Secondary-Structure-Driven Self-Assembly of Reactive Polypept(o)ides: Controlling Size, Shape, and Function of Core Cross-Linked Nanostructures

Article abstract of DOI:10.1002/anie.201702624

Achieving precise control over the morphology and function of polymeric nanostructures during self-assembly remains a challenge in materials as well as biomedical science, especially when independent control over particle properties is desired. Herein, we report on nanostructures derived from amphiphilic block copolypept(o)ides by secondary-structure-directed self-assembly, presenting a strategy to adjust core polarity and function separately from particle preparation in a bioreversible manner. The peptide-inherent process of secondary-structure formation allows for the synthesis of spherical and worm-like core-cross-linked architectures from the same block copolymer, introducing a simple yet powerful approach to versatile peptide-based core–shell nanostructures.

Full text of DOI:10.1002/anie.201702624

A Journal of the Gesellschaft Deutscher Chemiker
Angewandte
Chemie
International Edition
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Accepted Article
Title: Secondary Structure-Driven Self-Assembly of Reactive
Polypept(o)ides: Controlling Size, Shape and Function of Core
Cross-Linked Nanostructures
Authors: Kristina Klinker, Olga Schäfer, David Huesmann, Tobias
Bauer, Leon Capelôa, Lydia Braun, Natascha Stergiou, Meike
Schinnerer, Anjaneyulu Dirisala, Kanjiro Miyata, Kensuke
Osada, Horacio Cabral, Kazunori Kataoka, and Matthias Barz
This manuscript has been accepted after peer review and appears as an
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of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
published online in Early View as soon as possible and may be different
to this Accepted Article as a result of editing. Readers should obtain
the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.201702624
Angew. Chem. 10.1002/ange.201702624
Link to VoR: http://dx.doi.org/10.1002/anie.201702624
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10.1002/anie.201702624
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Secondary Structure-Driven Self-Assembly of Reactive
Polypept(o)ides: Controlling Size, Shape and Function of Core
Cross-Linked Nanostructures
Kristina Klinker+, Olga Schäfer+, David Huesmann, Tobias Bauer, Leon Capelôa, Lydia Braun,
Natascha Stergiou, Meike Schinnerer, Anjaneyulu Dirisala, Kanjiro Miyata, Kensuke Osada, Horacio
Cabral, Kazunori Kataoka, Matthias Barz*
Controlling the self-assembly of block copolymers in solution
offers a versatile and powerful method to create nanometer-
sized particles with defined and controllable geometry, size, and
functionality.[1–4] Size, shape and internal structure of nano-sized
objects obtained by aggregation of amphiphilic coil-coil block
copolymers in a block selective solvent in thermodynamic
equilibrium is mainly dictated by the ratio of the volume fraction
between the solvophilic and solvophobic block.[5] The self-
assembly of rod-coil block copolymers is additionally driven by
aggregation of the rod segment and the formation of
orientational order, which may lead to morphologies different
from those obtained from typical coil-coil polymers.[6–9] In
crystallization-driven self-assembly, different types of polymers
have been described as rod segments throughout the literature,
among them polyferrocenylsilane (PFS) block copolymers with
poly(dimethyl-siloxane) (PDMS), polyisoprene (PI) and
poly(lactic acid).[10–12] In contrast to this type of organization,
anisotropic nanomaterials can also be obtained by controlled
aggregation of peptide amphiphiles (PAs), a concept well-
sequence-defined peptides.[16–19] Approaches using ring-opening
polymerization of α-amino acid N-carboxyanhydrides (NCAs) to
create the peptidic rod-like segment have also been used to
stabilize micellar aggregates in vivo,[20] to induce aggregation of
oligomers or to create smart materials that can be switched from
rod-coil to coil-coil using external stimuli such as temperature,
pH and chaotropic agents.[21,22] However, approaches utilizing
synthetic polypeptides derived by NCA polymerization are
mostly restricted to α-helical motifs since synthesis of β-sheet
forming polymers is not trivial to achieve.[23] Therefore, directing
morphology of polymeric micelles by β-sheet formation of the
hydrophobic core-forming synthetic polypeptide has not yet been
realized.
Cysteine is a particularly interesting amino acid because of
its thiol side chain for potential bio-reversible modification and its
propensity to adopt a β-sheet conformation in solution.[24] Our
group has recently introduced S-alkylsulfonyl-protected cysteine
NCAs,[25] which enable controlled ring-opening polymerization,
yielding average chain lengths up to 30 repeat units of well-
established in supramolecular chemistry.[7,13–15] In these systems, defined
self-assembly is predominantly driven by secondary structure
formation of the peptide segment. It is noteworthy that β-sheet-
driven polypeptide self-assembly is so far mostly limited to
polymers
with
intrinsic
anti-paralell
ß-sheet
[*]
K. Klinker[+] O. Schäfer[+] Dr. D. Huesmann, T. Bauer, L. Capelôa, L.
Braun, M. Schinnerer, Dr. M. Barz
Institute of Organic Chemistry
Johannes Gutenberg University Mainz
Duesbergweg 10-14, 55128 Mainz, Germany
E-mail: barz@uni-mainz.de
K. Klinker[+]
Graduate School Materials Science in Mainz,
Staudinger Weg 9, 55128 Mainz, Germany
N. Stergiou
University Medical Center-Institute of Immunology
Johannes Gutenberg University Mainz
Langenbeckstrasse 1, Geb. 708, 55101 Mainz, Germany
Dr. A. Dirisala, Prof. Dr. K. Kataoka
Innovation Center of NanoMedicine
KAWASAKI Institute of Industrial Promotion
3-25-14, Tonomachi, Kawasaki-ku, Kawasaki 210-0821
Prof. Dr. K. Miyata
Department of Materials Engineering
Graduate School of Engineering, The University of Tokyo
7-3-1 Hongo, Bunkyo-ku, Tokyo 113–8656, Japan
Prof. Dr. K. Osada, Prof. Dr. H. Cabral,
Department of Bioengineering
Graduate School of Engineering, The University of Tokyo
7-3-1 Hongo, Bunkyo-ku, Tokyo 113–8656, Japan
Prof. Dr. K. Kataoka
Figure 1. A) Schematic representation of anti-paralell ß-sheet formation
and B) suppressed secondary structures by chaotropic agents (e.g.,
thiourea) C) Illustration of the proposed platform featuring the following
phases: (I) self-assembly of amphiphilic PSarn-b-PCys(SO2Et)m block
copolymers into different micellar morphologies directed by secondary
structure alignment, (II) subsequent core cross-linking by disulfides of
choice, yielding core cross-linked polymeric micelles (CCPM) or
Policy Alternatives Research Institute, The University of Tokyo
7-3-1 Hongo, Bunkyo-ku, Tokyo 113–8656, Japan
[+]
These authors contributed equally to this work.
nanonydrogels (NHG) and (III) disintegration of the system upon
reductive stimulus.
a
Supporting information for this article is available under:
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10.1002/anie.201702624
Angewandte Chemie International Edition
COMMUNICATION
conformation.[26] Employed as a building block in polypept(o)ides,
a unique material can be obtained that combines functionality,
stimuli-responsiveness, and secondary structure formation of
polypeptides with the stealth-like properties of the coil-forming
polypeptoid polysarcosine (PSar).[27] Keeping in mind that
secondary structure of proteins can be denatured by different
external stimuli,[28] the self-assembly of these block
copolypept(o)ides should be controllable by switching from rod-
coil block copolymers (intact β-sheet conformation, Figure 1A) to
coil-coil polymers by a chaotropic agent (disturbed β-sheet
conformation, Figure 1B).
Apart from constituting the driving force for self-assembly
(Figure 1CI), the S-alkyl sulfonyl cysteine block remains highly
reactive
towards
thiols
after
polymerization.[29]
This
chemoselective reactivity enables core cross-linking by
asymmetric disulfides (Figure 1CII), which seems suitable to
preserve the formed nanoparticle morphologies. PSarn-b-
PCys(SO2Et)m block copolymers can be obtained by NCA
polymerization using
a
bifunctional initiator as reported
previously (Scheme S1).[30] Block copolymers with differing block
length ratios and total block lengths were successfully prepared
with dispersity indices (Đ) between 1.2 - 1.4 (Table S1 and
Figure S1). Self-assembly of PSarn-b-PCys(SO2Et)m block
copolymers was investigated methodically by varying two major
factors contributing to the overall morphology: the composition of
the amphiphile as well as intact or suppressed secondary
structure by addition of the chaotropic agent thiourea (TU). The
percentage of the hydrophobic cysteine block was kept at 15
wt.% (P1 Xn/Xm = 407:28, P2 Xn/Xm = 265:17, determined by
1H NMR), and 30 wt.% (P3 Xn/Xm 97:15, determined by 1H NMR),
respectively. To obtain well-defined micelles, block copolymers
were first dissolved in N,N-dimethylacetamide (DMAc),
aggregated in the presence of 20 vol.% 1 mM acetate buffer
(Figure S2) and subsequently dialyzed against 1mM acetate
buffer to allow the formation of thermodynamically favored
structures. For ß-sheet suppression (Figure 1C II), 1 M TU in
DMAc and 10 mM in buffer was used. Upon solvent exchange,
micelles were core cross-linked by disulfides to stabilize the
formed aggregates (triethylenetetramine α,ω-di(cysteine)diamide
c2 was used throughout Figure 2 yielding nanohydrogels, see
also Table S2). As obtained morphologies were fixated by cross-
linking, size distributions remained fully preserved (Figure 3BI),
thus enabling detailed characterization of the particles using
dynamic light scattering (DLS), circular dichroism (CD),
transmission electron microscopy (TEM) and atomic force
microscopy (AFM).
Figure 2. AI) Illustrated core framework of worm-like particles with twisted
ß-sheets in the absence of a chaotropic agent. (II) Size range of the
obtained core cross-linked nanohydrogels 1,2, and 4 (DLS). (III) Illustrated
core framework of spherical particles with interwined polymer chains in
random coil conformation due to the presence of a chaotropic agent. B)
Shape control of nanohydrogels
1 and 2 (visualized by AFM/TEM)
modulated by presence or absence of secondary structure stabilization
(from P1, 15 wt.% of hydrophobic segment). C) Nanohydrogels from a
shorter block copolymer (P2, 15 wt.%) exhibit either a mixed morphology in
the presence of secondary structures 3 or small-scale spheres 4 (AFM) D)
Worm-like nanohydrogels 5 and 6 (AFM) obtained independently from
secondary structure stabilization due to a higher weight percentage of the
hydrophobic segment of P3 (30 wt.%).
As determined by cumulant analysis of DLS, P1 formed
structures with Dh = 77 nm (1) in the absence of TU and
considerably smaller structures with Dh = 51 nm (2) in presence
of TU (Figure 2AII, Table 1). From a theoretical point of view,
block copolymers with 15 wt.% hydrophobic block are expected
to form spherical micelles.[5] As briefly outlined above, the self-
assembly behavior of rod-coil polymers differs considerably from
that of coil-coil polymers. In the case of ß-sheet rods,
intermolecular interactions between rod chains dominate and an
enhanced packing of crystallizing chains can lead to the
formation of form-anisotropic /elongated objects. Moreover, ß-
sheets tend to arrange in twisted structures[31] also typical for
natural peptides, e.g. ß-sheet barrels (Figure 2AI).[32] Coil-coil
polymers on the other hand favor rounded interfaces to minimize
contact between the less soluble block and the solvent (Figure
2AIII). TEM and AFM were subsequently used to elucidate the
morphology of core cross-linked micellar aggregates (Figure 2B
and S3). TEM images were stained using uranyl acetate to
visualize the polycysteine core,[33] whereas AFM shows the
dimensions of the whole particle. Both methods demonstrate the
formation of worm-like structures 1 in the rod-coil case (absence
of TU) and of spherical micelles 2 in the coil-coil case (presence
of TU). The larger Dh of 1 observed in DLS can thus be
explained by a one-dimensional growth of twisted anti-parallel ß-
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10.1002/anie.201702624
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sheet alignments into worm-like micelles. The retained internal
ß-sheet structure was further confirmed using CD before and
after core cross-linking and purification (Figure S4). Worm-like
micelles show a negative Cotton effect at approx. 220 nm,
confirming the presence of ß-sheets throughout the particle
preparation. Reliable CD data of micelles in the presence of
thiourea could not be obtained due to high UV-absorbance of
thiourea at relevant concentrations (1 M - 10 mM). Polymer P2
with the same block-to-block ratio (15 wt.%), but decreased total
chain length leads to the formation of a mixture of spherical and
worm-like micelles 3 in the absence of TU and small spherical
micelles 4 (Dh = 29 nm, Figure 2AII, C and S5) in the presence
of TU. This coexistence of morphologies most likely originates
from the fact that synthetic block copolymers bear a certain
polydispersity, indicating that not all polymer chain have attained
the critical length to successfully participate in β-sheet
alignments necessary to form exclusively elongated structures.
Hence multi-angle DLS revealed an angle-dependent scattering
behavior of 3 indicating non-spherical and/or polydisperse
samples with a µ2 of 0.17, whereas 4 showed no angular
dependency typical for spherical and uniform particles with a µ2
of 0.06 indicating low polydispersity (Figure S6). Increasing the
percentage of the hydrophobic block to 30 wt.% in polymer P3
resulted in the formation of worm-like micelles 5 and 6 in both
the presence and the absence of thiourea, only differing slightly
in hydrodynamic diameter (Dh = 62 nm and 50 nm, respectively,
Figure 2D, S5 and S7). This behavior was anticipated for coil-
coil polymers with 30 wt.% of the hydrophobic block.[5] Our
findings underline the fact that β-sheet formation of the
polycysteine block in block copolypept(o)ides substantially
influences self-assembly behavior, since it forms a rigid rod
segment by intermolecular interactions that can be transformed
into coil conformation by the chaotropic agent thiourea.
Consequently, a PSarn-b-PCys(SO2Et)m block copolymer with a
hydrophobic ratio known to form spherical micelles combined
with a certain critical total length will form elongated structures if
undisturbed (rod-coil) and spherical structures when transformed
into a coil-coil polymer in the presence of TU.
Figure 3. A) Cross-linking of self-assembled nanostructures using the thiol-
reactivity of the S-ethylsulfonyl group with various dithiols and thus
introducing bio-reversible disulfide bonds in cross-linked polymeric micelles
(CCPM, yellow core) and nynohydrogels (NHG, blue core). BI) An identical
particle size is retained throughout the use of various cross-linkers, (II)
while core functionality is changed. CI) Adjusting core polarity of CCPMs
with lipoic acid-derived cross-linkers of varying side chains, (II) without
affecting particle size.
In addition to morphology control and covalent core cross-
linking, the S-alkylsulfonyl protective group in PSarn-b-
PCys(SO2Et)m block copolymers is particular attractive for the
construction of drug delivery systems. This approach can be not
only used to incorporate functionality into the core of micelles,
but leads to bio-reversible disulfide bonds (Figure 3A and S8).
Disulfide cross-linked micelles and polyplexes are known to be
stable in blood (10 μM glutathione (GSH)) but are rapidly
cleaved inside cells (10 mM GSH).[34,35] Therefore, this type of
cross-linking appears ideal to stabilize nanoparticles while they
are in circulation, but makes them responsive to a change in
redox potential once they enter cells in order to disintegrate and
release their cargo specifically.[36] In contrast to the disulfide
cross-linking frequently reported in literature, e.g. oxidation in
the presence of oxygen[37] or DMSO,[38] the cross-linking reaction
of CysSO2Et is thiol-selective, proceeds rapidly, and yields
asymmetric disulfides in aqueous solution.[26,29] Having
established that core cross-linking does not alter the size
distribution of micelles at optimized cross-linking conditions
(Figure 3BI, Table S2), it seems highly interesting to investigate,
if the adjustment or even the inversion of the core-polarity is
possible. In this case, the desired function of the carrier system
can be introduced in one single step by the cross-linker itself,
allowing for rapid production of nanoparticle libraries from one
precursor micelle. While hydrophobic cross-linkers (e.g.,
hexanedithiol c1) form core cross-linked polymeric micelles
(CCPMs), cationic cross-linkers (e.g., triethylenetetramine α,ω-
di(cysteine)diamide c2) invert core polarity and thus lead to the
Table 1. Particle characterization of core cross-linked polymeric micelles
(CCPM) and nanohydrogels (NHG).
particle
NHG 1
polymer
P1
thiourea
morphology
worm-like
spherical
mixed
crosslinker
-
cationic (c2)
cationic (c2)
cationic (c2)
cationic (c2)
cationic (c2)
cationic (c2)
hydrophobic (c1)
hydrophobic (c1)
cationic (c2)
NHG 2
P1
+
-
NHG 3
P2
NHG 4
P2
+
-
spherical
worm-like
worm-like
spherical
spherical
worm-like
NHG 5
P3
NHG 6
P3
+
+
+
-
CCPM 8
CCPM 10
CCPM 11
P2
P2
P1
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10.1002/anie.201702624
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ζ = 0.4 mV before and ζ = 0.5 mV after loading, Figure 4BI),
which is due to effective shielding of the PSar segment. The bio-
reversible nature of siRNA complexation was evaluated by
agarose gel electrophoresis using extra- and intracellularly
relevant glutathione (GSH) concentrations (Figure 4BII). In the
absence of GSH and with 10 μM GSH (extracellular), chol-
siRNA-loaded nanohydrogels remained stable, whereas siRNA
is released at 10 mM GSH (intracellular). The successful loading
of respective core-functionalized particles as well as cargo
release upon a reductive stimulus portray the last phase of the
proposed platform (Figure 1CIII).
In summary, our findings highlight the potential of secondary
structure-driven self-assembly to control size and shape of
polymeric nanostructures in combination with adjustable core
functionality by bio-reversible core cross-linking in a separate,
single post-polymerization step. The S-ethylsulfonyl protective
group for cysteine enables the controlled NCA ring-opening
polymerization, which is exploited here for the synthesis of
amphiphilic PSarn-b-PCys(SO2Et)m block copolypept(o)ides.
Their self-assembly behavior can be directed by switching the
conformation of the polycysteine segment from rod to coil, and
tune the polymer association by manipulating the secondary
structures. In addition to size and shape control, this approach
enables complete decoupling of function from the self-assembly
process, since polarity of the micellar core is introduced only
after particle formation using dithiol-containing cross-linkers.
This novel polypept(o)ide-based nanoparticle platform can be
used to generate nanoparticle libraries for various biomedical
applications, ranging from nucleic acid delivery (RNAi) over drug
delivery to imaging and may help to further elucidate the size
and shape dependency of circulation, bio-distribution and
cellular responses.
Figure 4: A) Core cross-linked polymeric micelle 8 with cross-linker c1
loaded with PTX 10 (I) showing uniform micelles prior and after loading as
well as (II) pronounced efficacy in vitro on TD47 cells. B) Cationic
nahohydrogel with cross-linker c2 prior (1) and after complexation of siRNA
(11) (I) exhibits
a near neutral ζ-potentials and (II) enabling stimuli-
responsive release at intracellular glutathione levels (10mM).
formation of cationic nanohydrogels (NHGs) (Figure 3BII). Since
both structures are derived from polypept(o)ides, we define them
as core cross-linked PeptoMicelle and NanoPeptoGel.
Successful core cross-linking was verified by gel permeation
chromatography in hexafluoro-2-propanol (HFIP), which is a
good solvent for both blocks (Figure S9). With core cross-linked
polymeric micelles and nanohydrogels as the core polarity
extremes, a library of lipoic acid-based cross-linkers c4a-k was
synthesized to fine-tune core polarity (Figure 3CI, Table S3). In
addition to simply altering polarity, residues R of the cross-linker
c4 can readily be adjusted to match, for example, a certain
cargo, to covalently attach drugs or imaging probes.[39,40] As
shown earlier, the cross-linking reaction (for c3 and c4a-k after
equimolar reduction with tris(2-carboxyethyl)phosphine (TCEP),
Scheme S2) is independent of the cross-linker's chemical nature
and results in particles 9a-g with comparable size distributions
(Figure 3CII, Table S2). These findings underline that
nanostructure formation is completely separated from chemical
Acknowledgements
The authors would like to Prof. Angelika Kühnle for providing
AFM facilities. D.H. would like to acknowledge the support by
the “Verband der Chemischen Industrie” (VCI) and the “Max
Planck Graduate Center with the Johannes Gutenberg-
Universität Mainz” (MPGC). K.K. acknowledges the financial
support from graduate school MAINZ and is a recipient of the
fellowship through the Excellence Initiative (DFG/GSC 266). O.S.
acknowledges support by the „Evangelisches Studienwerk e.V.
Villigst“.
functionality in this approach, which is
characteristic in a new material.
a very desirable
Furthermore, the function of both core cross-linked polymeric
micelles on the one hand and nanohydrogels on the other was
investigated. Spherical CCPMs (8) were formulated with the
hydrophobic chemotherapeutic agent paclitaxel (PTX), yielding
CCPMs 10 with Dh = 39 nm. Hydrodynamic diameters did not
change significantly before and after loading with paclitaxel
(Figure 4AI, Table S2). Successful loading and release was
confirmed indirectly by in vitro cell experiments using human
breast cancer cells (TD47 cells). While PTX-loaded CCPMs led
to a substantially decreased cell viability, comparable to free
paclitaxel, empty CCPMs were non-toxic (Figure 4AII). On the
other hand, worm-like nanohydrogels 1 enable complexation of
chol-siRNA at N/P ratios of 8 (Figure S10), resulting in
nanohydrogels 11 with Dh = 81 nm, once again with unchanged
size distributions (Figure S11, Table S2). Additionally, loaded
nanohydrogels exhibits near neutral ζ-potentials (surface
Keywords: reactive polypept(o)ides • micelles • self-assembly •
secondary structure • bio-reversible cross-linking
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10.1002/anie.201702624
Angewandte Chemie International Edition
COMMUNICATION
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COMMUNICATION
Kristina Klinker+, Olga Schäfer+, David
Huesmann, Tobias Bauer, Leon Capelôa,
Lydia Braun, Natascha Stergiou, Meike
Schinnerer, Anjaneyulu Dirisala, Kanjiro
Miyata, Kensuke Osada, Horacio Cabral,
Kazunori Kataoka, Matthias Barz*
Worms and spheres: Secondary
structure-driven self-assembly allows
for the synthesis of spherical and
worm-like
architectures
core
from
cross-linked
the same
amphiphilic block copolypept(o)ide.
Utilizing bio-reversible disulfide cross-
linking, core polarity and function is
adjusted independently from particle
Page No. – Page No.
Secondary Structure-Driven Self-
Assembly of Reactive
Polypept(o)ides: Controlling Size,
Shape and Function of Core Cross-
Linked Nanostructures
preparation, offering
a
versatile
biocompatible nanoparticle platform.
This article is protected by copyright. All rights reserved.