Development of a theranostic prodrug for colon cancer therapy by combining ligand-targeted delivery and enzyme-stimulated activation

Article abstract of DOI:10.1016/j.biomaterials.2017.11.019

The high incidence of colorectal cancer worldwide is currently a major health concern. Although conventional chemotherapy and surgery are effective to some extent, there is always a risk of relapse due to associated side effects, including post-surgical complications and non-discrimination between cancer and normal cells. In this study, we developed a small molecule-based theranostic system, Gal-Dox, which 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 was also evaluated, both in vivo and ex vivo, thus illustrating the potential of Gal-Dox as a colorectal cancer theranostic with great specificity.

Full text of DOI:10.1016/j.biomaterials.2017.11.019

Accepted Manuscript
Development of a theranostic prodrug for colon cancer therapy by combining ligand-
targeted delivery and enzyme-stimulated activation
Amit Sharma, Eun-Joong Kim, Hu Shi, Jin Yong Lee, Bong Geun Chung, Jong Seung
Kim
PII:
S0142-9612(17)30748-2
10.1016/j.biomaterials.2017.11.019
JBMT 18357
DOI:
Reference:
To appear in: Biomaterials
Received Date: 24 August 2017
Revised Date: 13 November 2017
Accepted Date: 17 November 2017
Please cite this article as: Sharma A, Kim E-J, Shi H, Lee JY, Chung BG, Kim JS, Development of
a theranostic prodrug for colon cancer therapy by combining ligand-targeted delivery and enzyme-
stimulated activation, Biomaterials (2017), doi: 10.1016/j.biomaterials.2017.11.019.
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Graphical Abstract
Herein, we developed a small molecule-based theranostic system, Gal-Dox, which 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 was also monitored, both in vivo and ex vivo, thus illustrating
the potential of Gal-Dox as a colorectal cancer theranostic with great specificity.

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Development of a theranostic prodrug for colon cancer therapy by
combining ligand-targeted delivery and enzyme-stimulated activation
Amit Sharma,‡,a Eun-Joong Kim,‡,b Hu Shi,‡,c Jin Yong Lee,c,* Bong Geun Chung,b,* and
Jong Seung Kima,*
a Department of Chemistry, Korea University, Seoul 02841, Korea.
b Department of Mechanical Engineering, Sogang University, Seoul 04107, Korea.
c Department of Chemistry, Sungkyunkwan University, Suwon 16419, Korea.
‡ These authors contributed equally to this work.
*Corresponding authors: jongskim@korea.ac.kr (J. S. Kim); bchung@sogang.ac.kr (B. G.
Chung); jinylee@skku.edu (J. Y. Lee).
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Abstract
The high incidence of colorectal cancer worldwide is currently a major health concern.
Although conventional chemotherapy and surgery are effective to some extent, there is
always a risk of relapse due to associated side effects, including post-surgical complications
and non-discrimination between cancer and normal cells. In this study, we developed a small
molecule-based theranostic system, Gal-Dox, which 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
was also evaluated, both in vivo and ex vivo, thus illustrating the potential of Gal-Dox as a
colorectal cancer theranostic with great specificity.
Keywords
Targeted Drug delivery, Colon cancer, Theranostic, β-Galactosidase, Doxorubicin
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1. Introduction
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 untreated,
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.[4–6] Surgery is accompanied by various
complications, including the formation of blood clots in the legs, bleeding at the surgical sites,
and damage to nearby organs [7,8]. Hence, there is an urgent need for the development of
new therapeutic strategies for CRC, with improved clinical outcomes.
Conventional therapeutic strategies, involving the systemic delivery 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 [9–16]. 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. Nano formulations usually take advantage of leaky vasculature within the tumor mass
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for preferential accumulation, termed as enhanced permeability and retention effects (EPR).
However, this significantly depends upon the state of angiogenesis and vascularization of
solid tumors [17–20]. 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 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, bioimaging, 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 activation, 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, β-galactosidase (β-gal) is known to be upregulated in various
cancer subtypes, including liver, lung, and ovarian cancers [23–25]. Several efforts have been
made to visualize the upregulated activity of β-gal real time in various preclinical cancer
models [24–26]. β-gal activity has also been utilized to activate various cancer drug delivery
formulations [27] and theranostic agents [28]. Considering cancer as a robust system, the
preferential targeting and cellular uptake behavior of each designed DDS can be influenced
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by several factors, including complex structural construct, inherent molecular features, and
variation in expression levels of both targeting and trigger modules [29–31]. 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 efficacy and toxicity profile of the incorporated drug
[30–31]. As detailed below, we focus on the β-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
asialoglycoprotein (ASGP) receptors, which play a significant role in transcriptional
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 β-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
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, 10uM working solution in phosphate saline buffer (PBS) buffer solution (pH=7.4,
37oC) containing 1% DMSO was used. Excitation was done at 480 nm with all excitation and
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emission slit widths at 5 nm. The total volume for β-Galactosidase response and pH
dependent response tests was fixed at 3.0 mL. For HPLC analyses, a reverse-phase column
(C18, 5mm, Waters) equipped YL9101S (YL-Clarity) instrument was used. A UV-vis detector
(480 nm) was used. The eluent used for each analysis was a water-acetonitrile gradient (0-30
min; acetonitrile from 5% to 85%) with a 1.0 mL/min flow rate.
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 µ-slide 8 well chamber (ibidi, Munich, Germany) at a density of 1 × 104 cells per
well. After overnight incubation, 10 ꢀM 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
Biosciences, 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 ꢀM Gal-Dox for 2 h. The
histogram plots were performed using FlowJo software (TreStar, Olten, Switzerland) and
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mean fluorescence intensity values were shown in the bar graph.
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 tetrazolium
bromide (MTT, Roche Diagnostics GmbH, Mannheim, Germany) 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.
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 procedures 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 5-10 mm in diameter, Dox or Gal-Dox was injected intravenously into the tail
vein in a single dose of 5 mg/kg. Whole-body fluorescence 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 routinely in paraffin, sectioned at 5ꢀm thickness,
mounted using with DAPI containing Vecta shield (Vector Laboratories, CA, USA). To
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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=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 information). Following a reported procedure,
galactosidase linker intermediate 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 group was 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
(Fig. S14–25).
To obtain a deeper insight into binding sites at the atomic level, docking and molecular
dynamics (MD) simulations were studied, including β-gal and Gal-Dox interactions. The
molecular binding site between Gal-Dox and β-gal, and the decomposed binding free
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energies, are shown in Fig. 1B and S2, respectively. The calculated binding free energy
between Gal-Dox and β-gal was found to be -20.22 kcal/mol. According to the binding
interaction calculation, four significant interacting residues in β-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 (Fig. S2–3).
Figure 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 β-gal enzyme. C, Fluorescence
enhancement of Gal-Dox (10 ꢀM) upon exposure to the β-gal enzyme (1 U/mL) (excitation
wavelength λexc. = 480 nm). D, Time-dependent fluorescence enhancement upon β-gal action (10 ꢀM,
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1 U/mL enzyme, 37°C). Fluorescence enhancement is directly related to cumulative Dox release. E,
RP-HPLC curves of Gal-Dox (10 ꢀM) treated with β-gal (1 U/mL) at 37°C for 2 h. F, Mode of
activation of Gal-Dox upon β-gal action.
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 chromatography, HPLC), suggesting sufficient
chemical stability (Fig. 1 and S4). Gal-Dox exhibited a weak fluorescence emission at 590
nm (excitation wavelength λexc. = 480 nm). Interestingly, exposure of Gal-Dox (10 µM) to β-
gal (1 U/mL) resulted in cleavage of the galactosidase moiety, followed by the 1,6-
elimination route to release free Dox with simultaneous fluorescence enhancement at 590 nm
(Fig. 1C,D). Consequently, we used the fluorescence enhancement at 590 nm as an activation
response, for the observation of direct Dox release in further studies. The Gal-Dox activation
and drug release mechanisms were also determined using mass spectroscopy (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 β-gal, a new peak appeared at 15.3 min,
corresponding to free Dox. Altogether, treatment of Gal-Dox with β-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,
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and amino acids (Fig. S5–6). These results suggested that Gal-Dox should exhibit high
stability under biological conditions. Gal-Dox exhibited distinct fluorescence enhancement
(590 nm) only upon treatment with the β-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 targeting 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 pronounced intracellular
fluorescence, while relatively low fluorescence signals were detected in HeLa cells, an ASGP
receptor-negative
Figure 2. Targeted cellular uptake of Gal-Dox through ASGP receptor-mediated endocytosis. HT-29,
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HepG2 and HeLa cells were incubated with 10 ꢀM 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 560~590 nm. Scale bar: 20 ꢀm.
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 β-gal
activity occurs (Fig. S8-9), 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 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 conducted by flow cytometry analysis. Initially, HT-29 cells were
pretreated 100 times with free galactose as a competitor, followed by the addition of Gal-Dox
at a concentration of 10 ꢀM with incubation for 2 h. There was no significant difference in
fluorescence intensity between HeLa cells treated with excess galactose and control untreated
cells, whereas the fluorescence intensity in HT-29 cells pretreated with galactose remarkably
decreased (Fig. S10). Altogether, 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
β-Galactosidase mediated activity of Gal-Dox
As Gal-Dox exhibited β-gal-triggered Dox release, we next confirmed enzyme-specific
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Gal-Dox activation via the reduction of β-gal activity within the cells. To knock down the
expression of β-gal, an siRNA strategy was attempted to investigate the activity of Gal-Dox
in the presence of the low level of endogenous β-gal within HT-29 cells. For this, HT-29 cells
were treated with various concentrations of β-gal siRNA for 48 h, and the optimal siRNA
concentration (50 nM) was determined by western blot analysis for further studies (Fig. 3A).
After β-gal expression interference by pretreated siRNA (50 nM) for 48 h, HT-29 cells were
incubated with 10 ꢀM of Gal-Dox for 0.5 h and 12 h. While fluorescence decreased in HT-29
cells with low β-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 comparison between the mean
fluorescence intensity of Gal-Dox-treated HT-29 cells, with and without siRNA knockdown
(Fig. 3C). Collectively, these siRNA silencing results suggest that the activation of Gal-Dox
could be achieved by upregulated β-gal-responsive triggering. This sophisticated system
permits simultaneous Dox release and fluorescence activation-based imaging, for greater
precision in monitoring the drug activation event and its location.
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Figure 3. Gal-Dox activation through β-galactosidase-mediated cleavage. (A) Western blot analysis
of β-gal knock-down by siRNA. HT-29 cells were transfected with β-gal siRNA (25 nM and 50 nM)
for 48 h. The silencing effect of siRNA was analyzed by western blot, using an anti-β-gal antibody
and β-actin antibody as a loading control. (B) After transfection of siRNA (50 nM) directed against β-
gal for 48 h, HT-29 cells were treated with 10 ꢀM Gal-Dox for 0.5 h, and examined with confocal
microscopy. Filter sets: Doxorubicin (λex : 480 / λem : 560~590), F-actin (λex : 495 / λem : 518 and
DAPI (λex : 358 / λem : 461) Scale bar: 20 ꢀm. (C) Flow cytometry analysis was conducted with HT-29
cells treated with 10 ꢀM Gal-Dox after transfection of 50 nM of β-gal siRNA.
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 tetrazolium bromide (MTT) assay, as its anti-proliferative
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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 ꢀM, 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.
Figure 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.
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
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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
Figure 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 ꢀm (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.
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performed 2d after administration. While the tumor-targeted accumulation of Gal-Dox was
continuously visualized by enhanced fluorescence signal up to 48 h in the HT-29 tumor, free
Dox was observed to slightly clear from the tumor 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. S12 C 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 reflects 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 activation strategies. The imaging properties and therapeutic efficacy of Gal-Dox
in colorectal cancer models have been successfully demonstrated both in vitro and in vivo.
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Gal-Dox is preferentially taken up by HT-29 cancer cells through ASGP receptor-mediated
endocytosis and was activated by elevated lysosomal β-gal enzyme. In particular, the
theranostic activation event is accompanied 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 possess 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 commercial product for intended physiological properties,
pharmacological profiles and biological response. Gal-Dox with simple 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.
5. 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 (NRF) funded by the Ministry of Science, ICT & Future
Planning (MSIP) of Korea.
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