Carrier-Free Delivery of Precise Drug–Chemogene Conjugates for Synergistic Treatment of Drug-Resistant Cancer

Article abstract of DOI:10.1002/anie.202006895

Combinatorial antitumor therapies using different combinations of drugs and genes are emerging as promising ways to overcome drug resistance, which is a major cause for the failure of cancer treatment. However, dramatic pharmacokinetic differences of drug

Full text of DOI:10.1002/anie.202006895

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A Journal of the Gesellschaft Deutscher Chemiker
Angewandte
Chemie
International Edition
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Accepted Article
Title: Carrier-Free Delivery of Precise Drug-Chemogene Conjugates for
Synergistic Treatment of Drug-Resistant Cancer
Authors: Lijuan Zhu, Yuanyuan Guo, Qiuhui Qian, Deyue Yan, Yuehua
Li, Xinyuan Zhu, and Chuan Zhang
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.202006895
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10.1002/anie.202006895
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RESEARCH ARTICLE
Carrier-Free Delivery of Precise Drug-Chemogene Conjugates for
Synergistic Treatment of Drug-Resistant Cancer
Lijuan Zhu,[a],[b] Yuanyuan Guo,[b] Qiuhui Qian,[b] Deyue Yan,[a],[b] Yuehua Li,[c] Xinyuan Zhu,[b] and
Chuan Zhang*[b]
[a]
Dr. L. Zhu, Prof. D. Yan
Institute of Molecular Medicine, Renji Hospital, School of Medicine, Shanghai Jiao Tong University
160 Pujian Road, Shanghai, 200217, China
[b]
Y. Guo, Q. Qian, Prof. D. Yan, Prof. X. Zhu, Prof. C. Zhang*
School of Chemistry and Chemical Engineering, Frontiers Science Center for Transformative Molecules, and State Key Laboratory of Metal Matrix
Composites, Shanghai Jiao Tong University,
800 Dongchuan Road, Shanghai, 200240, China
E-Mail: chuanzhang@sjtu.edu.cn
[c]
Dr. Y. Li
Department of Radiology, Shanghai Jiao Tong University Affiliated Sixth People’s Hospital, Shanghai Jiao Tong University School of Medicine
600, Yi Shan Road, Shanghai 200233, China
Supporting information for this article is given via a link at the end of the document.
Abstract: Combinatorial antitumor therapies using different
chemodrug combo or chemo/gene agent combo emerge as promising
ways to overcome the drug resistance, a major cause for the failure
of cancer treatment. However, dramatic pharmacokinetic differences
highly relies on the accurate molar ratio of the selected drugs.[7]
The latter approach involves the co-administration of chemodrug
and drug resistance related protein inhibitors, such as small
molecule inhibitors,[8] antisense oligonucleotides (ASOs),[9] small
of adopted drugs greatly impede the combinatorial antitumor practices, interfering RNAs,[10] which restores the sensitivity of tumor cells to
raising the great demand of developing appropriate drug delivery
systems (DDSs) for tumor treatment. By employing fluorescent
dithiomaleimide (DTM) as a linker, herein, we conjugate two paclitaxel
used chemodrug. As the drug resistance is mainly caused by the
evolutionary adaption of tumor cells to the drug-induced pressure,
reversing drug resistance at genetic level is more effective to treat
the drug-resistant cancer. However, intrinsic differences between
small molecular drugs and macromolecular gene therapeutics are
even more prominent, making the delivery of chemodrug/gene
combo more challenging. Although various materials, such as
cationic polymers,[11] micelles,[12] liposomes,[13] porous
organic/inorganic nanomaterials,[14] have been explored to serve
as co-delivery carriers, concerns regarding drug loading capacity,
stability, and biosafety remain in this field. As such, carrier-free
DDS emerges as a new fashion in drug delivery study due to the
avoidance of carrier-induced problems.[15] For instance,
amphiphilic drug-drug conjugate (ADDC)[16] or amphiphilic
supramolecular drug-drug (ASDD) pair[17] have been engineered
using two small molecular drugs with opposite solubility, which
further self-assemble into micellular nanoparticles for systemic
delivery. These strategies allow the delivery of selected drugs with
accurate molar ratio to achieve great synergistic therapeutic
effects, but usually with limited type of drug molecules. In contrast,
carrier-free DDS containing both chemo and gene therapeutics is
seldom explored so far.
Recently, we demonstrated a concept of chemogene that
integrated chemodrug into sequence-specific nuclei acids to
simultaneously perform as gene regulator and chemotherapeutic
agents.[18] However, chemogene alone is difficult to transfect the
cells and play its functions owing to its negatively-charged feature,
requiring us to construct a nanoformulation for delivery.[18]
Inspired by ADDC approach, we envision that conjugating a
hydrophobic drug and chemogene to form a precise drug-
chemogene conjugates (DCgCs) may not only overcome the
carrier issue, but also achieve drug/drug and drug/gene
combinatorial therapy to reverse the MDR. As a proof-of-concept,
floxuridine (FdU) incorporated chemogene with antisense
sequence that target P-gp gene is employed to conjugate with
PTX to form the DCgC. As shown in Scheme 1, two PTX
molecules are first connected to a fluorescent and azide-
(PTX) molecules with
a
floxuridine (FdU)-integrated antisense
precise
oligonucleotide (termed as chemogene) to form
a
macromolecular drug-chemogene conjugate. This PTX-chemogene
conjugate can self-assemble into spherical nucleic acid (SNA)-like
micellular nanoparticle as a carrier-free DDS, which effectively knocks
down the expression of P-glycoprotein first and subsequently releases
the FdU and PTX to exert a synergistic antitumor effect and greatly
inhibit the tumor growth. Our carrier-free DDS bearing drug/drug
synergy and chemo/gene synergy may shed light on reversing multi-
drug resistance of cancer.
Introduction
Multidrug resistance (MDR) greatly hinders the effectiveness of
chemotherapy in cancer research and clinical applications, which
are mainly caused by the adaptive gene expression of tumor cells
under stressed pressure.[1] For instance, P-glycoprotein (P-gp), a
typical drug efflux pump that is usually overexpressed in drug-
resistant cells, has proven to be a main factor to reduce the
intracellular drug accumulation, especially for the hydrophobic
drugs (e.g. vincristine, paclitaxel, doxorubicin), resulting in the
failure of killing the tumor cells.[2] To address this issue, two major
strategies have been developed to reverse the drug resistance,
including: combination use of different chemodrugs[3] and
synergistic administration of chemodrug/inhibitor combo.[4] For
the former case, different chemodrugs with diverse molecular
mechanisms could exert antitumor effects cooperatively and
improve the efficacy via preventing cancer cells to form
compensatory
resistance
mechanism.[5]
For
instance,
combination use of doxorubicin and -lapachone has been
confirmed to against the drug-resistant breast tumor.[6] However,
the effectiveness of drug combination strategy is limited and
1
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10.1002/anie.202006895
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RESEARCH ARTICLE
containing DTM linker. Then the PTX dimer is further conjugated
with chemogene through click chemistry. Owing to their
amphiphilic feature, the obtained DCgCs can self-assemble into
SNA-like micellular particles, which may exhibit improved
physiological stability[19] and high cellular transfection
capability.[20] Equipped with chemogene and PTX, the SNA-like
assemblies first will knock down the P-gp expression, and then
release the fluoride monophosphate (FdUMP) and PTX either by
enzymatic degradation[18,21] or glutathione reduction[22], which can
serve as active agents to synergistically inhibit the proliferation of
tumor cells. With precise molecular design of DCgC and
synergistic functions of its assembly, our carrier-free DDS exhibits
great potential to reverse the drug resistance and suppress the
growth of drug-resistant cancer.
was first synthesized by condensation of the fluorescent DTM and
PTX with mole ratio of 1:2 (Figure S1 to S12). The successful
synthesis of PTX dimer was confirmed by proton nuclear
magnetic resonance (1H-NMR, Figure S10), carbon nuclear
magnetic resonance (13C-NMR, Figure S11) and Fourier-
transform infrared spectroscopy (FTIR, Figure S12). Matrix-
assisted laser desorption/ionization (Maldi-ToF) gave an m/z
value at 2123.448, which could be attributed to
[C110H120N6O32S2+Na]+. Chemogene, anti-P-gp ASO, and
scramble oligonucleotide (T20) with amine terminals (Table S1)
were obtained by DNA solid-phase synthesis and further modified
with dibenzocyclooctyne (DBCO) terminals via the reaction of
amine
and
N-hydroxysuccinimide.
20%
Denaturing
polyacrylamide gel electrophoresis (PAGE, Figure S13)
demonstrated the successful synthesis of the DBCO modified
oligonucleotides. Then the PTX dimer with azide group was
connected to the oligonucleotide strands by cooper-free click
chemistry. Maldi-ToF in Figure S14 verified the successful
synthesis of PTX-chemogene.
Results and Discussion
First, the PTX-chemogene conjugate was synthesized as
shown in Scheme 1a. In detail, PTX dimer with an azide group
Scheme 1. (a) The design and synthetic route of PTX-chemogene conjugates for the construction of self-fluorescent carrier-free DDS. (b) Schematic representation
of PTX-chemogene assembly to reverse the drug resistance. SNA-like PTX-chemogene assembly could efficiently enter the cells and be degraded into PTX under
intracellular GSH environment to prevent cell proliferation and chemogene to suppress the P-gp expression. Subsequently, FdUMP released by enzymatic
degradation could further induce cancer cell apoptosis.
The obtained amphiphilic PTX-chemogene could self-
assemble into nanoparticles in aqueous solution. After assembly,
the product was verified by non-denaturing agarose gel
electrophoresis (1% w/w). As shown in Figure 1a and Figure S15,
a major sharp band with much lower mobility could be observed
on the gel for all PTX-conjugated oligonucleotides compared to
that of free oligonucleotide strands, indicating the formation of
large assembled structures with relatively narrow size distribution.
To evaluate the stability of the obtained DDS, critical micelle
concentrations (CMCs) of the PTX-oligonucleotide conjugates
2
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10.1002/anie.202006895
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RESEARCH ARTICLE
were determined by a previously reported method using Nile red
as a florescent probe.[23] The results showed that CMC values of
assembled PTX-T20, PTX-ASO, and PTX-chemogene, were 8.7,
10.2, and 7.5 M, respectively (Figure 1b). In addition, dynamic
light scattering (DLS) measurements revealed that the average
hydrodynamic diameters of micellular structures assembled by
PTX-T20, PTX-ASO, and PTX-chemogene, were 89 nm, 105 nm,
and 81 nm, with polydispersity index (PDI) of 0.172, 0.118, and
0.219, respectively (Figure 1c), which are suitable for systemic
delivery.[24] Meanwhile, cryogenic electron microscopy (Cryo-EM,
Figure 1d), transmission electron microscopy (TEM, Figure S16
and Figure S17), and liquid atomic force microscopy (liquid AFM,
Figure S18) showed that the self-assembled PTX-chemogene
had spherical shape with the sizes ranging from ~ 50-90 nm. All
these data revealed that the PTX-chemogene was successfully
assembled into nanosized DDS.
flow cytometry and CLSM results indicated the efficient cellular
uptake behavior of the obtained PTX-chemogene assembly,
implying its potential to be an effective carrier for both
chemodrugs and nucleic acid delivery.
Figure 2. Self-fluorescent property of PTX-chemogene assemblies and their
cellular uptake behaviour. (a) Fluorescence spectra of FAM-labelled anti-P-gp
ASO (red line) and PTX-chemogene (black line). (b) Flow cytometry analysis of
HeLa/PTX cells incubated with FAM-labelled free anti-P-gp ASO, and PTX-
chemogene at the same oligonucleotide concentration of 2 µM at 37 oC for
different time intervals, 0.1 h, 2 h, and 6 h respectively. (c) CLSM images of
HeLa/PTX cells incubated with FAM-labelled free anti-P-gp ASO (yellow), and
PTX-chemogene (yellow), at an oligonucleotides concentration of 2 µM at 37 oC
for 2 h. Cell nuclei (red) were stained with PI. (Scale bar: 50 mm).
Figure 1. Characterizations of the SNA-like micellular particles assembled by
PTX-T20, PTX-ASO, and PTX-chemogene conjugates. (a) Non-denaturing
agarose gel (1% w/w) electrophoresis analysis of PTX dimer (lane 1), Anti-P-gp
ASO (lane 2), PTX-ASO (lane 3), chemogene (lane 4), PTX-chemogene (lane
5), T20 (lane 6), PTX-T20 (lane 7). (b) The CMCs of PTX-T20, PTX-ASO, and
PTX-chemogene determined by using Nile red as a fluorescence probe. (c)
Hydrodynamic diameters of PTX-T20, PTX-ASO, and PTX-chemogene in
aqueous solution. (d) Cryo-EM images of micellular particles assembled by
PTX-T20, PTX-ASO, and PTX-chemogene (Scale bar: 200 nm).
It has been widely reported that P-gp protein overexpressed on
tumor cell membrane highly accounts for the reduction of
intracellular drug accumulation and the raise of drug resistance
for hydrophobic drugs.[26] To investigate whether our designed
PTX-chemogene assembly could reverse drug resistance through
down regulating the P-gp protein expression, quantitative reverse
transcription polymerase chain reaction (qRT-PCR) analysis and
western blot assay were conducted to evaluate the P-gp
expression at both mRNA and protein level. After treating with
PTX-chemogene and a series of control samples, including naked
ASO, PTX-T20 assembly, PTX-ASO assembly, and mock group,
PTX-chemogene caused a 67% knockdown of P-gp mRNA in
HeLa/PTX cells (Figure 3a), which was close to that induced by
PTX-ASO (73%) and relative P-gp mRNA level in HeLa cells
(87%). As a comparison, only 20% P-gp mRNA knockdown was
observed in naked ASO, which could be ascribed to the poor cell
uptake efficiency of naked DNA. Meanwhile, the P-gp mRNA
knockdown of PTX-T20 assembly treated HeLa/PTX cells (34%)
is much lower than those of PTX-chemogene and PTX-ASO
treated cells, indicating that the anti-P-gp sequence was crucial to
regulate the gene levels. Moreover, protein expression of P-gp
valuated by western blot assay is consistent with the results of
qRT-PCR analysis. P-gp protein expression was knocked down
by 45% in PTX-chemogene treated HeLa/PTX cells, which is
comparable to that of PTX-ASO treated cells (P-gp protein
knockdown efficiency (46%), as determined by band densitometry
(Figure 3b). In contrast, PTX-T20 treated cells showed 20%
DTM, produced by reaction of 2,3-dibromomaleimide and thiols,
possesses a strong green fluorescence, which can be used for
tracking the DDS.[25] We used both flow cytometry and confocal
laser scanning microscopy (CLSM) to evaluate the in vitro cellular
uptake efficiency of PTX-chemogene using carboxyfluorescein
(FAM) labeled free ASO as a control. As shown in Figure 2a, PTX-
chemogene assembly showed an excitation wavelength of 405
nm and emission wavelength of 530 nm, which is similar to the
fluorescence property of FAM-labeled ASO. Thus, PTX-
chemogene assembly can perform as a self-fluorescent DDS for
biomedical applications. Flow cytometry showed that the mean
fluorescent intensity of PTX-chemogene assembly treated drug-
resistant HeLa (HeLa/PTX) cells was over ten times higher than
that of ASO/FAM treated group after 6 h incubation (Figure 2b),
suggesting the rapid internalization of PTX-chemogene. Even
after 6 h incubation, free DNA strands (ASO/FAM) still could
hardly internalize the HeLa/PTX cells. Moreover, PTX-
chemogene assembly treated HeLa/PTX cells exhibited stronger
green fluorescence in the cytoplasm than that of ASO/FAM
treated group after 2 h incubation as shown in Figure 2c. Both
3
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