher DP (˃ 200),
resulting in polymers with lower MWs then expected (Figure S11).
GPC traces of the obtained racemic PBDLG polypeptide showed
multiple distributions at higher DP (˃ 400), suggesting
uncontrolled polymerization. These results thus collectively
demonstrated that the well-controlled and rate-accelerating
surface NCA polymerization was driven by interaction of
neighboring α-helices.
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As the interaction of α-helices can be affected by the
surrounding environment, the effect of solvent on the
polymerization was then investigated. In another chlorinated
solvent, chloroform (CHCl3), the polymerization still featured
excellent control as exemplified by the fast rate, accurate MWs,
and low dispersity (Figure S12 and Table S4). However, when
conducted in N, N-dimethylformamide (DMF), the polymerization
was poorly controlled, yielding polymers with low MW and
extremely broad distribution (Figure S13-14). Increasing the
[M]0/[I]0 ratio resulted in negligible peak shift in the GPC traces,
indicating the inability to get high-MW polypeptides. It was
because DMF is a strong polar solvent with high dielectric
constant, which could eliminate not only the hydrogen bonding
interactions between polypeptides and NCA monomers, but also
the electrostatic interactions resulting from the macrodipole
moments of α-helices.32, 38 Taken together, these results confirmed
that the surface NCA polymerization in non-polar solvents can
serve as a facile method to fabricate high-MW and monodisperse
polypeptides.
We next investigated whether the strategy can be utilized for
other NCA monomers. Firstly, we polymerized γ-(4-
propargyloxybenzyl)-L-glutamate N-carboxyanhydride (POBLG-
NCA) in DCM. FT-IR study confirmed the fast conversion of
POBLG-NCA (Figure S21), and GPC analysis showed that these
polymers had accurate MW and narrow dispersity (Figure S22
and Table S5). DLS analysis demonstrated narrow size
distributions of the unimolecular micelles (Figure S23).
Additionally,
benzyloxycarbonyl-L-lysine N-carboxyanhydride (Lys(Z)-NCA)
and γ-(4-allyloxylbenzyl)-L-glutamate N-carboxyanhydride
other
NCA
monomers
such as
Nε-
(AOBLG-NCA) can also be applied in the current system (Figure
S24-25). It therefore suggested that size-controllable, polypeptide-
based unimolecular micelles bearing functional motifs can be
readily fabricated.
In conclusion, we report an unprecedented NCA
polymerization with fast rates and superior polymerization control
from the dendritic polymer surface. Polymers with extremely high
MW and low dispersity can be easily synthesized in the one-pot
system. To the best of our knowledge, these are the highest MW
synthetical polypeptides that have been reported. These polymers
can form spherical unimolecular micelles with very narrow
dispersity. This methodology enables precise control of micelle
sizes by tuning the DP of polymers, a feat seldomly achieved in
polymeric micellar system. Monomers with reactive handles (e.g.,
alkynyl and alkenyl groups) can be utilized in this system,
enabling the design of functional unimolecular micelles. Due to
the intrinsic stability of unimolecular micelles and the unique
properties of polypeptides, this study provides a simple and robust
strategy for the design of stable and unimolecular polypeptide
nanoparticles, which will find promising applications in the field
of biomimetic supramolecular chemistry, nanotechnology and
biomedicine.
Figure 3. (a) Size distributions of various unimolecular
micelles. (b) Amplified DLS histogram of G5-g-PBLG400
micelles. (c) Diameters and dispersity values of the unimolecular
micelles determined by DLS (*theoretical sizes). (d) Cryo TEM
image of G5-g-PBLG400 micelles in DMF (scale bar = 200 nm).
ASSOCIATED CONTENT
Supporting Information
It was expected that such low-dispersity polymers can form
uniform spherical unimolecular micelles. In support of such
assumption, the sizes of the obtained polymers were determined
by dynamic light scattering (DLS) after dissolved in DMF. In
consistence with their low dispersity, the polypeptides initiated by
PAMAM initiators of different generations demonstrated
extraordinarily narrow size distributions (Figure 3a, 3b and S15).
In addition, sizes of the unimolecular micelles were closely
correlated to their MWs (Figure 3c), enabling the precise control
over the particle size by tuning the DPs of polymers. The sizes of
micelles were slightly bigger than the theoretical values at lower
Experimental procedures and characterizations are included in
supporting information. This material is available free of charge
via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
jianjunc@illinois.edu; lcyin@suda.edu.cn.
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Notes
The authors declare no competing financial interests.
ACKNOWLEDGMENT
This research was financially supported by National Science
Foundation (CHE 1709820), the Ministry of Science and
Technology of China (2016YFA0201200), the National Natural
Science Foundation of China (51873142, 51573123 and
51722305), 111 project, and Priority Academic Program
Development of Jiangsu Higher Education Institutions (PAPD).
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polymerization of polypeptide helices. Nat. Commun. 2019, 10.
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