Biomaterials 155 (2018) 145e151

Contents lists available at ScienceDirect

Biomaterials
journal homepage: www.elsevier.com/locate/biomaterials

Development of a theranostic prodrug for colon cancer therapy by

combining ligand-targeted delivery and enzyme-stimulated activation
Amit Sharma a, 1, Eun-Joong Kim b, 1, Hu Shi c, 1, Jin Yong Lee c, ***, Bong Geun Chung b, **,
Jong Seung Kim a, *
a
Department of Chemistry, Korea University, Seoul 02841, South Korea
b
Department of Mechanical Engineering, Sogang University, Seoul 04107, South Korea
c
Department of Chemistry, Sungkyunkwan University, Suwon 16419, South Korea

a r t i c l e i n f o a b s t r a c t

Article history: The high incidence of colorectal cancer worldwide is currently a major health concern. Although con-
Received 24 August 2017 ventional chemotherapy and surgery are effective to some extent, there is always a risk of relapse due to
Received in revised form associated side effects, including post-surgical complications and non-discrimination between cancer
13 November 2017
and normal cells. In this study, we developed a small molecule-based theranostic system, Gal-Dox, which
Accepted 17 November 2017
Available online 20 November 2017
is preferentially taken up by colon cancer cells through receptor-mediated endocytosis. After cancer-
specific activation, the active drug Dox (doxorubicin) is released with a fluorescence turn-on response,
allowing both drug localization and site of action to be monitored. The therapeutic potency of Gal-Dox
Keywords:
Targeted drug delivery
was also evaluated, both in vivo and ex vivo, thus illustrating the potential of Gal-Dox as a colorectal
Colon cancer cancer theranostic with great specificity.
Theranostic © 2017 Elsevier Ltd. All rights reserved.
b-Galactosidase
Doxorubicin

1. Introduction complications, including the formation of blood clots in the legs,

Colorectal cancer (CRC) is the third leading cause of cancer-
related deaths worldwide, with over one million new cases in
Europe and the US every year [1,2]. It is the second most common
cancer affecting women, after breast cancer, and the third most
common in men, after prostate and lung cancers, with the overall
risk for developing CRC being approximately 1 in 20 [3]. Typically,
cancers confined to the colon are curable; however, if left un-
treated, they spread to the regional lymph nodes and metastasize to
distant organs. Usually, the initial stages of the disease are curable
with surgical excision and combined chemotherapy. However, an
appreciable proportion of CRC patients in early stage treatment
remain clinically remissive for a prolonged period of time, followed
by approximately 50% chances of tumor recurrence with later
metastasis [4e6]. Surgery is accompanied by various
* Corresponding author.
** Corresponding author.
*** Corresponding author.
E-mail addresses: jinylee@skku.edu (J.Y. Lee), bchung@sogang.ac.kr
(B.G. Chung), jongskim@korea.ac.kr (J.S. Kim).
1
These authors contributed equally to this work.
bleeding at the surgical sites, and damage to nearby organs [7,8].
Hence, there is an urgent need for the development of new ther-
apeutic strategies for CRC, with improved clinical outcomes.
Conventional therapeutic strategies, involving the systemic de-
livery of antitumor drugs, cannot distinguish between normal cells
and proliferating cancerous cells, causing collateral damage to
healthy tissue. To overcome such a formidable challenge, several
targeted delivery systems have been developed, including small
molecule-based drug delivery systems (DDS), liposomes, polymeric
systems, aptamers, and inorganic nanoparticles [9e16]. Ideally,
certain criteria must be fulfilled for the successful development of a
drug delivery formulation. These include preferential targeting of
tumors, maximal accumulation in tumors in vivo, and finally, an
efficient drug release profile within the tumor, with minimal
leakage to contiguous normal cells. Nanoformulations usually take
advantage of leaky vasculature within the tumor mass for prefer-
ential accumulation, termed as enhanced permeability and reten-
tion effects (EPR). However, this significantly depends upon the
state of angiogenesis and vascularization of solid tumors [17e20].
In past years, the design of nanomedicines in cancer therapeutics
has been upgraded, by the incorporation of ligands that actively
target overexpressed receptors on tumors. However, there are still

https://doi.org/10.1016/j.biomaterials.2017.11.019
0142-9612/© 2017 Elsevier Ltd. All rights reserved.
146 A. Sharma et al. / Biomaterials 155 (2018) 145e151
certain hurdles to be overcome to realize the final goal of precision
medicine, including cost-effectiveness, pharmacokinetics, and
disease-driven formulations [20]. This is where a small molecule-
based combination diagnosis and therapy can play a significant
role. In the past 20 years, several small molecule-based DDS have
been designed and developed, for use in early diagnosis, bio-
imaging, and therapeutics [10,11,15]. DDS are usually decorated
with specific cancer targeting units, along with a drug activation
trigger moiety, linked to the chemotherapy drug. For drug activa-
tion, several stimuli-responsive modes have been reported,
including pH, elevated enzyme activity, redox status, light, and
temperature [14,15,21,22]. From a pharmaco-economic perspective,
suitable targeting units and drug activation modes can be chosen
according to the cancer subtype, and synthetically designed. This
requires the simultaneous development of new DDS with easier
accessibility and promising pharmacokinetics that can also be
translated to establish superior therapies for patients.
The lysosomal enzyme, b-galactosidase (b-gal) is known to be
upregulated in various cancer subtypes, including liver, lung, and
ovarian cancers [23e25]. Several efforts have been made to visu-
alize the upregulated activity of b-gal real time in various preclin-
ical cancer models [24e26]. b-gal activity has also been utilized to
activate various cancer drug delivery formulations [27] and thera-
nostic agents [28]. Considering cancer as a robust system, the
preferential targeting and cellular uptake behavior of each
designed DDS can be influenced by several factors, including
complex structural construct, inherent molecular features, and
variation in expression levels of both targeting and trigger modules
[29e31]. Moreover, the overall safety and efficacy of complex DDS
can be influenced by several parameters including biodistribution,
potential immune toxicities and intended targeting that need to be
addressed carefully in preclinical and clinical stages.
In exploring a new direction for the advancement of antitumor
chemotherapeutics, it is critical to understand the in vivo uptake
and drug activation behavior of a novel DDS. This is because the
optimal drug availability within a tumor is governed by DDS-
associated features, which can alter the overall therapeutic effi-
cacy and toxicity profile of the incorporated drug [30,31]. As
detailed below, we focus on the b-gal enzyme as a target to both
deliver and activate the DDS (Gal-Dox) for targeted drug delivery
in vitro and in vivo, in CRC tumor models. The galactose moiety of
Gal-Dox serves as an excellent targeting ligand for asialoglyco-
protein (ASGP) receptors, which play a significant role in tran-
scriptional regulation at both cellular and molecular levels [32,33].
Doxorubicin (Dox) was chosen as a model anticancer drug. Gal-Dox
showed a significant ability to target ASGP receptors on colon
cancer cells, and enhanced activation behavior via lysosomal b-gal
enzyme. We also validated the potential of Gal-Dox in a mouse
tumor model and demonstrated its potential for effective cancer
therapy.
2. Material and methods
2.1. UV/vis, fluorescence spectroscopy and high performance liquid
chromatography methods
A stock solution of Gal-Dox was prepared in DMSO and for
preliminary solution studies, 10 mM working solution in phosphate
saline buffer (PBS) buffer solution (pH ¼ 7.4, 37  C) containing 1%
DMSO was used. Excitation was done at 480 nm with all excitation
and emission slit widths at 5 nm. The total volume for b-Galacto-
sidase response and pH-dependent response tests was fixed at
3.0 mL. For HPLC analyses, a reverse-phase column (C18, 5 mm,
Waters) equipped YL9101S (YL-Clarity) instrument was used. A
UVevis detector (480 nm) was used. The eluent used for each
analysis was a water-acetonitrile gradient (0e30 min; acetonitrile
from 5% to 85%) with a 1.0 mL/min flow rate.
2.2. Cellular uptake studies by fluorescence imaging and flow
cytometry
To evaluate the intracellular delivery of Gal-Dox, confocal laser
scanning microscopy (CLSM) and flow cytometry were both
employed on HT-29, HepG2, and HeLa cells. Cells were seeded and
incubated in m-slide 8 well chamber (ibidi, Munich, Germany) at a
density of 1  104 cells per well. After overnight incubation, 10 mM
Gal-Dox was treated into the culture media of well for various time
points. All cells were fixed with 4% paraformaldehyde in PBS for
20 min at RT, following washing with phosphate-buffered saline
(PBS) for three times. Nuclei and F-actin were counterstained with
DAPI (Invitrogen, Molecular Probe, Eugene, OR, USA) and Alexa
Fluor 488 phalloidin (Invitrogen, Molecular Probe) diluted in 3%
BSA for 20 min, respectively. Furthermore, Lysotracker (Invitrogen,
Molecular Probe) was added to the HT-29 cells at a concentration of
50 nM for 30 min for the examination of co-localization with Gal-
Dox and lysosome. The fluorescence images were acquired using a
CLSM (LSM 710, Carl Zeiss, and Germany). To further verify the Gal-
Dox being targeted to ASGP receptor positive cell lines, the cellular
uptake was analyzed by flow cytometry (FACS Calibur, BD Bio-
sciences, USA). HT-29 and HeLa cells were incubated in 12-well
plate at a density of 4  105/well and pretreated with 1 mM of D-
galactose (Sigma-Aldrich, St. Louis, MO, USA). After incubation for
4 h, the culture media were discarded, and the harvested cells were
examined by a flow cytometry, following the treatment of 10 mM
Gal-Dox for 2 h. The histogram plots were performed using FlowJo
software (TreStar, Olten, Switzerland) and mean fluorescence in-
tensity values were shown in the bar graph.
2.3. In vitro anti-cancer assay
HT-29 and HeLa cells were plated to 96 well plates at a density of
1  104 cells per well for 12 h, and then treated with Dox and Gal-
Dox at various concentrations for 24 h. Then, the culture media
were replaced with fresh medium and the anti-cancer effect was
evaluated by a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazo-
lium bromide (MTT, Roche Diagnostics GmbH, Mannheim, Ger-
many) colorimetric assay. MTT was added and incubated for an
additional 2 h at 37  C. The MTT absorbance was measured at
570 nm using a microplate reader (EL800, Bio-Tek Instruments,
Winooski, VT, USA) after the treatment of solubilization buffer
(Roche Diagnostics) to dissolve formazan crystals. All experiments
were carried out with three replicates.
2.4. In vivo tumor targeting and anti-cancer effects
For in vivo tumor-targeted imaging and anti-tumor activity,
colorectal cancer xenografts were established by subcutaneously
inoculating six-week-old male BALB/c nude mice (Orient Bio,
Sungnam, Korea) with HT-29 cells. All in vivo experimental pro-
cedures involving animals were approved by the Korea University
Institutional Animal Care and Use Committee (IACUC). A total of
1  106 viable HT-29 cells were injected into the flanks of nude
mice after anesthetization. When the tumors reached about
5e10 mm in diameter, Dox or Gal-Dox was injected intravenously
into the tail vein in a single dose of 5 mg/kg. Whole-body fluores-
cence images were continuously monitored and the dissected
major organs (i.e, liver, kidney, heart, lung, tumor) were visualized
or analyzed after sacrificing at 6 h, 24 h, 48 h, using an in vivo
imaging system (Maestro, CRi Inc., Woburn, MA, USA). Moreover,
tumors were fixed in 4% paraformaldehyde solution, embedded
 A. Sharma et al. / Biomaterials 155 (2018) 145e151 147

routinely in paraffin, sectioned at 5 mm thickness, mounted using
with DAPI containing Vecta shield (Vector Laboratories, CA, USA).
To validate the therapeutic efficacy of Gal-Dox, HT-29-inoculated
xenograft mice were subcutaneously injected with Saline, and
3 mg/kg dose of free Dox and Gal-Dox (xenografts, n ¼ 5) four times
every other day. Tumor volumes and body weights were measured
every 3 day using a caliper and terminated at 36 days. For in vivo
toxicity study, blood samples were collected from all the mice for
the measurement of serum biochemical parameters including
blood urea nitrogen (BUN), lactate dehydrogenase (LDH), aspartate
aminotransferase (AST), and alanine aminotransferase (ALT) at day
30 after tumor inoculation. Also, one mouse from each group was
sacrificed, and main organs such as heart, lung, liver, spleen, and
kidney, were submitted for routine haematoxylin and eosin (H&E)
staining.
3. Results and discussion
3.1. Synthesis, fluorescence response, and mechanism of Gal-Dox
activation
Gal-Dox was synthesized by following a series of steps in good
to excellent yield (Fig. 1A and S1, detailed in the supporting infor-
mation). Following a reported procedure, galactosidase linker in-
termediate 2 was synthesized. Further alcohol activation by p-
nitrobenzyl chloroformate and base-mediated substitution with
free Dox resulted in the formation of intermediate 4. The acetyl
groups were de-protected with sodium methoxide in anhydrous
methanol and treated with cation exchange resin, resulting in the
formation of the final product, Gal-Dox. All synthesized
intermediates and products were well characterized by 1H/13C
nuclear magnetic resonance (NMR) and electrospray ionization
mass spectrometry (Figs. S14e25).
To obtain a deeper insight into binding sites at the atomic level,
docking and molecular dynamics (MD) simulations were studied,
including b-gal and Gal-Dox interactions. The molecular binding
site between Gal-Dox and b-gal, and the decomposed binding free
energies, are shown in Fig. 1B and S2, respectively. The calculated
binding free energy between Gal-Dox and b-gal was found to
be 20.22 kcal/mol. According to the binding interaction calcula-
tion, four significant interacting residues in b-galactosidase were
observed, specified as N90 (red), P501 (orange), H528 (blue) and
W987 (pink). Considering the calculated binding free energies
among whole residues, W987 showed the strongest interaction
with the Gal-Dox ligand. Details of the simulation are provided in
the supporting information (Figs. S2e3).
To obtain insight into the preliminary stability and activation
mode of Gal-Dox, the compound was incubated in phosphate
buffered saline (PBS, pH 7.4, 37  C) for 24 h. No significant
decomposition was observed during this period (determined by
fluorescence microscopy and high performance liquid chromatog-
raphy, HPLC), suggesting sufficient chemical stability (Fig. 1 and S4).
Gal-Dox exhibited a weak fluorescence emission at 590 nm (exci-
tation wavelength lexc. ¼ 480 nm). Interestingly, exposure of Gal-
Dox (10 mM) to b-gal (1 U/mL) resulted in cleavage of the galacto-
sidase moiety, followed by the 1,6-elimination route to release free
Dox with simultaneous fluorescence enhancement at 590 nm
(Fig. 1C and D). Consequently, we used the fluorescence enhance-
ment at 590 nm as an activation response, for the observation of
direct Dox release in further studies. The Gal-Dox activation and

Fig. 1. Chemical structure, fluorescence response and activation mode for Gal-Dox. A, Chemical structure of Gal-Dox. B, Molecular binding of Gal-Dox with the b-gal enzyme. C,
Fluorescence enhancement of Gal-Dox (10 mM) upon exposure to the b-gal enzyme (1 U/mL) (excitation wavelength lexc. ¼ 480 nm). D, Time-dependent fluorescence enhancement
upon b-gal action (10 mM, 1 U/mL enzyme, 37  C). Fluorescence enhancement is directly related to cumulative Dox release. E, RP-HPLC curves of Gal-Dox (10 mM) treated with b-gal
(1 U/mL) at 37  C for 2 h. F, Mode of activation of Gal-Dox upon b-gal action.
148 A. Sharma et al. / Biomaterials 155 (2018) 145e151
drug release mechanisms were also determined using mass spec-
troscopy (MS) and HPLC (Fig. 1E and S7). The HPLC profile showed a
peak with an elution time of 20.4 min, corresponding to Gal-Dox.
Upon treatment with b-gal, a new peak appeared at 15.3 min,
corresponding to free Dox. Altogether, treatment of Gal-Dox with
b-gal resulted in the complete release of active Dox, over a time
period of 2 h. Given the disparities in pH and the presence of other
bio-analytes between cancerous and normal healthy cells, we next
evaluated the fluorescence behavior of Gal-Dox, in the presence of
various bio-analytes, and at a range of pHs. No appreciable change
in the fluorescence signal of Gal-Dox was observed at different pHs,
or in the presence of various bio-analytes, including reductase,
GSH, H2O2, and amino acids (Figs. S5e6). These results suggested
that Gal-Dox should exhibit high stability under biological condi-
tions. Gal-Dox exhibited distinct fluorescence enhancement
(590 nm) only upon treatment with the b-gal enzyme.
3.2. ASGP receptor-targeted delivery of Gal-Dox into colorectal
cancer cells
The specific interaction between the galactose residue and ASGP
receptors has been used for hepatocyte-targeted DDS via receptor-
mediated endocytosis [34]. In this study, Gal-Dox was employed as
a theranostic prodrug against the intestinal ASGP receptors in colon
cancer [35]. To confirm the selective delivery of Gal-Dox to colon
cancer cells overexpressing ASGP receptors, we evaluated its tar-
geting ability both in HT-29 cells, a colon adenocarcinoma cell line,
and in HepG2 cells, a well-known cancer cell line bearing the ASGP
receptor. As shown in Fig. 2, HT-29 and HepG2 cells showed pro-
nounced intracellular fluorescence, while relatively low fluores-
cence signals were detected in HeLa cells, an ASGP receptor-
negative cell line (control) [36,37], even after 2 h incubation. Co-
localization experiments confirmed that Gal-Dox was specifically
localized with lysosomes of HT-29 cells, in which b-gal activity
occurs (Figs. S8e9), Especially, intracellular fluorescence was
clearly observed 30 min after treatment because of its targeting
ability for ASGP receptors, while free Dox was not detectable at the
Fig. 2. Targeted cellular uptake of Gal-Dox through ASGP receptor-mediated endocytosis. HT-29, HepG2 and HeLa cells were incubated with 10 mM Gal-Dox for 2 h, fixed with 4%
paraformaldehyde, and counterstained with DAPI and Alexa Fluor 488-Phalloidin to visualize nuclei (blue) and F-actin (green), respectively. Confocal microscopy images of Gal-Dox
(red) were obtained with a laser using an excitation wavelength of 480 nm and an emission wavelength of 560e590 nm. Scale bar: 20 mm. (For interpretation of the references to
colour in this figure legend, the reader is referred to the web version of this article.)
same time. Fluorescence in Gal-Dox-treated cells was reached a
plateau after 6 h (Fig. S8). To further verify the ASGP receptor-
targeted cellular uptake of Gal-Dox, a competition assay was con-
ducted by flow cytometry analysis. Initially, HT-29 cells were pre-
treated 100 times with free galactose as a competitor, followed by
the addition of Gal-Dox at a concentration of 10 mM with incubation
for 2 h. There was no significant difference in fluorescence intensity
between HeLa cells treated with excess galactose and control un-
treated cells, whereas the fluorescence intensity in HT-29 cells
pretreated with galactose remarkably decreased (Fig. S10). Alto-
gether, these results support that the cellular uptake of Gal-Dox can
be specifically achieved by ASGP receptor-mediated endocytosis. To
the best of our knowledge, this is the first report citing the specific
targeting properties of a small molecule-based prodrug system to
ASGP receptor-overexpressing colon cancer cells, through cellular
uptake via receptor-mediated endocytosis.
3.3. b-Galactosidase mediated activity of Gal-Dox
As Gal-Dox exhibited b-gal-triggered Dox release, we next
confirmed enzyme-specific Gal-Dox activation via the reduction of
b-gal activity within the cells. To knock down the expression of b-
gal, an siRNA strategy was attempted to investigate the activity of
Gal-Dox in the presence of the low level of endogenous b-gal
within HT-29 cells. For this, HT-29 cells were treated with various
concentrations of b-gal siRNA for 48 h, and the optimal siRNA
concentration (50 nM) was determined by western blot analysis for
further studies (Fig. 3A). After b-gal expression interference by
pretreated siRNA (50 nM) for 48 h, HT-29 cells were incubated with
10 mM of Gal-Dox for 0.5 h and 12 h. While fluorescence decreased
in HT-29 cells with low b-gal expression, we observed relatively
strong fluorescence in control siRNA-untreated cells (Fig. 3B and
Fig. S11). Enzyme-specific activation of Gal-Dox was further
confirmed by flow cytometric analysis, which provided a compar-
ison between the mean fluorescence intensity of Gal-Dox-treated
HT-29 cells, with and without siRNA knockdown (Fig. 3C). Collec-
tively, these siRNA silencing results suggest that the activation of
 A. Sharma et al. / Biomaterials 155 (2018) 145e151 149

Fig. 3. Gal-Dox activation through b-galactosidase-mediated cleavage. (A) Western blot analysis of b-gal knock-down by siRNA. HT-29 cells were transfected with b-gal siRNA
(25 nM and 50 nM) for 48 h. The silencing effect of siRNA was analyzed by western blot, using an anti-b-gal antibody and b-actin antibody as a loading control. (B) After transfection
of siRNA (50 nM) directed against b-gal for 48 h, HT-29 cells were treated with 10 mM Gal-Dox for 0.5 h, and examined with confocal microscopy. Filter sets: Doxorubicin (lex: 480/
lem: 560e590), F-actin (lex: 495/lem: 518 and DAPI (lex: 358/lem: 461) Scale bar: 20 mm. (C) Flow cytometry analysis was conducted with HT-29 cells treated with 10 mM Gal-Dox
after transfection of 50 nM of b-gal siRNA.

Gal-Dox could be achieved by upregulated b-gal-responsive trig-
gering. This sophisticated system permits simultaneous Dox release
and fluorescence activation-based imaging, for greater precision in
monitoring the drug activation event and its location.
3.4. Anticancer effects of Gal-Dox with cell selectivity
The cytotoxicity of Gal-Dox was evaluated in HT-29 and HeLa
cells using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazo-
lium bromide (MTT) assay, as its anti-proliferative activity can be
determined by selective cellular uptake via ASGP receptor-
mediated endocytosis. Both cells were treated with free Dox and
Gal-Dox at concentrations ranging from 1 to 200 mM, for 24 h.
Although Gal-Dox did not significantly reduce viability even at the
maximum dose in both cells, similar behavior was observed for
both free Dox- and Gal-Dox-treated HT-29 cells (Fig. 4A). However,
a remarkable difference in cytotoxicity was detected between free
Dox- and Gal-Dox-treated HeLa cells (Fig. 4B). The comparison of
IC50 values indicated that Gal-Dox-treated HT-29 cells possessed
approximately 3-fold more potent therapeutic effects than HeLa
cell (Table S1). These results demonstrate that targeted Gal-Dox
delivery via the overexpressed ASGP receptors in colon cancer cells
not only improved cellular uptake, but also enhanced its anticancer
effect.

Fig. 4. Anticancer effects of Gal-Dox in HT-29 cancer cells (A) and HeLa (B) cells. Both cells were treated with Gal-Dox and Dox as a positive control, at various concentrations for
24 h. Cell viability was then determined using the MTT assay.
150 A. Sharma et al. / Biomaterials 155 (2018) 145e151

3.5. In vivo/ex vivo diagnosis and chemotherapeutic in xenograft
mouse models
To investigate the antitumor activity of Gal-Dox, in vivo tumor-
targeting ability was initially confirmed in HT-29 tumor-bearing
mice. For bio-distribution studies using an in vivo fluorescence
imaging system, saline, free Dox and Gal-Dox were intravenously
injected into mice at a single dose of 3 mg/kg, and real-time whole-
body fluorescence imaging was performed 2d after administration.
While the tumor-targeted accumulation of Gal-Dox was continu-
ously visualized by enhanced fluorescence signal up to 48 h in the
HT-29 tumor, free Dox was observed to slightly clear from the tu-
mor site in 24 h post injection (Fig. 5 and S12A). After sacrificing the
mice 48 h later, the tumor tissues were harvested and subsequently
subjected to ex vivo fluorescence imaging (Fig. S12B). Collectively,
whole-body fluorescence imaging results were comparable to
those for the dissected tumor tissues (Fig. 5B) and biodistribution
studies (Fig. S12C and D) from saline-, free Dox-, and Gal-Dox-
treated mice. The in vivo therapeutic efficacy of Gal-Dox was also
evaluated in nude mice bearing HT-29 cell-inoculated tumors,
following intravenous administration of Gal-Dox (5 mg/kg, four
times every second day), and controls including saline and free Dox.
As shown in Fig. 5C, tumor growth was significantly retarded in
Dox- and Gal-Dox-injected xenograft mice, compared to saline-
injected mice. However, administration of Gal-Dox showed
remarkable tumor growth inhibition (53.1%) compared to free Dox
treatment (34.9%, see Fig. 5D). Although Dox has been known to
have several adverse effects such as cardiotoxicity, no significant
in vivo toxicities were observed during this study (Fig. S13). We
believe that this remarkable therapeutic response of Gal-Dox re-
flects its efficient tumor uptake behavior by the galactosidase unit,
which not only serves to improve cancer targeting, but also as an
efficient prodrug activation mode to deliver the active Dox.
4. Conclusions
In conclusion, a small molecule-based theranostic prodrug, Gal-
Dox, was successfully developed for colon cancer chemotherapy,
using receptor-mediated targeting and enzyme-responsive activa-
tion strategies. The imaging properties and therapeutic efficacy of
Gal-Dox in colorectal cancer models have been successfully
demonstrated both in vitro and in vivo. Gal-Dox is preferentially
taken up by HT-29 cancer cells through ASGP receptor-mediated
endocytosis and was activated by elevated lysosomal b-gal
enzyme. In particular, the theranostic activation event is accom-
panied by a simultaneous fluorescence turn-on response. This also
supports the additional possibility of monitoring both the site of
drug activation, and the therapeutic response towards the tumor,
during early stages of treatment. Compared to the pre-reported
multi-component DDS systems incorporating Galactosidase as
trigger, drugs and cancer-specific targeting units altogether in a
complex design, Gal-Dox with simple design possesses remarkable
tumor specific targeting and therapeutic potential in ASGP receptor
positive cell lines. Additionally, complexity of pre-designed DDS as
multi-component requires detailed design and orthogonal analysis
with reproducible scale-up process for achieving a consistent

Fig. 5. Tumor-targeted accumulation and in vivo antitumor activity of Gal-Dox in nude mice bearing HT-29 cancer xenografts. (A) In vivo fluorescence imaging of Gal-Dox-treated
HT-29-xenograft nude mice. Mice bearing HT29 subcutaneous xenografts were intravenously injected with 5 mg/kg of Gal-Dox and in vivo fluorescent images were obtained 48 h
after treatment using the Maestro in vivo imaging system. (B) Fluorescence images in paraffin-embedded tumor tissue. Scale bar: 20 mm (C) Tumor growth inhibition in the various
groups. HT-29-bearing mice were intravenously injected with saline, Dox, or Gal-Dox at a dose of 3 mg/kg, four times every second day. Tumor volume was measured twice per
week. Data are shown as mean ± SE. *: P < 0.05 versus Dox; ***: P < 0.001 versus saline, determined using Student's t-test. (D) Tumor growth inhibition after treatment with Dox
and Gal-Dox at 36 days.
 A. Sharma et al. / Biomaterials 155 (2018) 145e151
commercial product for intended physiological properties, phar-
macological profiles, and biological response. Gal-Dox with a sim-
ple design, can be accessed with ease synthetically from pharmaco-
economic point of view also. Hence Gal-Dox shows the potential as
an ideal theranostic system in future personalized cancer
chemotherapeutics.
Acknowledgements
This research was supported by the CRI project (No. 2009-
0081566, J.S.K.) and the Bio & Medical Technology Development
Program (No. 2015M3A9D7030461, B.G.C.) of the National Research
Foundation funded by the Ministry of Science, ICT and Future
Planning of Korea.
Appendix A. Supplementary data
Supplementary data related to this article can be found at
https://doi.org/10.1016/j.biomaterials.2017.11.019.
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