A supramolecular hydrogel as a carrier to deliver microRNA into the encapsulated cells

Article abstract of DOI:10.1039/c4cc00156g

A supramolecular hydrogel formed by dipeptide Gly-Ala linked with biphenyl-substituted tetrazole serves not only as a 3D matrix for live cells, but also as a carrier to deliver microRNA into the encapsulated cells. This journal is the Partner Organisations 2014.

Full text of DOI:10.1039/c4cc00156g

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A supramolecular hydrogel as a carrier to deliver
microRNA into the encapsulated cells†
Cite this: Chem. Commun., 2014,
50, 3722
Jinbo Li,‡ Romain Kooger,‡ Mingtao He, Xiao Xiao, Li Zheng and Yan Zhang*
Received 8th January 2014,
Accepted 12th February 2014
DOI: 10.1039/c4cc00156g
www.rsc.org/chemcomm
A supramolecular hydrogel formed by dipeptide Gly-Ala linked with
biphenyl-substituted tetrazole serves not only as a 3D matrix for live cells,
but also as a carrier to deliver microRNA into the encapsulated cells.
Supramolecular hydrogels formed by the self-assembly of bio-
compatible small-molecule hydrogelators have emerged as ‘‘smart’’
biomaterials.1 Recent examples using hydrogels formed by self-
assembled peptides have demonstrated diverse biomedical applica-
tions of these supramolecular hydrogels in drug delivery,2 controlled
release,3 tissue engineering4 and regenerative medicine.5 With concise
structural modification of synthetic peptides, supramolecular hydro-
gels that respond to ligand–receptor interaction,6 enzymes7 and
other bio-mimetic stimuli8 have been constructed. Exploration of
the bio-medical applications of these new types of supramolecular
hydrogels is of current research interest to us and to others.9
MicroRNAs (miRNAs) are endogenous small non-coding RNAs
of B22 nucleotides that regulate target gene expression at the post-
transcriptional level.10 Chemical–biological studies with miRNAs as
biological targets are emerging, with a focus on the detection,
delivery and regulation of disease-related miRNAs.11 It is important
to develop various delivery systems for the efficient delivery of
miRNA mimics into cells to study the biological functions of specific
miRNAs as well as to realize a therapeutic purpose.12 Due to the poor
cell permeability of oligonucleotides and their strong tendency to be
degraded in biofluids, most miRNA delivery strategies have involved
the association of miRNA with a carrier such as virus vectors,
liposomes and nanoparticles.13 While polymeric hydrogels have
been demonstrated to be effective carriers enabling miRNAs to enter
cells,14 the application of supramolecular hydrogels to the delivery of
miRNAs into cells has not been explored. Here we report a supra-
molecular hydrogel that acts not only as 3D culture media for live
Fig. 1 Schematic illustration of the dual-functional supramolecular hydrogel.
cells, but also as an effective carrier to deliver miRNAs into living
cells encapsulated inside the gel matrix (Fig. 1).
The chemical structure of the small-molecular hydrogelator
Tet-GA is shown in Fig. 2A. It contains a dipeptide Gly-Ala with
the N-terminus linked with a biphenyl-substituted tetrazole moiety. In
our previous work, we have used a biaryl-substituted tetrazole with the
o-allyloxy group at the N-phenyl ring to link with the N-terminus
of synthetic short peptides to construct photo-degradable supramole-
cular hydrogels.9a The biaryl tetrazole moiety when linked with short
peptides has proved to be able to provide intramolecular p–p stacking
and upon balancing with the hydrophilic interaction of the peptide
backbone; it also promoted the self-assembly process.9a Therefore we
synthesized 4-(2-phenyl-2H-tetrazol-5-yl)benzoic acid and linked it to
the N-terminus of dipeptides with different sequences. Hydrogelation
tests showed that only Tet-GA was able to form a stable hydrogel
under neutral pH (ESI†).
In phosphate buffered saline (PBS), Tet-GA was able to form a
transparent and stable hydrogel at concentrations higher than
1.5 mg mLꢀ1 (Fig. 2B). Transmission electron microscopy (TEM)
State Key Lab of Analytical Chemistry for Life Science, School of Chemistry and
Chemical Engineering, Nanjing University, Nanjing, 210093, P. R. China.
E-mail: njuzy@nju.edu.cn
† Electronic supplementary information (ESI) available: Experimental procedures
and supporting figures. See DOI: 10.1039/c4cc00156g
Fig. 2 (A) Chemical structure of Tet-GA. (B) Picture of the Tet-GA gel
(1.5 mg mLꢀ1 in PBS). (C) TEM image of the Tet-GA gel. Scale bar: 100 nm.
‡ J. Li and R. Kooger contributed equally to this work.
3722 | Chem. Commun., 2014, 50, 3722--3724
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possibility of realizing a dual-functional Tet-GA gel for 3D culture
and for miRNA delivery into the 3D cultured cells.
We used miR-122 that is a liver-specific miRNA and a tumor-
suppressor in liver cancers17 to test the delivery efficiency of
Tet-GA gel. Given that a major problem of miRNA delivery arises
from the instability of miRNA in biofluids, we examined whether
the miR-122 embedded in the Tet-GA gel was protected against the
degradation by fetal bovine serum (FBS) added on top of the gel.
The results indicated that the remaining miR-122 embedded in the
Tet-GA gel upon exposure to 5% FBS was comparable to that after
2 hours of incubation with 5% FBS in DMEM medium (Fig. S5,
ESI†). This made it possible for us to study the delivery of miR-122
into the cells 3D cultured for several days with FBS-containing
medium on top of the gel. Since nanofibers in supramolecular
hydrogel were able to mediate the delivery of oligomers and siRNAs
Fig. 3 (A) MTT assays of the viability of HepG2 cells after treatment with
Tet-GA at various concentrations for 1–3 days. Data are shown as mean ꢁ s.d.
(n = 3). (B) 3D fluorescent images of HepG2 cells, which were encapsulated
inside 3 mg mLꢀ1 Tet-GA gel for 24 hours and stained with calcein AM (the
cube size is 450 ꢂ 450 ꢂ 450 mm3). Images were taken every 0.2 mm in the
Z direction with a frame size of 450 ꢂ 450 mm2 in the XY plane, and a
3D projection was performed to get a 3D image.
images of the gel showed a fibrous network in the gel matrix into cells,15 it is also possible for Tet-GA gel matrix to mediate the
(Fig. 2C). The circular dichroism (CD) spectrum of the gel indicated intracellular delivery of miRNA into cells.
a b-sheet secondary structure of the self-assembled nanofibers
We then embedded miR-122 and HepG2 cells simultaneously
(Fig. S1, ESI†). Dynamic frequency and strain sweep of the gel inside the Tet-GA gel to study the intracellular delivery of miRNA in a
formed by Tet-GA at 3 mg mLꢀ1 showed that the storage modulus 3D manner. HepG2 cells were 3D cultured in the miR-122 containing
(G0) of the gel was at the kilopascal level (Fig. S2, ESI†), indicating the gel matrix for 24 hours. Then the gel matrix was degraded and the
gel was mechanically strong enough to encapsulate live cells for 3D released cells were collected. The expression level of miR-122 inside
culture. The hydrogelation properties of Tet-GA in Dulbecco’s the cells was quantified using qRT-PCR. HepG2 cells cultured in
Modified Eagle Medium (DMEM) in the presence of miRNA mimics Tet-GA gel without miR-122 were used as a control to exclude the
were similar to that in PBS (Fig. S3, ESI†).
possible changes in endogenous miRNA expression levels in response
The biocompatibility of the hydrogelator was then examined. to the gel matrix alone. Fig. 4 showed the relative miR-122 levels in
According to the MTT assay, the compound Tet-GA at different HepG2 cells cultured in different microenvironments for 24 hours.
concentrations ranging from 10 to 500 mM did not show obvious While addition of a 40 pmol miR-122 mimic into the medium for 2D
cytotoxicity in HepG2 cells even after 3 days of culture (Fig. 3A). The HepG2 cell culture in dishes did not change the miR-122 level inside
results indicated that the hydrogelator itself was highly biocompatible the HepG2 cells after 24 hours (Fig. 4A), the presence of same amount
with live cells. Next we tried to culture live cells in a 3D manner with of miR-122 in the 3D culture gel matrix promoted the endogenous
the Tet-GA gel as the 3D culture medium. A viscous solution of Tet-GA miR-122 level by more than 4-fold (Fig. 4B). Dose-dependence was
was first prepared by dissolving Tet-GA in DMEM aided by gentle also observed upon the delivery of miR-122 in the hydrogel matrix.
heating. Upon cooling down to room temperature, HepG2 cells were The presence of the 80 pmol miR-122 mimic in the hydrogel matrix
mixed with the viscous solution before it formed a hydrogel, which was able to promote the miR-122 level in the encapsulated cell by
allowed the encapsulation of the cells inside the matrix upon gel B10-fold. Using miR-122 mimics labeled with a Cy3 tag (Cy3-miR-122)
formation. The three dimensionally encapsulated cells were stained to incubate HepG2 cells embedded in the gel matrix, we were able to
with calcein AM and imaged using confocal microscopy (Fig. 3B). image the presence of the Cy3-miR-122 delivered inside the HepG2
The cells were 3D cultured for 24 hours and a live/dead assay was cells even after a short culture time of 4 hours (Fig. S6, ESI†).
performed using Calcein AM (green) and EthD-1 (red) to stain
Upon confirmation of the dual function of the Tet-GA gel both
living and dead cells respectively (Fig. S4, ESI†). The results as 3D culture media and as carrier to deliver miRNA, we next tried
indicated that over 95% of the cells encapsulated in the Tet-GA to assess whether the intracellular delivery of miR-122 was able to
gel were alive after 24 hours of 3D culture, which made it possible
for us to study the delivery of miRNA inside the gel matrix into the
encapsulated living cells.
Effective delivery of synthetic oligonucleotides into cells has
proved to be able to regulate endogenous miRNAs and realize
important bio-functions.11,12 The use of low-molecular-weight
hydrogels (LMWGs) has been reported to mediate the delivery of
oligomers and siRNAs into cells cultured in dishes.15 Besides,
peptides have also demonstrated the ability to mediate the intra-
cellular delivery of miRNA-29b for osteogenic stem cell differentia-
tion.16 Since most of the cells in living creatures grow in a 3D
microenvironment, we hope to investigate the delivery of miRNA
into cells encapsulated in peptide-based hydrogels that mimic the
3D microenvironment of normal cells. Therefore we explored the
Fig. 4 RT-PCR quantification of miR-122 expression levels after HepG2
cells were cultured in a dish (A) or 3 mg mLꢀ1 Tet-GA gel (B) with 40 pmol
or 80 pmol miR-122 for 24 hours. Data are shown as mean ꢁ s.d. (n = 3).
*P o 0.05.
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Fig. 5 (A) Schematic illustration of the luciferase assay system for monitoring the
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In summary, we demonstrated the first example of 3D miRNA
delivery into live cells mediated by a supramolecular hydrogel using
a dual-functional Tet-GA gel. The superior biocompatibility as well
as mechanical strength of the supramolecular hydrogel made it
possible to culture cells inside the 3D gel matrix. At the same
time, the miRNA encapsulated together with cells inside the short-
peptide based hydrogel matrix were delivered into the encapsulated
cells and subsequently repressed target gene expression. This short-
peptide based hydrogel thus provided a unique platform to study
miRNA delivery and function in live cells under a biomimetic 3D
microenvironment. Further exploration of the biological functions
of specific miRNA delivered into the 3D encapsulated cells is
underway in our group.
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We would like to acknowledge financial support from the
National Natural Science Foundation of China (21302093,
91213306, 21372115, 21121091). J. L. also acknowledges the
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3724 | Chem. Commun., 2014, 50, 3722--3724
This journal is ©The Royal Society of Chemistry 2014