A gadolinium-complex-based theranostic prodrug for: In vivo tumour-targeted magnetic resonance imaging and therapy

Article abstract of DOI:10.1039/c9cc01816f

Here we report a target-specific theranostic prodrug (1a) containing Gd-DOTA, biotin, and camptothecin (CPT) along with a disulfide self-immolative linker. This prodrug exhibits selective targeting towards tumour cells and tissues, stimuli-responsive controlled release, enhanced anticancer efficacy, and accurate diagnosis and real-time monitoring via contrast-enhanced magnetic resonance imaging (MRI).

Full text of DOI:10.1039/c9cc01816f

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A gadolinium-complex-based theranostic prodrug for in vivo
tumour-targeted magnetic resonance imaging and therapy
Zhaoxuan Yang,a‡ Hongyu Lin,a‡ Jiaqi Huang,a‡ Ao Li,a Chengjie Sun,a Jonathan Richmond,b and
Received 00th January 20xx,
Accepted 00th January 20xx
Jinhao Gaoa
DOI: 10.1039/x0xx00000x
www.rsc.org/
Here we report a target-specific theranostic prodrug (1a) such as coumarin, BODIPY, Rhodamine, and cyanine, suffer
containing Gd-DOTA, biotin, and camptothecin (CPT) along from poor water solubility, which is often a challenging issue to
with a disulfide self-immolative linker. This prodrug features address during the development of theranostic prodrugs by
selective targeting towards tumour cells and tissues, stimuli- integrating fluorescent dyes and chemotherapeutic drugs.
responsive controlled release, enhanced anticancer efficacy,
Magnetic resonance imaging (MRI) is a non-invasive
and accurate diagnosis and real-time monitoring via contrast- imaging technique widely used in clinical practice. MRI is able to
enhanced magnetic resonance imaging (MRI).
offer high-definition images of an entire organism with deep
17
penetration and detailed anatomical information.16,
Many types of treatment have been developed for various Therefore, theranostic agents based on MRI could achieve not
cancer, such as surgery, immunotherapy,1 targeted radiation,2, only sensitive and accurate diagnosis, but also accomplish in
3
and gene therapies.4 In spite of 100-year medical
vivo real-time monitoring, which provides invaluable
advancement since its discovery, chemotherapy remains the information for precise assessment of the therapeutic
frontline therapy for many types of cancer, used in conjunction outcomes and better understanding of the biodistribution and
19
with surgical treatment. However, the main challenge of pharmacokinetics of drugs.18,
Various MRI-based
chemotherapy is its inability to selectively eliminate tumour nanoparticulate theranostic systems have been developed.17, 20,
21
cells and leave healthy cells undamaged. Ideally, we would be
However, several barriers including homogeneity,
able to administer a drug in vivo at the perfect dose, resulting reproducibility, and pharmacokinetics need to be overcome
in selective damage to tumour cells only. In attempt to achieve during the translation of these systems from bench to bedside.
this, small conjugate-based theranostic agents have been In contrast, molecular theranostic conjugates with a precise
developed.5 These agents could selectively release structure, repeatable synthesis, and ease of adaptation, offer a
chemotherapeutic agents at tumour sites and achieve both more promising prospect in both preclinical and clinical
diagnosis and therapy, which meet the needs of personalized contexts.
and precise medicine.6, 7 Among these agents, theranostic
prodrugs based on fluorescence imaging have been extensively (Figure 1a), which contains a targeting agent biotin, a modified
studied.8-15 Fluorescence imaging can accomplish high gadolinium
(III)-1,4,7,10-tetraazacyclododecane-1,4,7,10-
Herein, we designed a molecular theranostic prodrug 1a
sensitivity at cellular level. However, due to its limited tetraacetate (Gd-DOTA) complex, and an anticancer drug
penetration and poor ability to provide anatomy details, it has camptothecin (CPT). Biotin receptors (sodium-dependent
difficulties in accurate tumour diagnosis and detailed multivitamin transporter, SMVT) are overexpressed on the cell
treatment monitoring.16 This severely restricts its application surface of many cancer cells, including HeLa and A549, which
in vivo. Moreover, many commonly used organic fluorophores, renders biotin appealing as a targeting unit to boost the drug
efficacy.22 Gd-DOTA is a commonly used contrast agent for
clinical MRI, offering considerable contrast enhancement which
a. State Key Laboratory of Physical Chemistry of Solid Surfaces, The Key Laboratory
significantly increases both the sensitivity and accuracy of
for Chemical Biology of Fujian Province and Department of Chemical Biology,
College of Chemistry and Chemical Engineering, Xiamen University, Xiamen
361005, China. *E-mail: jhgao@xmu.edu.cn
24
MRI.23,
Camptothecin (CPT), a potent topoisomerase I
inhibitor,25 is one of the most effective antitumour drugs with
promising developmental prospects. However, clinical
applications of CPT have been restricted due to its poor water
solubility and adverse side effects.26 The incorporation of highly
b. Chemical Nanoscience Laboratory, School of Natural and Environmental Sciences,
Newcastle University, Newcastle-Upon-Tyne, NE1 7RU, United Kingdom.
‡ These authors contributed equally to this work.
† Electronic Supplementary Information (ESI) available: The experimental details
and Figures S1-S3 See DOI: 10.1039/x0xx00000x
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hydrophilic Gd-DOTA would greatly improve the water solubility targeting component biotin is absent in 1b, while 1c lacks the
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of 1a and hence reduce the uptake via passive diffusion in disulfide bond, preventing the releaseDOoIf: 1C0.P10T39i/nC9rCeCd0u1c8i1n6gF
normal cells.27 At the same time, the increase in hydrophilicity environments. Detailed synthesis and characterization of 1a, 1b,
would also enhance biotin receptor-mediated endocytosis.28 1c and the intermediates are shown in the Electronic Supporting
The use of a disulfide self-immolative linker would further Information (Figure S1).
increase the selectivity as the reductive environment inside
The fluorescence of CPT was significantly quenched in the
tumour cells would facilitate the release of CPT. Last but not the prodrug 1a (Figure 1b), most likely due to the presence of
least, the fluorescence of CPT would be quenched due to the paramagnetic Gd-DOTA. Upon treatment with GSH, which
presence of paramagnetic Gd-DOTA,17 which offers
a
cleaves the disulfide bond and discharges CPT, the fluorescence
complementary approach to monitor drug release with a “turn- was recovered (Figure 1b). This was exploited to monitor the
on” fluorescence signal (Figure 1a) apart from the major means release of CPT. As shown in Figure 1c and 1d, the fluorescence
of diagnosis and monitoring via MRI.
intensity increased by six fold over a period of 4 h after 1a was
treated with excess GSH, clearly demonstrating the GSH-
mediated release process of CPT. In contrast, no appreciable
change in fluorescence for 1c, which lacks the cleavable
disulfide bond, was observed under the same conditions (Figure
S2). To further validate the disulfide cleavage, we performed
high performance liquid chromatography (HPLC) to trace the
products. Upon incubation with GSH, the original peak for 1a at
22 min disappeared and a new peak at 25 min appeared, which
was confirmed to be free CPT (Figure 1e). On the contrary,
treatment of 1c with GSH showed no apparent change. These
results reveal that the disulfide bond in 1a could be effectively
cleaved in reductive environments, which triggers a self-
immolative process and releases CPT.
To investigate the targeting ability of the prodrug 1a, we
took the advantage of the “turn-on” fluorescence of released
CPT. Fluorescence microscopy showed that the fluorescence
intensity of biotin receptor (BR) overexpressed cancer cells
(HeLa and A549) was much higher than that of normal cells
(MRC-5 and HL7702) after treatment with 1a for 15 min (Figure
2a), indicating the higher uptake of 1a for BR overexpressed
cancer cells (Figure S3). When HeLa and A549 cells were
incubated with biotin before treatment with 1a, the
fluorescence intensity was significantly attenuated (Figure 2b),
suggesting that biotin could block the uptake of 1a and the
uptake of 1a is via BR-mediated endocytosis. Moreover, A549
cells treated with 1a showed significantly higher fluorescence
intensity than the cells treated with 1b or free CPT (Figure 2c,
d), validating the excellent targeting ability of the prodrug 1a.
We also evaluated the contrast-enhanced performance of
1a for MRI. The longitudinal relaxivity (r1) of 1a in PBS was
calculated to be 8.2 mM-1s-1, which is significantly higher than
that of Gd-DOTA, a typical clinical CA with r1 of 4.9 mM-1s-1
Figure 1. (a) The mechanism for the releasing of CPT from 1a and the (Figure S4).29 The excellent T1 relaxivity of 1a could increase the
structures of control molecules 1b and 1c. (b) Fluorescence spectra of sensitivity of contrast-enhanced MRI, which provides a better
1a (5 µM) incubated with or without GSH (5 mM) in PBS (pH 7.4) for 3h. performance for drug monitoring. The in vitro cellular uptake of
(c) Fluorescence spectra of 1a (5 µM) incubated with GSH (5 mM) in PBS 1a was visualized by the use of an MRI scanner. We incubated
over time. (d) Fluorescence intensities of 1a (5 μM) incubated with A549 cells with 1a over a period of 12 h and found that the MR
different concentrations of GSH in PBS. (e) HPLC chromatograms of CPT, signal gradually intensified as the incubation time increased and
1a (10 µM) and 1c (10 µM) incubated with or without GSH (1 mM) in reached a maximum at 3 h (Figure 2e), strongly indicating that
PBS for 3 h.
the time dependent uptake of 1a may saturate at 3 h.
The cytotoxicities of 1a against both cancer cells and normal
RESULTS AND DISCUSSION
cells were assessed via MTT assays. As shown in Table 1, the IC50
We constructed 1a with
a
modular strategy using of 1a against BR overexpressed cancer cells, A549 and HeLa, was
epichlorohydrin as the central piece (Scheme S1), which lower than those of free CPT and 1b, indicating the enhanced
facilitates preparation of the controls, 1b and 1c (Figure 1a). The cytotoxicity of 1a (Figure S5). Notably, the IC50 of 1a against
2 | J. Name., 2012, 00, 1-3
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Table 1. Cytotoxicity evaluation of 1a, 1b, 1c, and CPT against
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HeLa and A549 cancer cell lines and MRCD-5O,I:H1L07.1700392/,C299C3CT0,18a1n6dF
WPMY-1 normal cell lines via MTT assays.
Cell lines
IC50 values
1b
1a
CPT
1c
A549 a
HeLa a
MRC-5 b
HL7702 b
293T b
0.41q
0.79q
2.76q
18.15q
1.34q
19.70f
31.00f
34.60f
>20.00q
>64.00q
>8.00q
1188.00f
5220.00f
3867.00f
16.81q
>8.00q
60.40f
114.30f
119.20f
19.82q
1.97 q
25.51f
35.80f
33.38f
WPMY-1 b
a Biotin overexpressed cancer cell lines, b normal tissue cell lines.
q concentration unit M, f concentration unit nM.

Figure 3. The heat map depicting gene expression profiling of A549 cell
samples from each group (PBS, 1a, or CPT treatments). Red and blue
colors indicate high and low relative expression levels, respectively.
Figure 2. (a) Bright-field and fluorescent images of biotin-receptor
overexpressed cancer cells (HeLa and A549, BR positive) and normal
cells (MRC-5 and HL7702, BR negative). Cells were incubated with 1a (10
µM) in PBS for 15 min. (b) Fluorescent images of biotin-receptor
overexpressed A549 and HeLa cells incubated with 1a (10 µM) in PBS
without or with pre-incubation of biotin (1.5 mM in DMEM with 10%
FBS) for 3 h. (c) Bright-field and fluorescent images of A549 cells
incubated with 1a, 1b, or CPT in PBS for 3 h. (d) Quantitative analysis on
the fluorescence intensities of (c). The data were presented as mean ±
SD (n = 5/group). (e) T1-weighted MR images of A549 cells incubated
with 1a (20 µM in DMEM with 10% FBS) over a period of 12 h.
To evaluate the theranostic function of 1a in vivo, we used
nude mice bearing A549 tumours as a model. For imaging
performance evaluation, mice were separated into two groups,
injected intravenously with 1a or 1b, and subjected to T1-
weighted MRI. Compared to mice injected with 1b, mice
injected with 1a showed a much brighter signal in the tumour
regions, especially after 1 h post administration (Figure 4a).
Quantitative analysis further confirmed that the signal-to-noise
ratio (SNR) of the tumour region of mice treated with 1a was
significantly higher than that of mice treated with 1b, which is
highly ascribed to the biotin mediated targeting ability of 1a. For
therapeutic efficacy assessment, mice were separated into
three groups and subjected to intravenous injection of PBS, 1a,
or free CPT as indicated (Figure 4c, Figure S7). There was a
significant difference in tumour growth between mice treated
with 1a and mice treated with free CPT, indicating the
remarkable tumour inhibition ability of 1a over CPT, which is in
agreement with the cell cytotoxicity evaluation. H&E staining
showed that the prodrug 1a had no apparent side effects on
normal tissues, but caused necrosis of tumour tissues (Figure
S8).
A549 was 0.41 M, only one half of that of free CPT.
Interestingly, the cytotoxicity of 1a against normal cell lines was
much lower than those of free CPT and 1b, suggesting superior
selectivity of 1a. The elevated cytotoxicity and selectivity
against cancer cells may be attributed to the introduction of the
targeting group biotin, which is consistent with the results of
cell uptake experiments (Figure 2, Figure S6). Gene expression
profiles (RNA-seq, Figure 3) further revealed that 1a could up-
regulate anti-oncogenes and down-regulate proto-oncogenes
and non-small cell lung cancer (NSCLC) associated genes, which
is similar to free CPT, indicating that the enhanced cytotoxicity
and selectivity of 1a over free CPT mainly arise from the biotin
mediated targeting.
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The prodrug 1a could selectively target biotin-receptor
overexpressed cancer cells with high uptake, effectively release
CPT in reducing environments, and kill cancer cells with low IC50
values. The excellent T1 contrast-enhanced performance of 1a
enables real-time monitoring of drug uptake in cells and
accurate diagnosis of tumour with high sensitivity. Moreover,
the prodrug 1a could achieve a considerable improvement on
tumour treatment with higher efficacy and reduced side effects.
All these features demonstrate the great potential of 1a for
accurate diagnosis and precise treatment of tumours. This
strategy could be easily adapted for other anticancer drugs and
would be enlightening for the development of more molecular
theranostic agents.
The authors acknowledge the research support from
National Natural Science Foundation of China (21771148,
21602186, 21521004, and 81430041), Natural Science
Foundation of Fujian Province of China (2018J01011), and
Fundamental Research Funds for the Central Universities
(20720170020, 20720170088, and 20720180033).
26.
27.
28.
29.
Conflicts of interest
There are no conflicts of interest.
Notes and references
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A molecular theranostic prodrug for treatment of tumour and real-time monitoring via
MRI in vivo was reported.