Hydrogean Peroxide Inducible Acid-Activatable Prodrug for Targeted Cancer Treatment

Article abstract of DOI:10.1002/cmdc.202100354

Because some of the potentially most useful boronic acids are inherently unstable in blood plasma and exhibit poor selective retention in tumours, 2-heterocyclic N-methyliminodiacetic acid (MIDA) boronates provide a stable, spacious and highly effective harbor for prodrug conjugates. Herein we report MIDA boronates in conjunction with naphthalene-based fluorophores as suitable compounds for tumour diagnosis by virtue of their remarkable specificity and uniform benchtop stability. The shielding group was found to be effective at imparting stability under physiological conditions (pH 7.4), with rapid release of the drug upon exposure to the acidic microenvironment of the tumor. This approach significantly enhanced the efficiency of drug release and was found to exhibit fewer side effects, thus indicating its great potential for precision therapeutics.

Full text of DOI:10.1002/cmdc.202100354

Accepted Article
Title: Hydrogen Peroxide Inducible Acid-Activatable Prodrug for
Targeted Cancer Treatment
Authors: Jun Liu, Jinhua Wang, Shuang Si, Jinyi Xu, and Peng Xue
This manuscript has been accepted after peer review and appears as an
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To be cited as: ChemMedChem 10.1002/cmdc.202100354
Link to VoR: https://doi.org/10.1002/cmdc.202100354
01/2020

10.1002/cmdc.202100354
ChemMedChem
RESEARCH ARTICLE
Hydrogen Peroxide Inducible Acid-Activatable Prodrug for
Targeted Cancer Treatment
Jun Liu*, Jinhua Wang, Shuang Si, Jinyi Xu, and Peng Xue
J, Liu, J, Wang, S, Si, J, Xu, P, Xue
College of Chemistry and Chemical Engineering
Hexi University
Zhangye City 734000, Gansu Province, P. R. China
E-mail: liujun3622849@163.com
Abstract: In view of some of the potentially most useful boronic acids
are inherently unstable in the blood plasma and poor selective
retention in tumours, 2-heterocyclic N-methylimino diacetic acid
(MIDA) boronates provide a safe and spacious harbor for the prodrug
conjugates with highly effective and stable platform. We herein
proposed that N-methyliminodiacetic acid (MIDA) boronates in
concurrent with naphthalene-based fluorophore by virtue of their
remarkable capacity and uniform benchtop stability for tumor
diagnosis. The shielding group was found to be effective in treating
the stability at physiological pH 7.4 but released rapidly to the acidic
microenvironment of the tumor. This approach was significantly
enhanced drug release efficiency and exhibited fewer side effects,
thus indicating its great potential for precision therapeutics.
these dramatic rate differences to the structure of the organic
fragment, since they are stable in the blood plasma. Thus, it is
important to achieve controlled shielding of loaded drugs in the
biodistribution and blood circulation.
We propose that nitrogen mustard as the encapsulating to
create a promising framework for overcoming the stability in the
blood circulation. The prodrugs of nitrogen mustard coupled with
boronic acid or arylboronate derivatives can be selectively
activated by H2O2 in tumor sites. After the internalization, the
prodrugs (L-M-CPT) would disintegrate and release drug CPT
rapidly due to the destruction of MIDA boronates under mildly
acidic conditions. Using this approach, the stability and stimulus
responsive properties of prodrug as stable surrogates can
synergistically achieve effectively prevented premature CPT
release and achieved prompt intracellular release during
intracellular trafficking (Scheme 1).
Introduction
The principle goal of tumor targeting capability is crucial for the
accurate diagnosis of tumors. Therefore, improving the
therapeutic efficacy of the acidic tumor microenvironment is
required before starting therapy.[1-5] The formidable challenge for
tumor therapy is how to achieve enhanced permeability and
improve the drug therapeutic efficacy with minimum systemic
toxicity due to the non specific distribution of small molecular
drugs in vivo. However, the severe side effects of many drugs,
including systemic instability, poor bioavailability, rapid
blood/renal clearance, lack of cancer specificity, and
uncontrollable drug release, have impeded the efficient
internalization and caused premature drug release in the blood
circulation.[6-8] There is an urgent need to inspire the development
of fluorescence systems to establish stable, efficient, and
multifunction platform for drug delivery in the cancer cells.
Evidence suggests that the aberrant production of H2O2 can
be a therapeutic advantage, because it is an important regulator
of signal transduction.[9-13] The aryl boronic acids and their esters
group (deprotection) as species are well-known to be cleaved by
H2O2.[14-17] This reactivity provides a chem-specific, biologically
compatible reaction method for detecting endogenous H2O2
production. However, liberation of the boronic acid groups is
usually hampered by high toxicity and low cellular uptake,
specifically, boronic acids are notoriously unstable, which
precludes therapeutic efficacy of heterogeneous tumor
environments.[18-21] To circumvent these limitations, we herein
report that 2-heterocyclic N-methylimino diacetic acid (MIDA)
boronates in concurrent with naphthalene-based fluorophore by
virtue of their remarkable capacity and uniform benchtop stability
for tumor diagnosis.[22-25] In contrast to other boronates or esters,
the effects of manipulating MIDA hydrolysis, which has enabled
Scheme 1. Schematic representation of the reaction of prodrugs
with H2O2.
Results and Discussion
To test the proposed the optical sensing behavior of prodrug L-M-
CPT toward H2O2 by the breakage of the MIDA boronate, the
fluorescence properties of probe were carried out under optimum
conditions. As shown in Figure 1a, upon addition of H2O2 solution,
the fluorescence intensities (λex =470 nm, λem = 523 nm) was
dependent of different concentrations of L-M-CPT. These
1
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10.1002/cmdc.202100354
ChemMedChem
RESEARCH ARTICLE
experimental results demonstrated that concentration-dependent
enhanced fluorescence (3 μM) could be selected as an
appropriate working concentration for monitoring the drug release
process with the employed prodrug.
To verify MIDA-induced destruction as illustrated in Scheme
1, fluorescence spectra were used to probe the change of the L-
M-CPT from“caged” to “uncaged” state upon activation with H2O2.
In these experiments, we investigated the spectral properties of
the prodrug at 37 °C in phosphate buffer (pH 7.4, 10 mM), as
shown in Figure 1a–d. In order to evaluate the ability of MIDA
boronates could be cleaved by H2O2, the L-M-CPT solution (3 μM)
was incubated with H2O2 from 0 to 80 eq at 37 °C. After the
addition of H2O2, the fluorescence intensity around 523 nm
exhibits an excellent change with the increasing analyte level,
which ascribed to the cleavage of MIDA boronates caused by
H2O2.
In addition, we also investigated the CPT release behavior
under different pH conditions through monitoring the fluorescence
changes of 3 μM L-M-CPT. Upon the addition of H2O2 (1.0 mM)
at pH = 6.0, the CPT was released with the response rate by the
fluorescent recovery. However, L-M-CPT exhibits no change by
monitoring its fluorescent intensity for at least 1 h at the condition
of pH 7.4 (Figure 1c). The above results indicated that L-M-CPT
could remain stable in blood circulation while release the active
drug quickly in an acidic environment due to the“caged” or
“uncaged” state of the boronates. Furthermore, in order to assess
the cleavage of MIDA, we evaluated linear relationships of the
drug release to the fluorescence intensity at the condition of pH
6.0. The results shown in Figure S13 indicate that the
fluorescence signal is linearly correlated with CPT release from
the L-M-CPT upon specific activation of H2O2.
Subsequently, in order to validate the pH-induced controlled
release behavior of L-M-CPT, in vitro drug release profile was
further investigated in PBS solutions at 37 °C with different pH
values, i.e. pH 6.0 and 7.4 respectively. As displayed in Figure 1d,
approximately 90% of CPT has been released from L-M-CPT after
being incubated at pH 6.0 for 40 min, whereas only 17.5% of the
drug was detected at the condition of pH 7.4. From this point of
view, L-M-CPT can maintain its integrity under neutral
physiological conditions (blood circulation environment) while
specifically trigger rapid release under acidic conditions (tumor
environment). In addition, the limit of detection (LOD) was
calculated to be 30 nM (S/N=3, Figure S13), indicating the MIDA
boronates of the prodrug can be effectively cleaved by H2O2. As
a control, we also tested the selectivity of L-M-CPT toward H2O2
upon incubation with the interfering substances, including metal
ions, ROS and RNS. As depicted in Figure S14, S15, the
fluorescence response of L-M-CPT showed only neglectable
increases after incubation with other biological biomarkers. The
above results indicated that L-M-CPT could deliver
chemotherapeutics under hydrogen peroxide situation,
suggesting that theranostic agent with high selectivity over
potential interference to detect H2O2 quantitatively.
Figure 1. a) Effect of probe concentration (H2O2 concentration: 240 μM) in PBS
(pH=6.0). b) The fluorescence emission spectra of L-M-CPT (3 μM) in the
presence of different concentrations of H2O2 at pH 6.0 (PBS). λex=470 nm. c)
Fluorescence intensity changes of L-M-CPT exposed at pH 7.4 or 6.0 for
different time periods. d) In vitro release profiles of CPT from prodrug in PBS
under different pH conditions. Error bars were calculated from three
independent experiments. Error bars represent ± SD (n = 3).
Subsequently, the H2O2-induced release of CPT was also
confirmed as monitored by ESI-MS analysis. From the mass
spectra, we observed molecular peaks corresponding to CPT
([M+ H] = 349.52) and residue ([M + H] = 684.3) as shown in
Figure S11. This is taken as evidence that the hydrolysis of MIDA
boronate can release active drug moiety present in L-M-CPT was
cleaved by H2O2. The above investigations suggest that the MIDA
boronate as an outstanding platform by virtue of the uniform
benchtop stability for in situ release of the corresponding unstable
boronic acids. The sensing mechanism of the MIDA boronate
ligand containing L-M-CPT responding to H2O2/ H+ was
summarized and displayed in Scheme 2.
Scheme 2. Proposed drug release mechanism of the reaction of
prodrugs with H2O2 at the tumor site.
To more precisely demonstrate the drug release process in
cancer cells, probes L-B-CPT and L-M-CPT were separately
incubated with HeLa cells and B16F10 cells. Figure 2 shows the
subcellular location of L-M-CPT and L-B-CPT with that of
lysosome indicator (Lyso-Tracker Deep Red) to examine the
feasibility of prodrug using folate receptor-enriched tumor cells at
37 °C. After 2.5 h of incubation, the staining of the green
fluorescence translocation to the lysosomes shown colocalization
with the red fluorescence of the labeled lysosomes with Pearson’s
correlation coefficient of 0.94, which indicated that drug release of
L-M-CPT by H2O2 as a result of the acidic microenvironment. The
acid-dependent CPT release sustained manner was attributed to
the acid-labile MIDA boronate ligand of drug delivery system in
2
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10.1002/cmdc.202100354
ChemMedChem
RESEARCH ARTICLE
real time. This behavior stands in marked contrast to what is seen
in the case of L-B-CPT and is thus consistent with the proposed
the instability in the blood circulation.
To demonstrate the biodistribution and tumor accumulation
behavior of the drug carriers, the mice were administered
intravenously with the L-M-CPT. As shown in Figure 4, the
prodrug exhibited strong fluorescence in tumor in the first hour,
revealing rapid distribution of the L-M-CPT via the blood
circulation and significant drug release by H2O2 at the tumor site
in vivo. To clearly ascertain the tissue distribution of L-M-CPT in
mice, the internal organs after anatomy, including heart, liver,
spleen, lung, kidney and tumor, were resected and imaged in vivo
after intravenous administration. Analysis of organ distributions
showed low fluorescent intensities in the liver, lung, and kidney.
In contrast, the tumor bestowed the strongest fluorescence,
indicating that majority of prodrug could successfully accumulated
and the CPT released by plentiful H2O2 at the tumor site. More
importantly, there were no selective accumulation in the heart,
indicating that the prodrug could effectively avoid the drug
cardiotoxicity. Together, the results collectively support the active
MIDA boronate ligands of L-M-CPT as vehicles exhibited
prominent tumor targeting capacity in tumor sites via receptor-
mediated endocytosis.
Figure 2. The intracellular trafficking of L-B-CPT (1.0 μM) and L-M-CPT (1.0
μM) by using Lyso-Tracker (colocalization Pearson correlation coefficients, L-
M-CPT: 0.94) in HeLa cells at 37 °C (λex = 488 nm, λem = 520–560 nm). The
intracellular trafficking of L-M-CPT (1.0 μM) in B16F10 cells at 37 °C.
Encouraged by the intracellular drug release behavior of L-
M-CPT used as drug carrier, the cell cytotoxicity of L-M-CPT was
investigated quantitatively via an MTT colorimetric assay. As
presented in Figure 3, both free CPT, L-B-CPT and L-M-CPT
were incubated with HeLa cells show dose-dependent cytotoxicity
at pH 7.4 and pH 6.0. With increased concentration of CPT, The
CPT-loaded L-M-CPT exhibited the highest cytotoxicity (IC50, 1.21
μg/mL) against HeLa cells at pH 6.0, which was ascribed to the
receptor mediated endocytosis and drug released with the
protecting MIDA boronate was removed in acidic environments. It
also can be observed that the CPT exhibited higher cytotoxicity
than prodrug due to the L-M-CPT has markedly reduced cellular
uptake as compared with free CPT, consistent with a previous
report. Overall, the prodrug exhibited significant anticancer
efficacy to cancer cells.
Figure 4. White light (a, c) and fluorescence (b, d) images of BALB/c-nude
tumor bearing mice injected with L-M-CPT. Dissected tumor region, major
organs (e) and the corresponding fluorescent images (f) form tumor-bearing
mice after injection with L-M-CPT, He = heart, Li = liver, Sp = spleen, Lu = lung,
Ki = kidney, Tu = tumor.
Enhanced tumor-specific accumulation of prodrug in vivo
offers opportunities to evaluate the antitumor effect, as observed
by measuring tumor size and subcutaneous inoculation into
HeLa-inoculated xenograft murine model every three days. Mice
were injected with 3 mg/kg of CPT and CPT-loaded prodrug at
days 3, 6, 9, 12, 15, 18 and 21. The tumor xenograft volume after
Figure 3. In vitro cell viability assay of L-M-CPT in HeLa cells at pH 7.4 and 6.0
for 48 h. Each error bar represents the standard deviation of 3 replicates.
3
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10.1002/cmdc.202100354
ChemMedChem
RESEARCH ARTICLE
administration of the prodrug, PBS and free CPT are shown in
Figure 5. Among the three groups, tumors treated with the L-M-
CPT demonstrated significant growth inhibition compared with
control group (PBS and free CPT). After 21 days, significant
reductions in tumor volume were monitored, indicates its target
specificity. In contrast, the tumors of the mice injected with free
CPT and PBS grew rapidly over time. Within the same time frame,
29.7-fold increase for free CPT and 46.6-fold increase for PBS.
The results revealed that folated-prodrug could enhance the
antitumor efficacy and low side effects in the acidic tumor
microenvironment, supporting the remarkable potential drug
release utility for effective diagnosis and therapy of cancer.
probe, described above, could provide a promising framework for
overcoming the stability in the blood circulation and achieving
drug release in acidic tumor environments.
Acknowledgements
We would like to acknowledge the financial support of the Young
Science and Technology Plan Project of Gansu Province
(20JR5RA193), and the General Program of the Key Laboratory
of Hexi Corridor Resources Utilization of Gansu Universities
(XZ2003), and the PhD Start-up Foundation of Hexi University of
China (KYQD2020010).
Keywords: hydrogen peroxide • MIDA • prodrug • cancer
treatment • tumor environments
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ChemMedChem
RESEARCH ARTICLE
Entry for the Table of Contents
To achieve controlled shielding of loaded drugs in the biodistribution and blood circulation, we propose that nitrogen mustard as the
encapsulating to create a promising framework for overcoming the stability in the blood circulation. The prodrugs of nitrogen mustard
coupled with boronic acid or arylboronate derivatives can be selectively activated by H2O2 in tumor sites. Using this approach, the
stability and stimulus responsive properties of prodrug as stable surrogates can synergistically achieve effectively prevented premature
CPT release and achieved prompt intracellular release during intracellular trafficking. Therefore, the probe, could provide a promising
framework for overcoming the stability in the blood circulation and achieving drug release in acidic tumor environments.
5
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