A low-molecular-weight supramolecular hydrogel of riboflavin bolaamphiphile for VEGF-siRNA delivery

Article abstract of DOI:10.1039/c2cc34466a

A low-molecular-weight hydrogel derived from riboflavin (vitamin B ₂) that self-assembles to provide supramolecular nanofibers, biocompatible material, can be used to deliver VEGF-siRNA efficiently into human cells by the endocytosis pathway, where the siRNA is functionalized.

Full text of DOI:10.1039/c2cc34466a

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COMMUNICATION
A low-molecular-weight supramolecular hydrogel of riboflavin
bolaamphiphile for VEGF-siRNA deliverywzy
Sachin Prakash Patil, Hyun Seok Jeong and Byeang Hyean Kim*
Received 16th April 2012, Accepted 20th July 2012
DOI: 10.1039/c2cc34466a
A low-molecular-weight hydrogel derived from riboflavin (vitamin B2)
that self-assembles to provide supramolecular nanofibers,
biocompatible material, can be used to deliver VEGF-siRNA
efficiently into human cells by the endocytosis pathway, where
the siRNA is functionalized.
Synthetic siRNA is an important tool for the specific reduction,
or silencing, of gene expression through RNA interference
(RNAi), allowing particular cellular defects to be addressed.1
Fig. 1 Chemical structures of RBAs synthesized from riboflavin (RF).
The effective delivery of siRNA in vivo remains a challenge,
inhibiting its full therapeutic potential. Current approaches for
the delivery of siRNA in vivo include the preparation of siRNA-
bioconjugates and the incorporation of siRNA into multifunc-
tional polymeric or lipidic nanodevices.2 Most of these techniques
involve the formation of nanoscale siRNA composites, which are
likely to disperse rapidly. Sustained intracellular release of siRNA
might be a useful approach toward maintaining the cytosolic
siRNA concentration above the critical threshold for longer
period of time. Moreover, adverse effects arising from saturation
of the RNAi pathway or unwanted immune activation might be
minimized if the intracellular siRNA concentration were to be
better controlled.
clearance from the body (due to gel–sol transitions). These
attractive features make supramolecular hydrogels promising
alternatives to polymeric hydrogels.
Herein, we synthesized riboflavin (RF) based amphiphiles
(RBAs). RF is an essential biomolecule (vitamin B2) and it is
involved in various biochemical processes.6 So RF-based
LMWGs may be useful for various biological applications as
well as for designing new fluorescent sensors.7 To selectively
append lipopolyamine units at the N-3 position of RF, we
protected all the OH groups as acetates and then treated the
protected RF with a synthetic precursor 7, NaI, and K2CO3 in
DMF (Fig. S1, ESIw).
After the deprotection of the tert-butyl group, we coupled
the COOH group of the RF derivative with various protected
polyamines (Fig. S1, ESIw), and then deprotected the acetates
(7 N NH3 in MeOH) to obtain 6a–c. Deprotection of the Boc
groups (25% TFA in MeOH) provided the desired RBAs 1a–c
(Fig. 1).
Recently, several polymeric hydrogels have been developed
for the intracellular sustained release of siRNA.3 Hydrogels
are typically biocompatible, making them suitable for a wide
range of applications.3–5 The main interest is the design of
low-molecular-weight hydrogels (LMWGs)4a–e that can be
employed as substitutes for polymeric gels in many applications,4,5
even though they possess unique properties.3 Gels formed through
self-assembly of small molecules in water can (i) respond rapidly to
external stimuli, (ii) exhibit inherent gel–sol reversibility (due to the
non-covalent nature of gel formation) and (iii) undergo ready
Compound 1a formed hydrogels at 1.6 wt% in water
(Fig. 2a) by gentle heating, whereas 1b provided a viscous solution
and 1c was soluble in water even at high concentrations. In
addition, 1a also produced hydrogels at 1.6 wt% in acidic
(pH = 5) and neutral (pH = 7.4) buffer solution by mild heating,
while at pH = 9 it gave viscous solution (Fig. S2A, ESIw). We also
examined the behaviour of 1a and 1b, in the presence and absence
of siRNA, in Opti-MEM prior to transfection. 1a/siRNA was
pelleted similar to that mentioned for polymeric hydrogels,3c
whereas 1b/siRNA was dispersed (Fig. S2B and S2C, ESIw).
RF has an intense fluorescence emission, which allowed us to
examine the temperature dependent fluorescence spectrum of the
hydrogel 1a. Fluorescence intensity of hydrogel 1a was increased
with an increase in temperature (up to 20–50 1C) and then
considerably decreased at higher temperatures (Fig. S3A, ESIw).
Department of Chemistry, Division of IBB, Pohang University of
Science and Technology, Pohang 790-784, Korea.
E-mail: bhkim@postech.ac.kr; Fax: +82 54 279 3399;
Tel: +82 54 279 2115
w Electronic supplementary information (ESI) available: Experimental
procedures, synthetic route and precursors for the RBAs, NMR, mass,
and elemental analysis data, images of hydrogels at different pH and
Opti-MEM, temperature dependent fluorescence spectrum, images of
electronic microscopy, gel retardation assay and confocal images of
live cells. See DOI: 10.1039/c2cc34466a
z The manuscript is dedicated to the memory of H. Gobind Khorana.
y This article is part of the ‘Nucleic acids: new life, new materials’
web-themed issue.
c
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Chem. Commun., 2012, 48, 8901–8903 8901

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Fig. 3 (A) Cellular uptake assay of Fl-VEGF-siRNA (100 nM) with
1a–c at molar ratios of 100, 200, and 500; CTL: untreated cell; Nat: VEGF-
siRNA; Nat(ꢀ): only siRNA; Nat(+): siRNA with lipofectamine2000;
(N = 3). (B) Drug inhibition assay of Fl-VEGF-siRNA (100 nM) with 1a
at a molar ratio of 500; si-G1a: Fl-VEGF-siRNA with 1a; NaN3–DOG:
sodium azide with 2-deoxyglucose; Chlor: chlorpromazine; Gen: genistein;
MbCD: methyl-beta-cyclodextrin; (N = 3).
Fig. 2 (a) Photograph of hydrogel 1a; (b) TEM images of xerogel of 1a
and (c) 1a with VEGF-siRNA. The length of both scale bars is 200 nm.
This significant change is also evidenced in Fig. S3B (ESIw). The
increase in fluorescence intensity was explained by thinning of
fibrils. At higher temperature the fibrils begin to melt and the
fluorescence intensity decreases rapidly. RF-based LMWG may
be used as a temperature responsive fluorescent material.
We used scanning electron microscopy (SEM) and transmission
electron microscopy (TEM) to investigate the supramolecular
structures formed by the xerogel of 1a. Low-magnification SEM
images revealed that 1a exhibited high porosity (Fig. S4, ESIw) and
a three-dimensional network structure suitable for cell growth.8
High-magnification of SEM images revealed that the xerogel
formed entangled supramolecular nanofibers, similar to the extra-
cellular matrices (ECMs) found in tissue (Fig. S5–S7, ESIw).8
TEM images of the xerogel of 1a show long extended, entangled
helical nanofibers similar to those in ECMs (Fig. 2b and
Fig. S8 and S9, ESIw).8 Whereas the xerogel of 1a/siRNA
(500 : 1 molar ratio) revealed nanofibers with small particles
of siRNA complexed with nanofibers (Fig. 2c and Fig. S10
and S11, ESIw). SEM and TEM data of 1a suggested that
RF-based LMWG may be useful for tissue engineering, treating
infectious wounds, and serving as antiseptic matrices.
equal to that of lipofectamine2000 at a siRNA concentration of
100 nM at a molar ratio of 500. As per our knowledge, this is the
first report on siRNA (100 nM concentration) delivery by
LMWG as well as siRNA delivery dependent on hydrogelation
property of LMWG. However, the Xu4f and Barthelemy4g groups
have reported the LMWG-mediated delivery of polyA10-oligomer
(1 mM) and the 17-oligomer (0.5 mM), respectively.
To study the internalization mechanism of the VEGF-
siRNA by LMWG 1a (si-G1a), HeLa cells (non-phagocytosing
cells9a) were treated with known biochemical inhibitors, then
treated with si-G1a and analyzed by cellular uptake assay
(Fig. 3B).9 Cells were incubated with NaN3–2-deoxyglucose
(NaN3–DOG) to study the energy dependence process. We noticed
that NaN3–DOG inhibited the uptake of si-G1a (76% compared
with non-treated cells) and indicated that internalization is an
energy dependent process. We used chlorpromazine to examine
clathrin-mediated endocytosis. The uptake of si-G1a was restricted
by chlorpromazine (81% compared with non-treated cells) and it
was assumed that clathrin-mediated endocytosis may be one of the
internalization pathways. We observed an inhibition of uptake of
si-G1a in the presence of methyl b-cyclodextrin (MbCD). MbCD
causes cholesterol depletion and thereby may affect both
clathrin- and caveolae-mediated pathway.9c Genistein was
used to inhibit caveolae-mediated endocytosis. It was shown
that Genistein has no significant effect on the internalization of
si-G1a and suggested that the internalization is likely to be a
non-caveolin lipid-raft mediated mechanism. A more detailed
study of the internalization mechanism of si-G1a is needed.
Next, we performed an ELISA to investigate the amount of
VEGF protein expressed by si-G1a (Fig. 4A). We assessed the
siRNA transfection results after 24 and 48 h, using a 500 : 1
molar ratio of the hydrogel 1a to siRNA, with the latter at
concentrations of 50 and 100 nM. Highest, approximately
50%, gene knockdown occurred specifically by the VEGF
siRNA release from hydrogel 1a; no siRNA knockdown
occurred from the scrambled sequence of VEGF-siRNA under
otherwise identical conditions. This suggested that, consistent
with confocal results (Fig. 4Bc and Bd), hydrogel 1a can
deliver siRNA into cytoplasm, leading to suppression of the
VEGF expression. Although the hydrogel of 1a provided
cellular uptake nearly equal to that of the lipofectamine2000,
its gene knockdown regulation was only 50%. These results
suggested that our hydrogel 1a would release siRNA in a time-
controlled manner to allow site-specific delivery of siRNA and
We used a gel retardation assay to examine the ability of the
RBAs to bind the VEGF-siRNA. The bolaamphiphiles 1a–c
formed complexes with the VEGF-siRNA at molar ratios of
160–200 (Fig. S12, ESIw). Although the RBAs 1a–c could all
bind with the VEGF-siRNA, only the hydrogel formed by 1a
mediated the delivery of the VEGF-siRNA. In other words,
siRNA delivery was based on the hydrogelation properties of 1a.
We used the WST-1 assay to assess the cytotoxicity of the
RBAꢀsiRNA complexes at different molar ratios. Fig. S13
(ESIw) compares the effects of the RBAs 1a–c on Hela cell line
proliferation, relative to that of the commercially available
transfectant lipofectamine2000. The synthesized RBAs did not
exhibit significant toxicity at their optimal concentrations for
transfection. Gratifyingly, the cytotoxicity of each of the RBAs
was lower than that of lipofectamine2000. In addition, more than
95% of the cells survived at all concentrations of the RBAs. Thus,
the hydrogel formed by 1a is a biocompatible material.
Next, we used a cellular uptake assay to examine the
internalization of a fluorescein-tagged VEGF siRNA (Fl-siRNA).
Notably, the cellular uptake provided by 1a was superior to that
of 1b, although the N/P ratios of 1b/siRNA and 1c/siRNA were
twice and thrice that of 1a/siRNA (Fig. 3A). Chemical structure
of compounds 1a, 1b and 1c differs by only amine containing
chains. However, only compound 1a formed supramolecular
hydrogels so its hydrogelation property might be responsible for
delivery of siRNA. Hydrogel 1a provided a cellular uptake nearly
c
8902 Chem. Commun., 2012, 48, 8901–8903
This journal is The Royal Society of Chemistry 2012

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used for the several biological applications such as tissue
engineering, bio-sensing, and effective delivery with controlled
release of drugs including siRNA.
This study was supported by EPB Centre (2012-0000534)
and the WCU Programme (R31-10105).
Notes and references
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Fig. 4 (A) ELISAs of the hydrogel of 1a with VEGF-siRNA [Nat]
and scrambled-VEGF-siRNA [Scr] at various concentrations and
incubation times; CTL: untreated cell; Nat(ꢀ): only siRNA;
Nat(+): siRNA with lipofectamine2000; (N = 3). (B) Live cell images
obtained using confocal microscopy; Fl-siRNA (100 nM) incubated
with (a and b) lipofectamine2000; (c and d) hydrogel 1a; (e and f) 1b;
(g and h) 1c; (i and j) only Fl-siRNA. siRNA/1a–c molar ratio, 1 : 500
[scale bar = 10 mm].
it requires more than 48 h to release all of its siRNA. We are
currently performing a systematic study of the time-controlled
release of siRNA mediated by the hydrogel of 1a, both in vitro
and in vivo.
We employed confocal microscopy to study the localization
of Fl-siRNA in HeLa cells, using the dye DAPI for nuclear
staining (Fig. 4B and Fig. S14, ESIw). We formulated each
RBA 1a–c with Fl-siRNA (100 nM) at a molar ratio of 500 : 1.
Fig. 4Bi and Bj show the absence of green fluorescence in the
cells treated with naked siRNA, confirming the lack of siRNA
uptake. Similar to the results of the cellular uptake assay, the
cellular uptake mediated by the hydrogel of 1a was superior to
those of 1b and 1c. Even when the N/P ratios of 1b/siRNA and
1c/siRNA were twice and thrice that of 1a/siRNA, they did
not mediate prominent siRNA internalization (Fig. 4Be–Bh).
As shown in Fig. 4Ba and Bb Fl-siRNA was delivered by
lipofectamine2000 and it is hypothesized that siRNA appeared
in the endosome (punctuate pattern), with localization in the
cytosol as well as the nuclei of the cell, similar to that provided
by most particulate carriers.2 It is generally believed that a
secondary triggering mechanism must occur in the endosome
to release the siRNA in the cytosol after being endocytosed;
most of these mechanisms are based on a drop in endosomal pH.
Fig. 4Bc and Bd confirm Fl-siRNA delivered by the hydrogel of
1a and it is assumed that the siRNA was not in the endosome
(uniform pattern or diffuse green fluorescence in cytoplasm), but
with significant localization in the cytosol of the cell, similar to
that provided by polymer-based nanomaterials.3e
This communication presents the first LMWG-based bio-
material, derived from RF, an essential biomolecule, that
mediated siRNA delivery into cells nearly equal to that of
lipofectamine2000. Most notably, delivery of siRNA was
affected by the hydrogelation properties of 1a. The hydrogel
1a delivered siRNA specifically into the cytosol of the cell by
the endocytosis pathway, where the siRNA was uniformly
diffused, and functionalized through the RNAi mechanism.1 We
observed siRNA activity occurring specifically from VEGF-
siRNA released from the hydrogel of 1a; no gene downregulation
occurred from the scrambled sequence. Biocompatible and
temperature sensitive fluorescent hydrogels from vitamin B2
forming entangled fibers similar to ECMs have potential to be
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c
This journal is The Royal Society of Chemistry 2012
Chem. Commun., 2012, 48, 8901–8903 8903