Responsive biomimetic networks from polyisocyanopeptide hydrogels

TLDR: These results show that to develop artificial cytoskeletal or extracellular matrix mimics, the essential design parameters are not only the molecular stiffness, but also the extent of bundling, and polyisocyanide polymers are readily modified, giving a starting point for functional biomimetic hydrogels with potentially a wide variety of applications.

Abstract: Mechanical responsiveness is essential to all biological systems down to the level of tissues and cells. The intra- and extracellular mechanics of such systems are governed by a series of proteins, such as microtubules, actin, intermediate filaments and collagen. As a general design motif, these proteins self-assemble into helical structures and superstructures that differ in diameter and persistence length to cover the full mechanical spectrum. Gels of cytoskeletal proteins display particular mechanical responses (stress stiffening) that until now have been absent in synthetic polymeric and low-molar-mass gels. Here we present synthetic gels that mimic in nearly all aspects gels prepared from intermediate filaments. They are prepared from polyisocyanopeptides grafted with oligo(ethylene glycol) side chains. These responsive polymers possess a stiff and helical architecture, and show a tunable thermal transition where the chains bundle together to generate transparent gels at extremely low concentrations. Using characterization techniques operating at different length scales (for example, macroscopic rheology, atomic force microscopy and molecular force spectroscopy) combined with an appropriate theoretical network model, we establish the hierarchical relationship between the bulk mechanical properties and the single-molecule parameters. Our results show that to develop artificial cytoskeletal or extracellular matrix mimics, the essential design parameters are not only the molecular stiffness, but also the extent of bundling. In contrast to the peptidic materials, our polyisocyanide polymers are readily modified, giving a starting point for functional biomimetic hydrogels with potentially a wide variety of applications, in particular in the biomedical field.

Figures

Table 1 | Comparison of structural and mechanical properties between hydrogels based on polyisocyanide P2b and neurofilaments at similar concentrations.2

Figure 2 | AFM analysis of polymers and gel. a, AFM image of isolated polymer chains of P2b. b, AFM image of bundles of P2b from gels. c, Statistical height histograms of both isolated chains and bundles. Both show similarly narrow distributions: chain height h0 = 0.46 ± 0.13 nm and bundle diameter hB = 1.21 ± 0.16 nm.

Figure 4 | Stiffness of the gel vs stiffness of a polymer. Calculated G0 of P2b as a function of lp,0 and N at c = 1 mg mL−1 from eq. 3 by substituting lc for ξ with the experimental data (○) of P2b (c = 1 mg mL−1) and the corresponding calculated data (■) using N = 9.1, at different temperatures. The dotted line at G0 = 1 kPa is a guide to the eye. The inset shows the calculated σc with lp,0, (eq. 4), which is independent of N as well as the calculated (■) and experimental (○) data points at T = 30, 40 and 50 °C.

Figure 3 | Rheological analysis of polyisocyanopeptide gels. a, Moduli Gʹ and Gʹʹ as a function of temperature T for P2b and P3b at c = 1.0 mg mL−1. The arrows indicate the onset of the transition. b, Differential modulus Kʹ as a function of stress σ. c, Data scaled with the plateau modulus G0 and the critical stress σc. d, Single chain persistence length lp,0 as a function of T of P2b between 10 and 60 °C measured by

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2
Responsive Biomimetic Cytoskeletal Networks from Polyisocyanopeptides Hydrogels
Paul H.J. Kouwer
1†
*, Matthieu Koepf
1†
, Vincent A.A. Le Sage
1
, Maarten Jaspers,
1
Arend M. van
Buul
1
, Zaskia Eksteen
1
, Tim Woltinge
1
, Erik Schwartz
1
, Heather J. Kitto
1
, Richard Hoogenboom
1,3
,
Stephen J. Picken
2
, Roeland J.M. Nolte
1
, Eduardo Mendes
2
, and Alan E. Rowan
1
*.
Author affiliations
1
Radboud University Nijmegen, Institute for Molecules and Materials, Department of Molecular
Materials, Heyendaalseweg 135, 6525 AJ Nijmegen, The Netherlands.
2
Delft University of Technology, Department of NanoStructured Materials, Julianalaan 136, 2628
BL Delft, The Netherlands.
3
Current address: Supramolecular chemistry group, Department of Organic Chemistry, Ghent
University, Krijgslaan 281-S4, 9000 Ghent, Belgium.
†
These authors contributed equally to this work.
* Correspondence to: a.rowan@science.ru.nl, p.kouwer@science.ru.nl
Abstract
Responsive hydrogels applied in the biomedical area show great potential as synthetic extracellular
matrix mimics and as host medium for cell growth. The hydrogels often lack the characteristic
mechanical properties that are typically seen for natural gels. Here, we demonstrate the unique
responsive and mechanical properties of hydrogels based on oligo(ethylene glycol) functionalized
polyisocyanopeptides. These stiff helical polymers form gels upon warming at concentrations as low
as 0.006 %-wt polymer, with materials properties almost identical to those of their intermediate
filaments, a class of cytoskeletal proteins. Using a combination of macroscopic rheology and
molecular force microscopy the hierarchical relationship between the macroscopic behaviour of
theses peptide mimics has been correlated with the molecular parameters.

3
Material scientists often look to the natural world for both inspiration and instruction on how to
develop responsive materials. Mechanical responsiveness is essential to all biological systems down
to the level of tissues and cells. Tissues are responsive to deformation, can generate force and, in
turn, force can affect cell and tissue formation. Cell mechanics are governed by the cytoskeleton,
composed of stiff microtubules, actin filaments and intermediate filaments. The latter constitute
family of fibrous α-helical proteins, which, at the appropriate aqueous conditions, self-assemble into
semiflexible bundles with diameters (d
B
) around 10 nm and typical persistence lengths (l
p
) around 1
μm
1
.
Recently, detailed theoretical and experimental studies on cytoskeletal materials have elucidated the
strongly nonlinear mechanical properties of gels of these biological fibres
2
, and have helped to
highlight the fundamental parameters which control the mechanical response of entire cells
3
. A
simple examination of these biomaterials yields an often recurring design motif: a high l
p
, correlated
strongly to the helical architecture of the individual fibrils and the bundles they form. As far as we
know, there are no synthetic equivalents to these biogels that show similar mechanical behaviour.
Here, we present a hydrogel with mechanical properties that nearly completely overlap with those of
IFs and, moreover, by using appropriate network theories, we explain its hierarchical assembly
behaviour at different length scales.
The material is based on synthetic polyisocyanopeptides (PICs)
4
, which possess a 4
1
(four
repeat units per turn) β-helical architecture, in which a hydrogen bond network has developed
between the peptidic side groups parallel to the polymer backbone
5
, resulting in exceptionally stiff
chains; some polymers are amongst the stiffest manmade materials known to date with an l
p
up to
200 nm
6
. This precise architectural definition has been utilised in their application in electronic
applications
7
. We found that a family of water-soluble thermoresponsive oligo(ethylene glycol)
functionalized PICs is able to gel water with an extraordinary high efficiency
8-10
. Such hydrogel
materials are very attractive in, for instance, the fields of drug delivery
11
, regenerative surgery
12
and
advanced stimuli-responsive systems
13,14
.
N
N
H
O
O
O
O
C
Ni
(
C
l
O
4
)
2
m
N
N
H
O
O
O
O
m
N
H
O
O
O
O
O
O
N
H
O
O
O
O
O
O
N
H
O
O
O
O
O
O
1
2
3
m
=
2
m
=
3
m
=
4
P
1
P
2
a
P
2
b
P
3
a
P
3
b
m
=
2
m
=
3
m
=
3
m
=
4
m
=
4
A
C
B
n
=
2
600
n
=
4
2
00
n
=
11
000
n
=
7
00
n
=
7
5
00
n
t
o
l
u
e
n
e

4
Figure 1 | Oligo(ethylene glycol) substituted polyisocyanopeptides. a, Synthesis of the polymers – the
degree of polymerisation is estimated from AFM experiments. b, Representation of the hydrogen-bond
network that stabilises the secondary helical structure for P2. c, Schematic illustration of the 4
1
β-sheet helix.
The arrow represents the peptide substituents.
The hydrogels are composed of oligo(ethylene glycol) functionalised polyisocyanopeptides
P1-P3. These polymers were obtained through a nickel(II)-catalysed polymerization of di-, tri-, and
tetraethylene glycol functionalized isocyano-(D)-alanyl-(L)-alanines 1-3 (Fig. 1) using a previously
described procedure.
15
Variation of the catalyst to monomer ratio allowed us to tune the molecular
weights of the polymers, which were determined by atomic force microscopy (AFM) experiments
(see Supplementary). The hydrogen-bonded 4
1
helical structure of the polymer backbone was
confirmed by infrared (IR) and circular dichroism (CD, Supplementary Fig. S1) spectroscopies. In
aqueous solution and in the gel phase the secondary structure of the polymer is stable up to 70 °C as
shown with CD experiments (Figs. S2, S3). The combination of the densely-packed helical structure
and the strong intramolecular hydrogen-bonded network gives rise to stiff polymer chains
6
, which
could be readily visualized with AFM after drop casting or spin coating from a dilute solution onto
mica (Fig. 2a and Fig. S4).
0.0 0.5 1.0 1.5 2.0
0
10
20
30
40
50
Frequency
Height (nm)
Isolated polymer
Polymer bundle
C
Figure 2 | AFM analysis of polymers and gel. a, AFM image of isolated polymer chains of P2b. b, AFM
image of bundles of P2b from gels. c, Statistical height histograms of both isolated chains and bundles. Both
show similarly narrow distributions: chain height h
0
= 0.46 ± 0.13 nm and bundle diameter h
B
= 1.21 ± 0.16
nm.
Thermal analysis of dilute aqueous solutions of P2b and P3b showed the formation of
transparent hydrogels upon heating at 18 and 44 ºC, respectively
16
. The sol-gel phase transition was

Frequently asked questions

Q1. What contributions have the authors mentioned in the paper "Responsive biomimetic cytoskeletal networks from polyisocyanopeptides hydrogels" ?

Here, the authors demonstrate the unique responsive and mechanical properties of hydrogels based on oligo ( ethylene glycol ) functionalized polyisocyanopeptides. 

Q2. What is the process of bundling in polyisocyanopeptides gels?

For gels based on actin or IFs, bundling is controlled by additives, ranging from binding proteins19 to divalent metal ions20, whilst bundle formation in the polyisocyanopeptides gels is thermally activated. 

Q3. What is the effect of the entropic desolvation of the ethyleneglycol?

Upon heating P2 and P3, the entropic desolvation of the ethyleneglycol arms gives rise to more hydrophobic chains that separate from the aqueous solution. 

Q4. What is the role of the helical polyisocyanide backbone?

The helical polyisocyanide backbone plays a crucial role in providing an intrinsically stiff backbone and controlling the bundling process. 

Q5. How do the polymers behave at the transition temperature?

low molar mass polymers P2a and P3a precipitate at the transition temperature, in line with what has been observed for flexible (co)polymers. 

Q6. What is the effect of the stress on the gels?

Unlike many synthetic polymers, the cytoskeletal proteins (IFs and actin) and other stiff biopolymers show a strong, and well-defined, nonlinear stress response after a critical stress σc is applied to the gels27. 

Q7. How did the authors determine the molecular weights of the polymers?

15 Variation of the catalyst to monomer ratio allowed us to tune the molecular weights of the polymers, which were determined by atomic force microscopy (AFM) experiments (see Supplementary). 

Q8. What is the common design motif of hydrogels?

A simple examination of these biomaterials yields an often recurring design motif: a high lp, correlated strongly to the helical architecture of the individual fibrils and the bundles they form. 

Q9. What temperature was the plateau modulus at different concentrations?

In the experimentally accessible window in the gel phase (30 °C > T > 50 °C) the plateau moduli at different concentrations showed an exponential increase in with T (Figure 3F). 

Q10. How does the sol-gel transition temperature affect the ethylene glycoltail?

The sol-gel transition temperature, rheologically determined as the onset of the step in Gʹ at frequency ω = 6.2 rad s−1 (f = 1 Hz, Supplementary Fig. S12), shows little dependence on the polymer concentration c. 

Q11. How is the absolute value of G0 correlated to c?

The absolute value of G0, however, is strongly correlated to c. Analysis showed a power law behaviour, G0 ∝ cn with coefficients n of 2.2 and 2.7 for P2b and P3b, respectively. 

Q12. How has the relationship between the macroscopic behaviour of theses peptide mimics been?

Using a combination of macroscopic rheology and molecular force microscopy the hierarchical relationship between the macroscopic behaviour of theses peptide mimics has been correlated with the molecular parameters. 

Q13. What is the effect of a broad range frequency sweep in the gel phase?

A broad range frequency sweep in the gel phase (Fig. S10) corroborates that the crosslinks formed at the LCST are permanent in nature. 

Q14. How do the authors explain the mechanical properties of hydrogels?

the authors present a hydrogel with mechanical properties that nearly completely overlap with those of IFs and, moreover, by using appropriate network theories, the authors explain its hierarchical assembly behaviour at different length scales.