An oxaliplatin(iv) prodrug-based supramolecular self-delivery nanocarrier for targeted colorectal cancer treatment

Article abstract of DOI:10.1039/c8cc07858k

A redox-responsive supramolecular nanocarrier was constructed from the self-assembly of spermine modified cyclodextrin and oxaliplatin prodrug. The nanocarrier could preferentially accumulate in polyamine transporter over-expressing HCT116 cells, releasing drugs under a reducing intracellular environment to maximize anticancer treatment.

Full text of DOI:10.1039/c8cc07858k

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An oxaliplatin(IV) prodrug-based supramolecular
self-delivery nanocarrier for targeted colorectal
cancer treatment†
Cite this: Chem. Commun., 2018,
54, 12762
Received 1st October 2018,
Accepted 18th October 2018
b
Wei Qi Lim,ab Soo Zeng Fiona Phua,b Hongzhong Chenb and Yanli Zhao
*
DOI: 10.1039/c8cc07858k
rsc.li/chemcomm
A redox-responsive supramolecular nanocarrier was constructed at the tumor site.7 To become pharmacologically active, Pt(IV)
from the self-assembly of spermine modified cyclodextrin and prodrugs need to undergo intracellular reduction discriminatingly
oxaliplatin prodrug. The nanocarrier could preferentially accumulate in cancer cells containing high concentrations of endogenous
in polyamine transporter over-expressing HCT116 cells, releasing reducing agents.8 This feature could reduce side effects to normal
drugs under a reducing intracellular environment to maximize anti- cells, as the activation of the active drug is selective in cancer cells.
cancer treatment.
In this work, we report the fabrication of self-assembled
nanoparticles through the host–guest complexation between
Platinum-based anticancer drugs are among the most widely spermine-functionalized b-cyclodextrin (CD-spermine) and
used chemotherapeutics for the treatment of colorectal cancer. cholesterol-conjugated oxaliplatin(IV) (oxliPt(IV)-chol) for colorectal
Despite their success, these drugs often suffer from some cancer-targeted and redox-responsive drug delivery (Scheme 1).
drawbacks such as poor tumor selectivity and accumulation Each component in the nanocarrier had an irreplaceable function,
as well as dosage-dependent drug resistance, thus limiting their i.e., oxaliplatin(IV) as a prodrug, spermine as a targeting ligand,
applications to some extent.1 To address these problems, and cholesterol and b-cyclodextrin for the formation of an inclu-
considerable research progress had been made to develop sion complex with inherent amphiphilicity for the self-assembly.9
sophisticated drug carriers for targeted delivery to tumors.2 As a natural biomolecule involved in cellular biochemical path-
However, the low drug loading capacity, potential systemic ways such as signalling and protein biosynthesis, polyamine
toxicity, complex fabrication, and related metabolism concern spermine exhibits favourable biocompatibility and minimal side
of these multifunctional nanocarriers have hampered their effects.10 Colorectal cancer cells were found to overexpress poly-
clinical translations toward practical uses.3
amine transportation receptors in order to scavenge polyamines
Carrier-free Pt(IV) prodrug delivery systems are a novel kind from exogenous sources for sustaining rapid cell division.11
of therapeutics.4 Such prodrugs are easily prepared by introdu- Hence, spermine could function as an active targeting ligand
cing two axial ligands to a Pt(II) drug to not only tune the redox
potential of the platinum core to Pt(IV) but also change
the hydrophobicity/hydrophilicity balance.5 The amphiphilic
molecular prodrugs could in turn form nanostructures that
act as both carriers and cargoes.6 Compared to traditional
carrier-based drug delivery systems, such carrier-free self-
delivery nanosystems demonstrate much higher drug loading
capacity and eliminate the toxicity concerns of the drug carriers,
whilst having the nanoscale advantage of the enhanced perme-
ability and retention (EPR) effect to achieve passive accumulation
a NTU-Northwestern Institute for Nanomedicine, Interdisciplinary Graduate School,
Nanyang Technological University, Singapore
b Division of Chemistry and Biological Chemistry, School of Physical and
Mathematical Sciences, Nanyang Technological University, 21 Nanyang Link,
Scheme 1 Schematic illustration of (a) self-assembly of CD-spermine
and oxliPt(IV)-chol for the formation of CD-spermine: oxliPt(IV)-chol nano-
particles and (b) redox-responsive drug release in cancer cells.
Singapore 637371. E-mail: zhaoyanli@ntu.edu.sg
† Electronic supplementary information (ESI) available: Synthesis and characterization
details. See DOI: 10.1039/c8cc07858k
12762 | Chem. Commun., 2018, 54, 12762--12765
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for enhanced accumulation of the nanocarrier in colorectal
cancer cells.
The oxliPt(IV)-chol prodrug as a guest molecule was prepared
by reacting ethylenediamine-modified cholesterol with oxliPt(IV)–
COOH (Schemes S1 and S2, ESI†). The chemical structures of
the intermediates and final product were fully characterized via
several analytical techniques (Fig. S1 and S2, ESI†). CD-spermine
as the host molecule was attained by reacting excess spermine
with tosylated b-cyclodextrin (Scheme S3, ESI†). Its structure was
well characterized with 1H NMR and mass spectrometry.
A decrease in the zeta potential from ꢀ20.1 mV of b-cyclodextrin
to ꢀ6.8 mV of CD-spermine also indicated the successful synthesis
of CD-spermine (Fig. S3, ESI†). Such changes could be attributed
to the reduction of the anionic hydroxyl groups on b-cyclodextrin
after the conjugation with spermine that carries highly positive
amino units.
Before preparing the self-assembled nanoparticles, we inves-
tigated whether the molecular prodrug could undergo the
reduction to give active oxaliplatin(II) species. High-performance
liquid chromatography (HPLC) and liquid chromatography-mass
spectrometry (LC-MS) were employed to study the reduction
reaction products of oxliPt(IV)-chol incubated with sodium
ascorbate. The reduction of the prodrug was expected to lead to
Fig. 1 (a) Job’s plot indicating a 2 : 1 binding ratio between CD-spermine
and oxliPt(IV)-chol, (b) 1H NMR comparison of CD-spermine and
CD-spermine: oxliPt(IV)-chol complex in D2O/DMSO-d6 (5/5, v/v),
(c) plot of concentration-dependent optical transmittance changes of
oxliPt(IV)-chol at 240 nm in the nanoparticle formation, and (d) TEM image
of CD-spermine: oxliPt(IV)-chol nanoparticles.
the dissociation of the axial cholesterol ligand and produce active effect (Fig. 1b). This result demonstrated that the cholesterol
oxaliplatin (Fig. S4, ESI†). HPLC monitoring revealed that the moiety of the prodrug was included into the hydrophobic cavity
reaction outcome was indeed consistent with our expectation. of the cyclodextrin host to form a supramolecular complex.
A peak corresponding to oxliPt(IV)-chol (retention time: 17 min) Based on the obtained binding ratio, CD-spermine: oxliPt(IV)-
disappeared after the reduction with sodium ascorbate, whilst chol nanoparticles were prepared by mixing oxliPt(IV)-chol in
there was an emergence of a new peak corresponding to oxliPt(II) DMSO with CD-spermine aqueous solution. The critical aggrega-
(retention time: 9.5 min). In addition, the LC-MS spectrum of the tion concentration (CAC) of the complex was determined to be
reaction solution confirmed the presence of the active oxaliplatin 64.9 mM by monitoring the change in the optical transmittance
drug and cholesterol ligand after the reduction reaction. These at 240 nm upon increasing the concentration of oxliPt(IV)-chol
results indicate that our designed molecule could indeed function (Fig. 1c). The as-prepared CD-spermine: oxliPt(IV)-chol nano-
as a redox-responsive prodrug. It is pharmacologically inactive particles were then characterized via transmission electron
and requires reductive elimination to achieve the active form.
microscopy (TEM), and the obtained images revealed a well-
The cytotoxicity of oxaliplatin arises from the formation of dispersed spherical morphology (Fig. 1d). The nanoparticles
an adduct with DNA nucleobases, specifically binding to the N7 exhibited a narrow size distribution with an average hydro-
guanine.12 Hence, the ability of the released oxaliplatin drug dynamic diameter of 130 nm as measured by dynamic light
binding with DNA was investigated. 50-GMP was used to stimu- scattering (Fig. S6, ESI†). The oxaliplatin encapsulation efficiency
late the interaction mechanism of the drugs with DNA, where was 22.7 wt% as measured by inductively coupled plasma-optical
the adduct products were studied by HPLC and LC-MS (Fig. S5, emission spectrometry (ICP-OES).
ESI†). Taking the result of the direct reaction of oxaliplatin(II)
To evaluate the redox-triggered drug release properties of
with 50-GMP as the reference, HPLC peaks at the retention the CD-spermine: oxliPt(IV)-chol nanoparticles, the cumulative
times of 8.4 min and 10.2 min were found, corresponding drug release of Pt from the nanoparticles was studied using
to the adduct of oxaliplatin(II) with one and two 50-GMP, ICP-MS (Fig. 2). 5 mM sodium ascorbate solution was used to
respectively. The same peaks appeared for the reaction between simulate the reducing environment inside cancer cells.13 In the
the reduced oxliPt(IV)-chol prodrug and 50-GMP, indicating that absence of sodium ascorbate, there was only around 25% Pt
the released platinum species from the prodrug was most likely detected after 24 h, suggesting that the nanoparticles were
oxaliplatin(II) and could possibly exhibit similar behavior in vitro. relatively stable under physiological conditions. In comparison,
To prepare CD-spermine: oxliPt(IV)-chol nanoparticles, we first in 5 mM sodium ascorbate, the released amount of Pt drasti-
quantitatively investigated the binding stoichiometry between cally increased to 63%, illustrating that the CD-spermine:
CD-spermine and oxliPt(IV)-chol. A Job’s plot was obtained to oxliPt(IV)-chol nanoparticles could be an effective drug delivery
1
determine the host–guest inclusion ratio of 2 : 1 (Fig. 1a). H NMR system. The encapsulated Pt(IV) prodrug would only be released
spectra of CD-spermine revealed changes in the chemical shifts of in the presence of enough amount of reducing agents in the
characteristic protons before and after binding with 0.5 equivalent intercellular matrix of cancer cells. This feature ensured minimal
of oxliPt(IV)-chol, attributing to the complexation-induced shielding drug leakage before the cellular uptake for reduced side effects.
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Subsequently, the anticancer efficacy of the nanoparticles
was evaluated via 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT) assay. The cytotoxicity of CD-spermine alone on
HCT116 cells was first evaluated and the obtained results showed
low cytotoxicity. Hence, CD-spermine would have a minimal effect
on the cytotoxicity of the nanocarrier system (Fig. S8, ESI†). Then,
oxliPt(II) and CD-spermine: oxliPt(IV)-chol nanoparticles with
various Pt concentrations from 1 to 100 mM were incubated
with HCT116 cells for 24 h and 48 h. As shown in Fig. 3b, the
CD-spermine: oxliPt(IV)-chol nanoparticles exhibited both concen-
tration- and time-dependent cytotoxicity. The nanoparticles
presented higher cytotoxicity to HCT116 cells as compared to free
oxliPt(II) possibly due to enhanced cellular accumulation of the
nanoparticles. The internalization of the drug-containing nano-
Fig. 2 Cumulative platinum drug release profile from CD-spermine: particles via the transporter-mediated process is more efficient
oxliPt(IV)-chol nanoparticles in PBS solution (pH 7.4) and in 5 mM sodium
than simple molecular diffusion of the free platinum drug within
the same incubation time. Thereby, a better cell killing effect was
ascorbate solution.
observed for the nanoparticles. ICP-MS results of the cellular Pt
uptake showed that the accumulation of CD-spermine: oxliPt(IV)-
The cellular uptake of CD-spermine: oxliPt(IV)-chol nano-
chol nanoparticles was about 30-fold greater than that of oxliPt(II)
particles was evaluated using fluorescein isothiocyanate (FITC)-
at 12 h incubation (Fig. S9, ESI†), verifying that the enhanced
labelled nanoparticles. Fig. 3a shows the confocal laser scan-
endocytosis of the nanoparticles was due to its nanosize and
ning microscopy (CLSM) images of colorectal cancer HCT116
targeting effect of spermine. At a longer incubation time, however,
cells after the incubation with the nanoparticles at 2 h and 18 h.
the therapeutics effect of CD-spermine: oxliPt(IV)-chol nano-
After 2 h of incubation, a little green fluorescence was observed
inside the cells, implying that the nanoparticles hardly entered
the cells. On the other hand, the enhanced cellular FITC
fluorescence after 18 h of incubation was evident that the
particles (IC50 = 41.2 mM at 48 h) was not significantly better than
that of the free oxaliplatin (IC50 = 37.7 mM at 48 h). This
observation might be the result of the time-dependent drug
release characteristics of the nanoparticles. Despite the enhanced
CD-spermine: oxliPt(IV)-chol nanoparticles could be internalized
cellular uptake, the nanoparticles had to undergo the reduction of
with time. Flow cytometry also confirmed that the uptake of the
the Pt(IV) species to its active Pt(II) form and subsequent dissocia-
nanoparticles was time-dependent (Fig. S7, ESI†).
tion and diffusion from the nanoparticles before inhibiting the
cell growth. Moreover, it was plausible that not all of oxliPt(IV)-chol
prodrug was reduced under these conditions, offsetting the effect
of increased nanoparticle accumulation and mitigating the corres-
ponding cytotoxicity in vitro.
To confirm the targeting ability of the spermine ligand, FITC
labelled CD-NH2: oxliPt(IV)-chol nanoparticles as a control
group were also prepared for comparison. It was anticipated
that the cellular uptake of these nanoparticles would be lower
as the ethylenediamine ligand of CD-NH2 cannot be recognized
by the polyamine transporter, thereby reducing the rate of
cellular internalization. As shown in Fig. S10, ESI,† the fluores-
cence intensity of FITC was stronger in the CLSM images of the
CD-spermine: oxliPt(IV)-chol treated cells than those treated
with the control nanoparticles, indicating that the spermine
modification could promote cellular uptake probably through
the transporter-mediated pathway. Furthermore, the HCT116 cell
viability after incubating with CD-spermine: oxliPt(IV)-chol nano-
particles at 24 h and 48 h was shown to be lower than that of
CD-NH2: oxliPt(IV)-chol nanoparticles (Fig. 4a). The higher cyto-
toxicity of the CD-spermine: oxliPt(IV)-chol nanoparticles could be
due to their increased cellular uptake via polyamine transporter-
Fig. 3 (a) CLSM images of HCT116 cells incubated with FITC-labelled
CD-spermine: oxliPt(IV)-chol nanoparticles. Cell nuclei were stained
mediated endocytosis. In addition, competitive inhibition of the
polyamine transporter was carried out by pretreating cells with
by H33342. Scale bar: 50 mm. (b) In vitro cytotoxicity of oxliPt(II) and
spermine prior to the incubation with CD-spermine: oxliPt(IV)-chol
nanoparticles. As observed in Fig. 4b and c, the fluorescence
CD-spermine: oxliPt(IV)-chol nanoparticles incubated with HCT116 cells
for 24 and 48 h.
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National Research Foundation Investigatorship (No. NRF-
NRFI2018-03).
Conflicts of interest
There are no conflicts to declare.
Notes and references
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In summary, redox-responsive supramolecular prodrug
nanoparticles have been successfully constructed via host–
guest interaction. OxliPt(IV)-chol and CD-spermine not only
serve as the building units, but also as the cargo and an active
targeting component, respectively. Thus, the supramolecular
nanoparticles prepared using this unique strategy integrate
the advantages of active targeting, prodrug, and carrier-free
systems, while simplifying the assembly complexity. The
prodrug could be released under a reductive cellular micro-
environment, allowing for controlled drug release. Importantly,
in vitro experiments have confirmed that the CD-spermine:
oxliPt(IV)-chol nanoparticles offered comparable cytotoxicity as
free platinum drugs and provided specificity to cancer cells
based on their redox responsiveness and targeting capability.
This work presents an alternative strategy to the development
of smart supramolecular nanocarriers, showing great potential
in the field of controlled drug release and delivery toward
cancer treatment.
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This research was supported by the Singapore Academic
Research Fund (No. RG11/17 and RG114/17) and the Singapore
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