Rapid formation of a supramolecular polypeptide-DNA Hydrogel for in situ three-dimensional multilayer bioprinting

Article abstract of DOI:10.1002/anie.201411383

A rapidly formed supramolecular polypeptide-DNA hydrogel was prepared and used for in situ multilayer three-dimensional bioprinting for the first time. By alternative deposition of two complementary bio-inks, designed structures can be printed. Based on their healing properties and high mechanical strengths, the printed structures are geometrically uniform without boundaries and can keep their shapes up to the millimeter scale without collapse. 3D cell printing was demonstrated to fabricate live-cell-containing structures with normal cellular functions. Together with the unique properties of biocompatibility, permeability, and biodegradability, the hydrogel becomes an ideal biomaterial for 3D bioprinting to produce designable 3D constructs for applications in tissue engineering.

Full text of DOI:10.1002/anie.201411383

Angewandte
Chemie
DOI: 10.1002/anie.201411383
Biofabrication
Hot Paper
Rapid Formation of a Supramolecular Polypeptide–DNA Hydrogel for
In Situ Three-Dimensional Multilayer Bioprinting**
Chuang Li, Alan Faulkner-Jones, Alison R. Dun, Juan Jin, Ping Chen, Yongzheng Xing,
Zhongqiang Yang, Zhibo Li, Wenmiao Shu,* Dongsheng Liu,* and Rory R. Duncan
Abstract: A rapidly formed supramolecular polypeptide–
DNA hydrogel was prepared and used for in situ multilayer
three-dimensional bioprinting for the first time. By alternative
deposition of two complementary bio-inks, designed structures
can be printed. Based on their healing properties and high
mechanical strengths, the printed structures are geometrically
uniform without boundaries and can keep their shapes up to
the millimeter scale without collapse. 3D cell printing was
demonstrated to fabricate live-cell-containing structures with
normal cellular functions. Together with the unique properties
of biocompatibility, permeability, and biodegradability, the
hydrogel becomes an ideal biomaterial for 3D bioprinting to
produce designable 3D constructs for applications in tissue
engineering.
lular matrices (ECM), thus providing a structural and physical
support for cells similar to that of a natural environment.[3] To
date, non-covalently cross-linked hydrogels from natural
products, including alginate, chitosan, collagen, matrigel,
gelatin, and agarose, have been used in vitro as scaffold
materials for bioprinting;[4] however, significant limitations,
including the thermal triggering of hydrogel formation,
shrinking-induced shape deformations, and a lack of respon-
siveness and tailorability hinder their further application in
3D bioprinting with living cells. Alternatively, covalently
cross-linked hydrogels from synthetic products, including
poly(ethylene glycol) (PEG), polypeptides, poly(N-isopropyl-
acrylamide), and pluronics, have emerged as appealing
candidates because of their clear molecular structures with
possibilities to fine-tune their responsive properties.[5] How-
ever, several drawbacks, such as harsh reaction conditions,
a lack of specific biodegradability and biocompatibility, and
the inability of self-healing between layers, have limited their
applications in in situ multilayer 3D bioprinting with living
cells. Therefore, the development of novel bioprintable
scaffold materials that overcome the above-mentioned lim-
itations is urgently needed, but remains challenging. DNA is
an excellent building scaffold to construct versatile devices
and materials,[6] especially DNA hydrogels, which possess
several advantages, such as designable responsiveness (e.g., to
the pH value, temperature, the presence of enzymes and
aptamers, or light),[7] non-swelling/non-shrinking properties,
biodegradability, and the permeability of nutrients.[7f] Pre-
viously reported applications, such as cell-free protein pro-
duction,[8] covers for single-cell capture and release,[7f] and ion
detection,[7d,9] were only based on some of these properties.
However, applications that combine all of these unique
properties and fulfill the requirements for 3D bioprinting
have not been explored to date.
B
ioprinting has attracted wide-spread attention in tissue
engineering as a powerful fabrication method to design and
create tissue-like structures.[1] Selecting a suitable scaffold
material to be used as the bio-ink is one of the critical issues of
bioprinting.[2] Hydrogels have been widely explored as scaf-
fold materials owing to their similarities to natural extracel-
[*] C. Li, Dr. J. Jin, Dr. Y. Xing, Prof. Z. Yang, Prof. D. Liu
Key Laboratory of Organic Optoelectronics & Molecular Engineer-
ing of the Ministry of Education
Department of Chemistry
Tsinghua University, Beijing 100084 (China)
E-mail: liudongsheng@tsinghua.edu.cn
A. Faulkner-Jones, Dr. A. R. Dun, Dr. W. Shu, Prof. R. R. Duncan
Institute of Biological Chemistry, Biophysics and Bioengineering
Heriot-Watt University, Edinburgh EH14 4AS (UK)
E-mail: w.shu@hw.ac.uk
Dr. P. Chen, Prof. Z. Li
Beijing National Laboratory for Molecular Sciences (BNLMS)
Institute of Chemistry, Chinese Academy of Sciences
Beijing 100190 (China)
Herein, we report the first method for rapid in situ
multilayer 3D bioprinting with DNA-based hydrogels as bio-
inks. As shown in Scheme 1, the DNA hydrogel contains two
components: A polypeptide–DNA conjugate (bio-ink A) and
a complementary DNA linker (bio-ink B). The mixing of bio-
ink A and bio-ink B in a desired molar ratio leads to rapid
in situ hydrogel formation (within seconds) owing to DNA
hybridization. By alternative deposition of bio-ink A and bio-
ink B in the programmed position, designed 3D structures
containing viable and functional living cells could be con-
structed. The resultant hydrogel combines favorable proper-
ties of both the polypeptide and DNA components, that is, it is
responsive to proteases and nucleases, leading to full biode-
gradability and programmability of the hydrogel networks
under physiological conditions.
[**] We thank the National Basic Research Program of China (973
program, 2013CB932803), the National Natural Science Foundation
of China (91427302, 21421064), the NSFC–DFG joint project
TRR61, and the Beijing Municipal Science & Technology Commis-
sion for financial support. This project was also partly supported by
the Sino–UK Higher Education Research Partnership for PhD
Studies Scheme funded by the British Council and the Ministry of
Education in China and EPSRC (EP/M506837/1) and MRC awards
to R.R.D. (MRC_G0901607) and by the Wellcome Trust
(WT074146). The MRC (MRC_MR/K01563X/1) Edinburgh Super-
Resolution Imaging Consortium (ESRIC) provided the microscope
platforms, technical assistance, tissue culturing facilities, and
analysis packages required for the acquisition and analysis of
fluorescence imaging data summarized in this article.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201411383.
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Scheme S1).[7i] On average, five to six ssDNA motifs were
conjugated to each polypeptide backbone (Figure S1), result-
ing in a sufficient number of crosslinking points. In contrast,
bio-ink B is a double-stranded DNA (dsDNA) containing two
“sticky ends” with exactly the same sequences (Figure S2),
which are complementary to those of the ssDNA motifs
grafted onto the polypeptides in bio-ink A (for the exact
sequences, see Table S1). It was found that a mixture (5 wt%)
of the two bio-inks with a 1:1 molar ratio of sticky ends in 1 ꢀ
TBE buffer (pH 8.3, NaCl concentration: 200 mm; TBE =
Tris-borate-EDTA) rapidly changed from a fluidic solution
into an optically transparent supramolecularly cross-linked
network within seconds, namely a polypeptide–DNA hydro-
gel (Figure 1a). Notably, the hydrogel could be formed under
physiological conditions (e.g., [NaCl] = 150 mm, PBS buffer,
pH 7.4, 378C; see Figure S3), which is desirable for applica-
tions where the hydrogels have to be formed in situ with living
cells. The hydrogel formation was further characterized using
a shear rheometer, and G’ was found to be significantly higher
than G’’ over the entire frequency range (Figure 1b),
indicating that the hydrogel had indeed been formed as
designed. The G’ value was determined to be very high (ca.
5000 Pa), resulting in a self-supported and free-standing
hydrogel up to the millimeter scale (Figure 1a).
Scheme 1. 3D bioprinting of the polypeptide–DNA hydrogel to fabri-
cate arbitrarily designed 3D structures. Bio-ink A (blue): polypeptide–
DNA, bio-ink B (red): DNA linker. The DNA sequences of bio-ink A
and bio-ink B are complementary, and hybridization will cause cross-
linking, leading to hydrogel formation (pink). The formed hydrogels
are responsive to both proteases and nucleases, resulting in the full
on-demand degradation of the hydrogel networks after printing.
The unique two-component mixing strategy and the rapid
hydrogel formation can fulfil the requirements for 3D
bioprinting, which is a promising free-form fabrication
method to produce tissue-like structures.[4e] Therefore, equal
amounts of bio-ink A (polypeptide–DNA, 6 wt%, 100 mL)
and bio-ink B (DNA linker, 2 mm, 100 mL) were loaded into
two separate printing cartridges of a microvalve-based 3D
bioprinter, which was able to print in 3D human pluripotent
stem cells whilst maintaining cell viability and function.[10] By
alternatively printing microdroplets of bio-ink A and bio-ink
B on the same spot, immediate contact and mixing of the
printed nanoliter droplets led to rapid in situ hydrogel
formation by DNA hybridization. Different 3D tissue-like
patterns and structures with desired scales and dimensions
could be constructed with the bioprinter based on this
bottom-up assembly strategy. Figure 2a shows a printed
array of hydrogel droplets with an increasing gradient of
sizes with blue dye added for visualization. The smallest
printed hydrogel droplet was estimated to have a diameter of
approximately 500 mm, a thickness of 80 mm, and a volume of
60 nL. The formation of the 3D printed hydrogel was very
rapid (completed within 1 s), which is significantly faster than
hydrogel formation after manual mixing (e.g., within several
seconds). This is probably facilitated by the small volume of
the printed droplets and hence smaller diffusion distances.
Interestingly, the volume and scale of one hydrogel droplet
can be precisely controlled, and the locations of and distances
between different hydrogel droplets can be geometrically
tuned by designing the required programs. Owing to cross-
linking by the rigid DNA duplex, the printed hydrogel
droplets show no obvious shrinking or swelling phenomena,
avoiding the possibility of shape deformation after printing.
Furthermore, hydrogel droplets with 20 layers of printed inks
were found to be mechanically strong enough to be manip-
ulated physically (Figure 2a, inset). Apart from the droplet
As illustrated in Figure 1a, the hydrogel can be fabricated
by a two-component mixing strategy: Bio-ink A is a polypep-
tide–DNA conjugate, which was synthesized by grafting
multiple single-stranded DNAs (ssDNAs) onto a polypeptide
backbone by a copper(I)-catalyzed click reaction between
azide–DNA and poly(l-glutamic acid240-co-g-propargyl-l-glu-
tamate20) (p(LGA240-co-PLG20), Mw = 34060, PDI 1.4) follow-
ing an established method (see the Supporting Information,
Figure 1. a) Preparation of the polypeptide–DNA hydrogel by the two-
component mixing of bio-inks A and B. b) Rheological characterization
of a hydrogel (5 wt%). The frequency sweep was carried out between
0.05 and 100 Hz at a fixed strain of 1% at 258C.
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a promising printing material for the fabrication of complex
3D constructs with precise inner structures.
Furthermore, we used the hydrogels in cell printing, which
offers the possibility to build 3D structures of cells (Fig-
ure 3a).[1a] AtT-20 cells (an anterior pituitary cell line) were
chosen as the model cell line.[11] On addition of the AtT-20
cells, the bio-inks maintained them in a stable and homoge-
nous cell suspension, preventing the cell settlement and
aggregation that commonly impede cell printing. This may be
an unexpected advantage of the specific viscosity and surface
tension of the bio-inks, which not only help maintain the cells
in a suspension, but also meet the stringent requirements in
terms of fluid properties needed for bioprinting with a nozzle-
based technique. We performed a thorough characterization
of the viability and function of the AtT-20 cells after printing
within the 3D hydrogel. Figure 3b shows a 3D stack of printed
AtT-20 cells in a hydrogel stained with fluorescein diacetate
(FDA); a live/dead assay gave a viability of 98.8 Æ 1.4%
(mean Æ SD), indicating that the printing process apparently
caused negligible damage to the cells. To test the biological
functions of the printed cells, we also monitored single cells in
the hydrogel stained with Lysotracker Red at a high reso-
lution over time, revealing intracellular acidic compartments
(in red; Figure 3c). These intracellular acidic compartments
(including lysosomes and large dense-cored vesicles) were
visualized within the cytosol of a printed AtT20 cell using an
inverted wide-field microscope (Figure 3d) and were tracked
over time (Figure 3e). The organelle dynamics indicated that
the printed AtT-20 cells were viable, had a normal 3D
morphology, and exhibited various cellular functions, includ-
ing proton pump activity, metabolic turnover, and membrane
Figure 2. 3D printing of polypeptide–DNA hydrogels into 3D structures
with a blue dye added for visualization. a) An array of printed droplets
with an increasing number of layers (up to 5 layers). Inset: Hydrogel
structure with 20 layers. b) The letters “THU” printed with five layers.
c,d) A triangle with ten layers, which was printed in five minutes.
d) Rhe printed hydrogel structure is strong enough to be picked up
with tweezers.
arrays, designable hydrogel structures,
such as the letters “THU” with five
layers (Figure 2b) and a simple trian-
gle with ten layers (Figure 2c) were
3D printed, demonstrating the ability
of our system to print arbitrary 3D
structures. One layer of the triangle
could be prepared in 30 s, indicating
that our printing strategy is a rapid
fabrication method for millimeter-
sized objects. The triangular hydrogel
structure (Figure 2d) could be manip-
ulated without collapse, indicating
that the mechanical strength of the
3D printed hydrogel is strong enough
to support its printed shapes. Further-
more, the printed structures are opti-
cally transparent and geometrically
uniform without obvious boundaries
Figure 3. 3D bioprinting of polypeptide–DNA hydrogels with AtT-20 cells. a) Cell-printing process
based on ink-jet techniques. b,c) 3D stacks of AtT-20 cells printed in a hydrogel with FDA staining
in green (gridlines: 50 mm; b) or Lysotracker Red staining in red (gridlines: 5 mm; c). d) A single
cell was imaged using wide-field microscopy, a cross section of the cells shows acidic compart-
ments. Scale bar: 1 mm. e) Dynamic organelles were tracked and trajectories from inside the cell
in (d) are shown. The tracked organelles are shown as red spheres and tracks as colored lines
with displacements indicated by gray arrows. The color bar and the colors of the tracks represent
between the printed layers. This is due
to the fact that the contacting layers of
the hydrogel can further merge and
heal together based on the supra-
molecular dynamic cross-linking of
DNA hybridization. All of these
unique properties make our hydrogel the time (0–50 s). Gridlines: 1 mm.
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trafficking.[12] Next, cell cultures of two types of cells (AtT-20
and HEK-293 cells) were monitored for a prolonged period of
time, which showed that the cells maintained their high
viability (Figure S5), demonstrating that the hydrogel is
mechanically strong enough to provide physical support for
the encapsulated cells, non-toxic to cells, and permeable for
nutrients, which are desirable properties for the long-term use
of cell cultures.
We then studied the biodegradability of our hydrogel,
which is a crucial and desirable property for bioprinting. It
was found that protease (endoproteinase Glu-C, 30 U) can
degrade the polypeptide backbone, which leads to the
collapse of the hydrogel (5 wt%; Figure 4, tube 2) after
was demonstrated to produce structures containing viable
cells with normal cellular functions. As the hydrogel is also
biocompatible with cells, permeable to nutrients, and biode-
gradable, it is a new promising printable biomaterial for the
fabrication of complex three-dimensional tissue-like con-
structs in tissue engineering.
Received: November 24, 2014
Revised: December 26, 2014
Published online: && &&, &&&&
Keywords: biodegradability · DNA structures · hydrogels ·
.
polypeptides · supramolecular chemistry
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Figure 4. Enzymatic degradation of the polypeptide–DNA hydrogels.
Tubes 1 and 2: Protease responsiveness. The hydrogel (5 wt%, 10 mL)
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Communications
Biofabrication
C. Li, A. Faulkner-Jones, A. R. Dun, J. Jin,
P. Chen, Y. Xing, Z. Yang, Z. Li, W. Shu,*
D. Liu,* R. R. Duncan
&&&& — &&&&
Rapid Formation of a Supramolecular
Polypeptide–DNA Hydrogel for In Situ
Three-Dimensional Multilayer Bioprinting
Printed DNA hydrogel: The rapid forma-
tion of a supramolecular polypeptide–
DNA hydrogel with self-healing proper-
ties and a high mechanical strength is
described. The hydrogel was used for
in situ multilayer three-dimensional bio-
printing and cell printing, which pro-
duced structures that contain living cells
with normal cellular functions, and is fully
biodegradable by proteases or nucleases.
6
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Angew. Chem. Int. Ed. 2015, 54, 1 – 6
These are not the final page numbers!