Automated modular synthesis of Aptamer-drug conjugates for targeted drug delivery

Article abstract of DOI:10.1021/ja4117395

Aptamer-drug conjugates (ApDCs) are promising targeted drug delivery systems for reducing toxicity while increasing the efficacy of chemotherapy. However, current ApDC technologies suffer from problems caused by the complicated preparation and low control

Full text of DOI:10.1021/ja4117395

Communication
pubs.acs.org/JACS
Open Access on 01/31/2015
Automated Modular Synthesis of Aptamer−Drug Conjugates for
Targeted Drug Delivery
RuoWen Wang,† Guizhi Zhu,† Lei Mei,† Yan Xie,† Haibin Ma,† Mao Ye,† Feng-Ling Qing,§
and Weihong Tan*,†
†Molecular Sciences and Biomedicine Laboratory, State Key Laboratory for Chemo/Biosensing and Chemometrics, College of
Chemistry and Chemical Engineering and College of Biology, Collaborative Innovation Center for Chemistry and Molecular
Medicine, Hunan University, Changsha 410082, China
§Key Laboratory of Organofluorine Chemistry, Shanghai Institute of Organic Chemistry, Chinese Academy of Science, 345 Lingling
Lu, Shanghai 200032, China
S
* Supporting Information
antibodies, some aptamers themselves can be used as
macromolecular drugs.7 Compared with antibodies, aptamers
are more stable as dry powders or in solution, less toxic, easier
to modify for diverse purposes, and more economical to
produce. Hence, aptamers have been exploited as exceptional
targeting elements for cancer cells and have been widely studied
in targeted cancer therapy.8
ABSTRACT: Aptamer−drug conjugates (ApDCs) are
promising targeted drug delivery systems for reducing
toxicity while increasing the efficacy of chemotherapy.
However, current ApDC technologies suffer from
problems caused by the complicated preparation and low
controllability of drug−aptamer conjugation. To solve
such problems, we have designed and synthesized a
therapeutic module for solid phase synthesis, which is a
phosphoramdite containing an anticancer drug moiety and
a photocleavable linker. Using this module, we have
realized automated and modular synthesis of ApDCs, and
multiple drugs were efficiently incorporated into ApDCs at
predesigned positions. The ApDCs not only recognize
target cancer cells specifically, but also release drugs in a
photocontrollable manner. We demonstrated the potential
of automated and modular ApDC technology for
applications in targeted cancer therapy.
Aptamer−drug conjugates (ApDCs) have been developed to
exploit aptamer-based targeted drug delivery.8a,e,f ApDCs/
ADCs are similar to molecular trains (MT) in structure, and
drugs are manually loaded into the “vehicles” (aptamers/
antibodies) and selectively transmitted to target cells.
Compared to ADCs, ApDCs present unique advantages,
including vehicle economy and procedural simplicity, because
aptamers are relatively small in size and can be chemically
synthesized. For aptamer−drug coupling, current ApDC
technologies largely rely on the noncovalent association of
drug and specific DNA sequences,8g or complicated and less
efficient organic synthesis.8a These technologies suffer from
complicated preparation, low controllability of site-specific drug
conjugation on vehicles, low synthesis yield in some cases, low
drug loading capacity and the accompanying high cost and low
spatiotemporal controllability in drug release. For instance, only
a limited number of drug copies can be conjugated onto one
aptamer strand in ApDCs, resulting in low drug loading
capacity. This is also the case for antibody vehicles, which can
deliver only a very few drug molecules in each ADC. Moreover,
the preparation of these ApDCs or ADCs is typically
complicated: aptamer and antibody “vehicles” are prepared
first, followed by the chemical reactions to conjugate drugs and
the vehicle manually.3,8,9
raditional anticancer chemotherapeutic drugs affect both
T
cancer cells and healthy cells without selectivity, resulting
in severe side effects.1 Targeted drug delivery is promising to
reduce drug toxicity in healthy cells and diminish side effects,
thereby improving therapeutic efficacy.2 Active targeted therapy
exploits the specific recognition ability of targeting elements
(e.g., antibodies and aptamers) to disease tissue. For instance,
antibody−drug conjugates (ADCs) combine less selective
cytotoxic drugs with antibodies, so that ADCs can selectively
target and kill disease cells, including cancer cells, with reduced
side effects.3 Several ADCs have received marketing approval
by the FDA, and more ADCs are in clinical trials.3b,d,4 This
demonstrates the feasibility of targeted therapy by conjugating
less selective drugs with molecular targeting elements that can
selectively recognize target disease sites.
Solid-phase synthesis technology represents a paradigm of
automated and highly controllable molecular synthesis.10 This
technology is able to generate DNA from individual
phosphoramidite building blocks (A, T, C, and G) with high
efficiency, providing a platform for automated and sequence-
predesigned DNA synthesis. Inspired by this technology, we are
interested in the development of therapeutic modules that can
be integrated into ApDCs, as well as the downstream
In addition to antibodies, aptamers comprise another class of
molecular targeting elements for selective disease tissue
recognition in targeted therapy. Aptamers are single-stranded
oligonucleotides that can specifically recognize their targets,
including live cancer cells, and bind to the targets with high
affinity.5 DNA aptamers targeting cancer cells can be generated
by cell-SELEX (systematic evolution of ligands by exponential
enrichment) technology developed by our group.6 Similar to
Received: November 28, 2013
Published: January 31, 2014
© 2014 American Chemical Society
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Communication
automated and modular ApDC preparation by solid-phase
synthesis technology. Here we report the design and synthesis
of the next-generation of ApDCs for targeted drug delivery, by
first developing a drug-incorporated module and then using the
solid-phase synthesis technology to automatically integrate
aptamers and drug modules. As shown in Figure 1A, the
and the backbone of MT to achieve photocontrollable drug
release.13
The synthesis of phosphoramidite D is started with 3-amino-
1,2-propanediol, which was selectively protected with Cbz and
TBS groups providing compound 1 in 72% yield in two steps
(Scheme 1). Removal of N-Cbzgroup followed by coupling of
Scheme 1. Synthetic Route of Phosphoramidite 8 as a
Module for DNA Synthesis
Figure 1. (A) Automated and modular synthesis of ApDCs from
phosphoramidites A, T, C, G, and D. (B) Structural features of
phosphoramidite D.
amine 2 with benzoyl chloride 3 gave nitrobenzyl bromide 4.
The nucleophilic substitution of 4 with 3-N-benzoyl 5-FU (5)
provided compound 6 in 52% yield. The solution of compound
6 in MeCN/H2O was submitted to UV light (365 nm)
irradiation, and the results demonstrated efficient photocontrol-
lable release of the drug moiety (see Supporting Information
(SI)). Deprotection of O-TBS groups by hydrochloride
solution in ethanol gave diol 7 in 90% yield, which was
converted into phosphoramidite 8 in two steps following
standard protocols.14
Using the modular phosphoramidite 8, any number of drug
moieties can be automatically conjugated with an aptamer in a
chained mode at predesigned sites on a DNA synthesizer.
Aptamer sgc8 was chosen as a model for the automated and
modular synthesis of ApDCs with a high capacity of drugs at
predetermined positions. Sgc8 can bind to target protein PTK7,
which is overexpressed on target HCT116 colon cancer cells,
but not on nontarget Ramos lymphoma cells.6a,15 To test the
efficacy of the MTs for targeted drug delivery using
phosphoramidite 8, we synthesized two ApDCs, MT-I (sgc8-
(PC-5FU)) and MT-II (sgc8-(PC-5FU)5) with one and five
5FU units, respectively, at the 5′-end of the aptamer (Table S1
(SI)). We also synthesized an ApDC sgc8-5FU incorporated
with 5FU from commercially available phosphoramidite, in
which no PC-linker was integrated. A random-sequence DNA-
drug conjugate (LIB, Table S1 (SI)) was also prepared as a
control.
aptamer moiety on one end serves as the locomotive of the
MT, and the tandem drug modules serve as the “boxcars”. Each
boxcar contains a drug (D) molecule, which was first integrated
into phosphoramidite as a therapeutic module phosphoramidite
D (Figure 1B). In addition, we incorporated spatiotemporal
controllability of drug release into the therapeutic module
through a photocleavable chemical linkage of drugs and
vehicles. The ApDCs can be automatically prepared from
modules D, A, T, C, and G on an automated DNA synthesizer
in a tailor-designed manner (Figure 1A). In this way, multiple
drug moieties can be conjugated onto one aptamer at
predesigned positions and drug loading capacity. The
automated conjugation of aptamers with drugs is operationally
simple and highly efficient. It also improves vehicle economy
and achieves spatiotemporally controllable drug release.
To realize the automated and modular synthesis of ApDCs,
the key is the design and synthesis of the therapeutic module:
phosphoramidite D. As shown in Figure 1B, phosphoramidite
D is composed of three parts: solid phase synthesis
functionalities, drug moiety, and a linker between. For synthesis
simplicity, we chose glycol as the backbone instead of ribose.11
Various drug molecules can be covalently incorporated into the
phosphoramidite as prodrugs, but subtle protecting strategy will
be required for those with nucleophilic functionalities such as
hydroxyl and amino groups. We chose Fluorouracil (5-FU), a
simple and widely used anticancer drug for treatment of many
types of cancers, including colorectal cancer and pancreatic
cancer,12 as a model in this study to test our idea. Between the
backbone and drug, a functional linker is necessary for efficient
and controllable drug release (as the results of ApDC sgc8-5FU
demonstrated). In this design a nitrobenzene derivative was
selected as the photocleavable linker (PC-linker) between drug
We then evaluated the specific recognition ability of MTs to
target cancer cells. To do so, biotinylated MTs were used in
combination with a streptavidin-PE-Cy5.5 (Phycoerythrin)
conjugate dye for a cell binding assay. In this assay, flow
cytometry was used to monitor the fluorescence intensities of
cells, with aptamer sgc8 as a positive control and random
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sequences (LIB) as a negative control. As shown in Figure 2A,
compared to the negative control, the fluorescence enhance-
cence intensity, demonstrating the specificity of MT-II
recognition and internalization into target cancer cells. The
ability of MTs to be internalized into target cancer cells
provides the basis of efficient targeted drug delivery.
Once MTs were validated for selective cancer cell
recognition and internalization, they were subsequently
evaluated for targeted therapy. The cytotoxicity was evaluated
using an MTS assay. The cytotoxic efficacy of 5FU released
from DNA−drug conjugates was first demonstrated using a
short oligonucleotide incorporated with phosphoramidite 8
(T(PC-5FU)3T), which was either irradiated with UV to
release 5FU (precleaved) before treatment of cells, or irradiated
by UV after incubation with cells (Figure S1 (SI)). It is worth
noting that UV irradiation was optimized and did not cause
dramatic cytotoxicity under our experimental conditions,
removing the concerns of side effects caused by UV
overexposure. The selective in vitro cytotoxicity induced by
MT-II was then studied. Free 5FU, LIB, and sgc8-5FU
(prepared from commercially available 5FU phosphoramidite,
without PC linkers between 5FU and backbones) were used as
controls. Specifically, target HCT116 cells were treated with
free 5FU, LIB, sgc8-5FU, and MT-II, respectively, at a series of
different 5FU equivalent concentrations. The resultant cells
were incubated for 2 h to allow specific binding and
internalization of drug or drug carriers into cells, prior to
washing and UV exposure for 1 h. The capability of cell
proliferation was evaluated using an MTS assay. While neither
LIB nor sgc8-5FU induced appreciable cytotoxicity, both free
5FU and MT-II showed dose-dependent cytotoxicity in target
HCT-116 cells (Figure 3). This demonstrated the robust
Figure 2. (A) Flow cytometric results indicating the selective
recognition abilities of MTs and sgc8-5FU to target HCT116 cells,
but not nontarget Ramos cells. Sgc8 was used as a positive control, and
LIB as a negative control. All probes were labeled with biotin at the 3′
ends. (B) Confocal miscroscopy images demonstrating the specific
binding and internalization of MT-II into target HCT116 cells, as
shown by strong fluorescence intensity of the membrane and
cytoplasm in cells treated with MT-II, in contrast to negligible
fluorescence intensity of cells treated with LIB. Both probes were
labeled with Cy5 at the 3′ ends. Cells were stained with Hoechst
33342 for nucleus identification.
ment of cells incubated with MTs indicates these MTs
maintained their specific binding ability to target HCT116
cells. The fluorescence intensities of cells bound with either
MTs or sgc8-5FU were weaker than those bound with sgc8,
presumably because the 5FU moiety influences aptamer−target
interaction. In contrast to specific binding to target cells,
neither LIB nor MTs bound to Ramos cells (Figure 2A),
indicating the selectivity of MTs for cancer cell recognition.
The maintenance of selective recognition ability of MTs to
target cancer cells provides the basis for downstream
application of targeted drug delivery.
To achieve efficient anticancer drug delivery, drug carriers
should be internalized into cancer cells. It has been reported
that many aptamers are capable of internalization into cancer
cells through various pathways. In particular, sgc8 was reported
to be internalized into target CEM T-cell leukemia cells.16 We
then studied whether MTs could be specifically internalized
into target cancer cells. In this study, MT-II and LIB were
labeled with fluorophore Cy5 and incubated respectively with
HCT116 cells in a cell culture incubator for 3 h. Cells were
stained with Hoechst 33342 to localize the nucleus and washed
prior to confocal microscopy observation. Results in Figure 2B
showed that strong fluorescence was identified on the cell
membrane or in the cytoplasm of HCT116 cells treated with
MT-II, indicating that MT-II not only bound to target
HCT116 cells, but was also internalized into cancer cells. In
contrast, cells treated with LIB displayed negligible fluores-
Figure 3. MTS results showing the specific and dose-dependent
cytotoxicity induced in target HCT116 cells by 5FU delivered via
MTs. Free 5FU, LIB, and sgc8-5FU were used as controls.
cytotoxic efficacy of 5FU delivered by MT-II into target cells
and photocontrollably released from these molecular trains at
target cancer cells, as well as the specificity of cytotoxicity
mediated by the aptamer moiety in the MT system.
In conclusion, we have developed MTs as an automated and
modular ApDC technology. Using this technology, anticancer
drugs can be synthesized into ApDCs at predesigned positions
and are capable of photocontrollable release in targeted cancer
therapy. This technology takes advantage of the well-
established solid-phase synthesis platform, in which a novel
therapeutic module, a phosphoramidite incorporated with an
anticancer drug, can be incorporated into ApDCs in a tailored
manner. 5FU was used as a model drug in this study to
demonstrate the principle. It is worth noting that this
phosphoramidite was further functionalized with the ability of
photocontrollable drug release. Our flow cytometry study
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ApDCs, and confocal microscopy results further showed the
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targeted cancer therapy.
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ASSOCIATED CONTENT
■
S
* Supporting Information
Procedures and additional data. This material is available free of
charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
tan@chem.ufl.edu
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by the National Key Scientific
Program of China (2011CB911000), the Foundation for
Innovative Research Groups of the NSFC (NSFC 21221003
and NSFC 21327009), the China National Instrumentation
Program (2011YQ03012412), and the Project Sponsored by
the Scientific Research Foundation for the Returned Overseas
Chinese Scholars, State Education Ministry.
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