Enhancing antitumor efficacy of nucleoside analog 5-fluorodeoxyuridine on her2-overexpressing breast cancer by affibody-engineered DNA nanoparticle

Article abstract of DOI:10.2147/IJN.S231144

Background: Chemotherapy, as an adjuvant treatment strategy for HER2-positive breast cancer, can effectively improve clinical symptoms and overcome the drug resistance of therapeutic monoclonal antibodies. Nucleoside analogues are a class of traditional c

Full text of DOI:10.2147/IJN.S231144

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O R I G I N A L R E S E A R C H
Enhancing Antitumor Efficacy of Nucleoside
Analog 5-Fluorodeoxyuridine on
HER2-Overexpressing Breast Cancer by
Affibody-Engineered DNA Nanoparticle
This article was published in the following Dove Press journal:
International Journal of Nanomedicine
Chao Zhang1
Background: Chemotherapy, as an adjuvant treatment strategy for HER2-positive breast
1
cancer, can effectively improve clinical symptoms and overcome the drug resistance of
Mengnan Han
Fanghua Zhang1
therapeutic monoclonal antibodies. Nucleoside analogues are a class of traditional che-
Xueli Yang1
motherapeutic drugs that are widely applied in adjuvant therapy. However, there are many
critical issues that limit their clinical efficiency, including poor selectivity and stability,
Jie Du1
severe side effects and suboptimal therapeutic efficacy. Hence, this work aims to develop
Honglei Zhang1
a new DNA nanocarrier for targeted drug delivery to solve the above problems.
Wei Li1
Methods: Four 41-mer DNA strands were synthesized and 10 FUdR molecules were attached
Shengxi Chen2
to 5ʹ end of each DNA strand by DNA solid-phase synthesis. An affibody molecule was
1College of Chemistry and Environmental
Science, Key Laboratory of Chemical
Biology of Hebei Province, Laboratory of
connected to the end of polymeric FUdR through a linker in one of the four strands. The affibody-
FUdR-tetrahedral DNA nanostructures (affi-F/TDNs) were self-assembled through four DNA
strands, in which one vertex was connected to an affibody at the end of a polymeric FUdR tail and
three vertices were only polymeric FUdR tails. In vitro cellular uptake of affi-F/TDNs was
examined visually with confocal fluorescence microscopy and flow cytometry, and the cytotoxi-
city of affi-F/TDNs against cancer cells was investigated with MTT assay. Cell apoptosis was
detected by Annexin V-FITC/PI double staining method. Using NOD/SCID (Mus Musculus)
mice model, the targeted killing efficacy of affi-F/TDNs was also evaluated.
Medicinal Chemistry and Molecular
Diagnosis of the Ministry of Education,
Hebei University, Baoding 071002,
People’s Republic of China; 2Biodesign
Center for BioEnergetics, Arizona State
University, Tempe, AZ 85287, USA
Results: The drug-loading of FUdR in affi-TDNs was 19.6% in mole ratio. The in vitro
results showed that affi-F/TDNs had high selectivity and inhibition (81.2%) for breast cancer
BT474 cells overexpressing HER2 and low toxicity in MCF-7 cells with low HER2 expres-
sion. During the in vivo application, affi-F/TDNs displayed good stability in the blood
circulation, achieved specific accumulation in tumor region and the best antitumor efficacy
(inhibition ratio of 58.1%), and showed excellent biocompatibility.
Conclusions: The affibody-DNA tetrahedrons, as a simple and effective active targeting
delivery nanocarrier, provided a new avenue for the transport of nucleoside antitumor drugs.
Keywords: 5-fluorodeoxyuridine, targeting therapy, her2, breast cancer, affibody, DNA
nanoparticle
Correspondence: Honglei Zhang; Wei Li
College of Chemistry and Environmental
Science, Key Laboratory of Chemical
Biology of Hebei Province, Laboratory of
Medicinal Chemistry and Molecular
Diagnosis of the Ministry of Education,
Hebei University, Baoding 071002,
People’s Republic of China
Introduction
Breast cancer is a global disease with the highest incidence among women worldwide,
and according to the 2018 Global Cancer Statistics data, new breast cancers globally
account for 24.2% of all new malignant tumors, and the mortality rate of breast cancer
accounts for 15%.1 Amplification of HER2 gene occurs in 25–30% of breast cancers
Tel/Fax +86 312 592 9009
Email zhanghonglei@hbu.edu.cn;
liweihebeilab@163.com
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http://doi.org/10.2147/IJN.S231144

Zhang et al
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and results in high levels of HER2 protein expression.2 Thus,
DNA nanoparticles as drug carriers have the advan-
the higher expression of HER2 in cancer cells than normal tages of convenient synthetic process, excellent mechan-
cells and the accessibility of its extracellular domain make ical rigidity, and structural stability.20 Meanwhile, DNA
HER2 an attractive target for developing targeted therapeutic
strategies. Many studies have proved that the antibody thera-
pies against HER2 (Trastuzumab, Pertuzumab, and
Ertumaxomab) can significantly improve the therapeutic
effect and overall survival rate.3 However, due to their large
molecular weight and humanized construction, monoclonal
antibodies have limitations in clinical application, including
limited ability to penetrate cells and tissues, the possibility of
causing an immune response, and the high cost. Moreover,
40–60% of patients do not respond to the treatment or
develop primary and secondary drug resistance to antibody
therapy.4,5 To overcome these limitations, small peptide
mimics of antibodies have become attractive alternatives
because they are smaller in size and can be easily engineered
and modified for specific biological activities.6 Affibody
molecules are the small engineered protein with 58-amino
acid residues and three α-helix bundle domains, which can
bind a range of different proteins, such as insulin, TNF-a,
EGFR, and HER2.7 Unlike antibodies, HER2-binding affi-
body molecules have the advantages of much smaller size
(~7.0 vs ~150.0 kDa), faster tumor-targeting ability, more
well-defined structure, and easier site-specific modification.
In addition, chemotherapy is often used clinically as an
adjuvant therapy for antibody therapy to overcome anti-
body resistance.8–11 The typical antitumor nucleoside ana-
log, 5-fluorouracil (5-FU), as a component of adjuvant
chemotherapy of trastuzumab has been widely applied
and achieved satisfactory therapeutic effects against
HER2 overexpressing breast cancer.12–14 It produces activ-
ity by incorporating DNA or RNA to inhibit cellular divi-
sion, and by covalently binding to thymidylate synthase
(TS) to inhibit the synthesis of deoxythymidine monopho-
sphate (dTMP) and cause cell death (Figure S1).15
Although 5-FU has been proved to be a successful ther-
apeutic agent, there still exist a lot of problems in clinical
application, such as short half-life (approximately 8–20
mins), poor selectivity, and severe side effects. To circum-
vent these drawbacks of 5-FU, a variety of organic and
inorganic drug delivery systems with nanomaterials have
been tested as the alternative approaches to systemic drug
administration, such as hydrogels, liposome, Metal-
Organic Frameworks (MOFs), boron nitride nanotubes
and so on.16–19 However, considering potential toxicity,
lack of tissue specificity, and unsatisfactory clinical
results, there are still many defects to be solved.
also possesses excellent biocompatibility, biodegradability,
and low cytotoxicity. Recently, based on the structural
similarity to thymidine (T), Mou21 and Jorge22 incorpo-
rated 5-fluorodeoxyuridine (FUdR, metabolite of 5-FU)
into DNA polyhedra and DNA origami, respectively, by
covalent ligation using DNA solid-phase synthesis tech-
nology, which exhibited an enhanced cytotoxicity related
to conventional 5-FU and FUdR. However, the processes
of constructing DNA polyhedra and DNA origami are
tedious and complicated, and the drug-loading rate is
low. In addition, the lack of active targeting also prevents
the nanodrug from being adequately taken up by cell, and
reduces their antitumor efficacy.
In our previous studies, an antibody-like DNA-affibody
nanoparticle was constructed using affibody molecule as
a targeting ligand and DNA tetrahedron as a scaffold. Then,
this nanoparticle was employed for transporting doxorubicin
and cisplatin to specific cancer cells through non-covalent
bonding, respectively, to enhance the targeting of drug
treatment.23,24 However, their main issues are the difficulty in
accurately controlling drug loading and the leak of chemother-
apy drugs during in vivo transport.25 These are the main
obstructions in the development and clinical application of
this kind of nanodrugs. Thus, in this study, a new affibody-
DNA tetrahedron nanoparticle loaded with FUdRs by covalent
bonds was prepared. The nano-structural drug contained one
DNA tetrahedral core, four polymeric FUdR oligonucleotides
tails and an affibody molecule attached to one end of
a polymeric FUdR oligonucleotides tail for targeting HER2.
These nanodrugs were prepared by a facile bioconjugation
process and DNA self-assembly (Scheme 1A), and had definite
drug-loading ratio (19.6%) and stable nanostructure. Then, the
potency of nanodrugs was also tested in vitro and in vivo. They
exhibited an excellent selectivity and inhibition for HER2-
overexpressing breast cancer. Based on the above findings
and results, the antibody-like DNA-affibody nanoparticle can
be used as a template to design the similar nucleoside nano-
drugs for targeting therapy of HER2-overexpressing tumor.
Materials and Methods
Materials and Chemicals
5-Fluorodeoxyuridine was purchased from Alfa Aesar
(Shanghai, China). 4,4ʹ-Dimethoxytrityl Chloride (DMT-
Cl, 97%) N,N-Diisopropylethylamine (DIPEA, 97%) and
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Scheme 1 (A) Schematic illustration of preparation procedure of affi-F/TDNs. FUdR was converted into its phosphoramidite and linked to the 5ʹ end of each DNA strand
one by one by solid-phase synthesis. The 5ʹ-NH2 labeled DNA strand, A13F-NH2, was conjugated with affibody via EMCS and then self-assembled into affi-F/TDNs with
other three DNA strands. (B) Schematic illustration of the HER2 receptor-mediated tumor-targeting delivery of affi-F/TDNs in vivo and in vitro. The affi-F/TDNs were
injected into blood vessels and accumulated in HER2-overexpressing breast tumor sites. Then, they specifically bound to HER2 receptor on the surface of breast cancer cells
and were internalized into cancer cell via receptor-mediated endocytosis. The affi-F/TDNs were degraded in lysosomes by intracellular nucleases to release FUdRs.
2-Cyanoethyl N,N,N’,N’-tetraisopropylphosphordiamidite
Synthesis of FUdR Phosphoramidite
(95%) were obtained from Macklin Inc. (Shanghai,
China). N-(ε-malemidocaproyloxy) succinimide ester
(EMCS) was purchased from Aladdin (Shanghai, China).
Ni-NTA agarose, and Sephadex G-25 were obtained from
GE Healthcare (Piscataway, USA). Imidazole, sodium
chloride, sodium acetate, polyacrylamide, tris base, acetic
acid, ethylenediaminetetraacetic acid (EDTA), magnesium
chloride, ethanol, and DAPI were obtained from Sangon
Biotech (Shanghai) Co., Ltd (China). Amicon ultracentri-
fugal filters were purchased from Merck Millipore Ltd
(Darmstadt, Germany). RPMI 1640 medium, DMEM
medium, MEBM medium, trypsin, 3-(4,5-dimethylthia-
zol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT),
antibiotic-antimycotic (100×) and fetal bovine serum
(FBS) were purchased from Wisent Biotechnology
To integrate FUdR into DNA strands, FUdR was first
converted into its corresponding phosphoramidite form,
and the detailed synthesis was performed according to
a previously reported procedure with slight modification
(Scheme S1).26 In brief, FUdR (2.46 g, 10 mmol) and
DMT-Cl (3.38 g, 10.0 mmol) were dissolved in 35 mL
of anhydrous pyridine, and stirred at room temperature
under a positive pressure of argon for 6 hrs. The reaction
mixture was concentrated by rotary evaporator and puri-
fied by column chromatography. The obtained FUdR-
DMT (2.75 g, 5 mmol) and DIPEA (0.78 g, 6 mmol)
were dissolved in 30 mL of anhydrous CH2Cl2. Ten milli-
liters of 2-Cyanoethyl N,N,N’,N’-tetraisopropylphosphor-
diamidite (1.21 g, 5.1 mmol) in anhydrous CH2Cl2 was
added dropwise to the reaction solution. The reaction was
(Nanjing) Co. Ltd (China). All chemicals were used with- stirred overnight at room temperature under a positive
out further purification.
pressure of argon. The reaction mixture was diluted with
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50 mL of CH2Cl2, washed with saturated brine, and the NKFNKEMRNAYWEIALLPNLNNQQKRAFIRSLYDD-
organic phase was dried over anhydrous Na2SO4, filtered, PSQSANLLAEAKKLNDAQAPKVDC.
According to the standard cloning and expression pro-
cedures, the modified affibody molecule ZhcHER2:342 was
expressed in E. coli BL21 cells. After the pelleted bacterial
cells were harvested and broken, the purified ZhcHER2:342
molecules were obtained through affinity chromatography.
The synthesis process of FUdR-DNA-affibody chimera
was shown in Scheme S2. The DNA strand A13F-NH2
(261.6 µg, 16.5 nmol) was dissolved in 200 µL of phosphate-
buffered saline (PBS) and treated with 20 µL of 50mM
EMCS in dimethyl sulfoxide. The reaction mixture was
incubated at 37°C for 3 hrs and stopped by the addition of
22 µL of 3 M NaOAc. Next, 600 μL of pre-cooled ethanol
was added to the reaction mixture and incubated at −20°C for
30 mins, and then the mixture was centrifuged at 15,000 g for
30 mins. After the resulting precipitate was washed twice
with 70% ethanol, it was dissolved in 200 μL of PBS buffer
and treated with 1500 μg (180 nmol) affibody in 1000 μL of
PBS buffer for 3 hrs at room temperature. Then, the reaction
mixture was purified on a Capto DEAE column (1 mL, GE
Healthcare), which was eluted with Tris-HCl (10 mM, pH
8.0) buffer containing 0.5–1.0 M NaCl. The purified compo-
nents were analyzed by 10% native polyacrylamide gel elec-
trophoresis. The gel was run at 100 V for 1 hr and stained
with GelStain (Sangon Biotech (Shanghai) Co., Ltd, China).
The eluted fraction of the previous step was further purified
using a HisTrap HP column (1 mL, GE Healthcare). Finally,
the product was eluted with 50 mM Tris-HCl, pH 8.0, con-
taining 300 mM NaCl and 150 mM imidazole. Aliquots of
each fraction were analyzed by 10% native polyacrylamide
gel electrophoresis. The pure A13F-affibody was concen-
trated using Amicon ultracentrifugal filters (MW cutoff 10
kDa) and stored at 4°C.
and concentrated by rotary evaporator. Purification by
silica gel flash chromatography (discontinuous gradient
of ethyl acetate/methanol (1:0–5:1) with 1% triethylamine
by volume as eluant) afforded pure product (yield 78%).
1H NMR data of FUdR phosphoramidite follow: 1H NMR
(600 MHz, DMSO): δ 11.87 (1H, s), 7.94 (1H, t, J = 6.6
Hz), 7.40 (2H, t, J = 7.2 Hz), 7.32–7.23 (7H, m), 6.88 (4H,
J = 8.4 Hz, J =3.0 Hz), 6.17–6.12 (1H, m), 4.54–4.48
(1H, m), 4.06–3.99 (1H, m), 3.74 (3H, s), 3.73 (3H, s),
3.71–3.59 (2H, m), 3.58–3.53 (1H, m), 3.51–3.46 (1H, m),
3.32–3.28 (1H, m), 3.25–3.19 (1H, m), 2.76 (1H, t, J =
11.4 Hz), 2.64 (1H, t, J = 12Hz), 2.44–2.39 (1H, m),
2.37–2.27 (1H, m), 1.13 (3H, J = 6.6 Hz), 1.11–1.09
(6H, m), 0.98 (3H, d, J = 6.6 Hz); 13C NMR (600 MHz,
DMSO): δ 158.1, 157.1, 156.9, 148.9, 144.6, 135.3, 135.2,
129.7, 127.8, 127.6, 118.9, 118.7, 113.2, 85.9, 85.8, 84.5,
58.4, 58.2, 55.0, 42.6, 42.5, 24.3, 19.8.
Solid-Phase Synthesis of FUdR-Containing
DNA Strands
The synthesis of FUdR-containing DNA strands (FUdR-
DNA) was carried out by our laboratory in collaboration
with TsingKe Biological Technology. All DNA sequences
(listed in Table S1) were synthesized on a Dr. Oligo 96
Oligo Synthesizer (Biolytic Lab Performance, Inc.
Fremont, USA) using the standard solid-phase phosphor-
amidite methodology. The FUdR-DNA strand used to link
a targeting ligand was an amino-modified DNA strand,
A13F-NH2. After synthesis, the obtained FUdR-DNA
strands were cleaved and deprotected from the CPG col-
umn, and then precipitated overnight in cold ethanol solu-
tion at −20°C. After the supernatant was removed by
centrifugation, the FUdR-DNAs were dissolved with
0.1 M triethylamine acetate (TEAA) and purified by
reversed phase HPLC using a BioBasic4 column (5 µm,
4.6ⅹ250 mm, Thermo Fisher Scientific, USA). Finally,
the obtained FUdR-DNA strands were characterized by
LCMS-2020 (Shimadzu, Japan) and quantified using
Nanodrop 2000c (Thermo Fisher Scientific, USA).
Preparation of Tetrahedral DNA
Nanostructures
The sequences of all single-stranded DNAs used to form
DNA tetrahedron were shown in Table S1. Four DNA
strands, A13 (5 nmol), B13 (5 nmol), C13 (5 nmol) and
D13 (5 nmol) were mixed in 2 mL TM buffer (10 mM
Tris-HCl, pH 8.0, 12 mM MgCl2). The solution was
quickly heated to 95°C for 10 mins, then cooled down
to 25°C for 20 mins to form TDNs. Four FUdR-DNA
strands, A13F (5 nmol), B13F (5 nmol), C13F (5 nmol)
Synthesis and Purification of FUdR-DNA-
Affibody Chimera
The sequence of the modified affibody molecule and D13F (5 nmol), were mixed and the FUdR-
ZhcHER2:342 used in this study was MIHHHHHHLQVD tetrahedral DNA nanostructures (F/TDNs) were
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constructed by the method described above. The affi-F/ concentration of 10% (v/v) and 1% (v/v), respectively.
TDNs were prepared by mixing A13F-affibody (10
nmol), B13F (10 nmol), C13F (10 nmol) and D13F (10
nmol), and the mixture was incubated 75°C for 15 mins.
It was then cooled to room temperature. The obtained
DNA nanoparticles were analyzed by 10% native poly-
acrylamide gel electrophoresis. The gel was run at 100
V for 1 hr and stained with GelStain.
MCF-7 breast cancer cells (ATCC HTB-22, low expres-
sion of HER2) were cultured in DMEM containing 10%
(v/v) FBS and 1% (v/v) penicillin-streptomycin solution.
The human breast epithelial MCF10A cells were cultured
in MEBM with 10% (v/v) FBS, 20 ng/mL human epider-
mal growth factor (EGF), 100 ng/mL cholera toxin,
0.01 mg/mL bovine insulin, and 500 ng/mL hydrocorti-
sone. The cells were placed at 37°C in a humidified atmo-
sphere containing 5% CO2.
Atomic Force Microscopy (AFM)
Characterization
For F/TDNs and affi-F/TDNs imaging, 10 µL samples (10
nM) were deposited onto a freshly peeled mica surface for
5 mins. Next, 40 µL of TAE/Mg2+ buffer (50 mM Tris,
20 mM acetic acid, 2 mM EDTA, 12 mM MgCl2, pH 8.0)
was added in the mica and then dried in air at room
temperature. The samples were imaged with AFM
(Agilent Technologies, 5500 AFM/SPM System, USA).
Targeted Uptake Evaluation by Confocal
Laser Scanning Microscopy
BT474 (1x105 cells/well) and MCF-7 cells (1x105 cells/
well) were separately planted into glass bottom microwell
disks at 37°C. When the cell confluency reached about
70%, the cells were treated with FAM-B13F, F/TDNs,
and affi-F/TDNs (equivalent to 5 µM FAM), respectively,
for 1 hr. The culture solution was carefully removed and
the cells were washed 3 times with ice-cold PBS.
Thereafter, the cells were fixed with 4% formaldehyde
for 20 mins at room temperature and washed 3 times with
ice-cold PBS again. Finally, the cell nuclei were stained
using 2.5 μg/mL DAPI for 30 mins at 37°C, followed by
rinsed with PBS for 2 times. The fluorescent images were
obtained using a Zeiss laser scanning confocal micro-
scope (Zeiss LSM 810, Germany). All images were
recorded and the target cells were counted using a 40×
oil objective.
Dynamic Light Scattering (DLS)
The hydrodynamic sizes of the affi-F/TDNs were measured
with Nanobrook Omni (Brookhaven Instruments Corporation,
USA). The concentration of samples used for the DLS analysis
was 500 nM.
Stability Analysis of Affi-F/TDNs
Solutions of affi-F/TDNs and single-strand FUdR-DNA
(unified to100 μg/mL) were separately mixed with non-
heat-inactivated FBS of equal volume and incubated at 37°
C. After incubation of 0, 1, 2, 4, and 8 hrs, the mixtures
were run on a 10% native polyacrylamide gel electrophor-
esis and stained with GelStain.
Targeted Uptake Evaluation by Flow
Cytometry
BT474 (1×105 cells/well) and MCF-7 cells (1×105 cells/
well) were seeded in glass bottom microwell disks in 2 mL
of medium with 10% FBS for 24 hrs under a humidified
5% CO2 atmosphere at 37°C, respectively. Afterward, the
medium was removed carefully and the cells were washed
twice with PBS. FAM-B13F, F/TDNs, and affi-F/TDNs
(equivalent to 5 µM FAM) were added to the culture
In vitro Release Study of FUdR from
Affi-F/TDNs
In order to prove that FUdR can be successfully released
from affi-F/TDNs in cells, a simulated enzymatic release
test was carried out in vitro. DNase II is widely found
inside the animal cells rather than plasma as an acid
endonuclease. Thus, affi-F/TDNs were incubated with
DNase II at a concentration of 20 U/mL with 8 hrs in medium separately, and the cells were treated for 2 hrs.
acetate buffer (pH 4.5) at 37°C.
Subsequently, the medium was removed and the cells were
washed with PBS three times. The cells were digested with
0.05% trypsin-EDTA and harvested by centrifugation. The
flow cytometry was performed on a BD FACS Calibur
(BD, Franklin Lakes, USA). The medium without DNA
Cell Cultures
BT474 breast cancer cells (ATCC HTB-20, overexpression
of HER2) were cultured in RPMI-1640 culture medium
with FBS and penicillin-streptomycin solution at the nanostructures was used as control.
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tumor size reached about 500 mm3, 100 µL of 20 µM
Cy5.0 labeled F/TDNs and affi-F/TDNs were injected into
mice by tail vein injection, respectively. The fluorescence
signals were recorded at 1, 2, 4, 8, and 12 hrs after
administration using an IVIS Lumina Series III
(PerkinElmer, USA) with excitation at 630 nm and emis-
sion at 670 nm. Then, to understand the distribution of
affi-F/TDNs in various tissues and organs, the mice treated
with affi-F/TDNs were sacrificed, and the tumors and
major organs (hearts, livers, spleens, lungs, and kidneys)
were excised and imaged with the same system.
In vitro Cytotoxicity
Exponentially growing BT474, MCF-7, and MCF-10A cells
were harvested and plated in 96-well plates at a concentration
of 5×103 cells/well. After the cells were incubated at 37°C
for 24 hrs, the culture medium was replaced with 100 µL of
fresh medium containing different concentrations of affibody
molecule, TDNs, FUdR, F/TDNs, and affi-F/TDNs, and the
cells were incubated for 4 hrs. Then, the cells were washed
with PBS thoroughly, and incubated in fresh medium for an
additional 72 hrs. Afterward, 10 µL of MTT (5 mg/mL) were
added to each well and the plates were incubated at 37°C for
4 hrs. The supernatant was discarded, and 100 µL of DMSO
was added to each well. The absorbance was determined at
490 nm. The cell viability was expressed as the percentage of
viable cells relative to the PBS-treated control group. Data
were reported as the mean of three independent experiments.
In vivo Antitumor Study
After the tumor reached about 100 mm3, the mice were
randomly divided into four groups, and received 0.2 mL of
PBS, FUdR, F/TDNs, and affi-F/TDNs containing equiva-
lent FUdR concentration (a dose of 10 mg/kg bw) via tail
vein injection. The nanodrugs were injected once every 3
days and three times in a row. The tumor volumes and
body weights of the mice were monitored and recorded
every 3 days. The tumor volume was calculated according
to the formula: V(mm3) = (length x width2)/2. On day 27,
the mice were sacrificed and the tumors were extracted
and weighed. The tumor growth inhibition ratio (IR) and
the ratio of tumor weight to body weight (tumor/body
weight) were calculated by the following formulas.
Apoptosis Analysis by Flow Cytometry
The cell apoptosis was evaluated by flow cytometry ana-
lysis using Annexin V-FITC/PI apoptosis analysis kit
(Absin, Shanghai, China) according to the manufacturer’s
instruction. Briefly, BT474, MCF-7, and MCF-10A cells
were seed in six-well plates and allowed to grow for 24
hrs, then exposed to a medium containing free FUdR, F/
TDNs, and affi-F/TDNs (a dose equal to 10 μM FUdR)
with a subsequent 12 hrs incubation. The cells were sub-
jected to apoptosis analysis using flow cytometry. Both
early apoptotic (Annexin V-FITC+/PI−) and late apoptotic
(Annexin V-FITC+/PI+) cells were included in cell apop-
tosis determinations.
IRð%Þ¼ð1ꢀTRTV=CRTVÞꢁ100%; RTV ¼Vt=V0
Tumor=body weight ¼Wtumor=Wbody
where TRTV and CRTV were defined as the mean value of
the relative tumor volume (RTV) of the test group and the
mean value of RTV of the control group, V0 and Vt were
defined as the tumor volume on day 0 and the day that the
value was recorded, and Wtumor and Wbody were defined as
weight of tumor and mice after the experiment, respec-
tively. Then, the collected tumors and major organs of
mice were fixed with 4% paraformaldehyde and cut into
5 μm thick slices for hematoxylin-eosin (HE) staining.
Animal Models
All animal procedures were performed in accordance with
the Guidelines of the Care and Use of Laboratory Animals
at Hebei University and experiments were approved by the
Animal Ethics Committee of Hebei University. In order to
construct a xenograft model, female NOD/SCID (Mus
Musculus) mice, 4–6 weeks old, were purchased from
Beijing Vital River Laboratory Animal Technology Co.,
Ltd. (China), and continued to be fed until their body
weight reached about 20 g. BT474 cells (1×108 cells)
were injected subcutaneously into the right coastal region
of mice to establish the tumor model.
Statistical Analysis
All samples were prepared and tested in triplicates or
more. Data were presented as mean standard deviation
(SD). The statistical significance of differences between
groups was determined by the Newman-Keuls analysis.
The differences were considered significant for *P<0.05,
In vivo Biodistribution Study
The tumor-targeting ability of affi-F/TDNs was assessed and highly significant for **P<0.01 and extremely signifi-
on the BT474 tumor xenograft mouse model. When the cant for ***P<0.001.
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excess affibody molecule and unreacted FUdR-DNA strands,
respectively (Figures S7 and S8). UV-vis spectroscopy was
used to verify the conjugation of affibody molecule to FUdR-
DNA strand via the measurement of the absorbance between
220 nm and 300 nm. Figure 1C shows that the absorption
spectrum of A13F-affibody was significantly different from
single affibody molecule and FUdR-DNA strand. A13F-
affibody was identified by SDS-PAGE, and the gel was treated
with GelRed and Coomassie Brilliant Blue R250, which
stained the DNA and protein, respectively (Figure 1D).
These results suggested that DNA strand and affibody mole-
cule were coupled and pure FUdR-DNA-affibody chimeras
were obtained by efficient purification process.
Results and Discussion
Preparation of FUdR-DNA Strands for
DNA Tetrahedron Nanoparticle
For delivering the cytotoxic anticancer drug FUdR, DNA
tetrahedron nanoparticle was selected as the drug carrier in
our study. Four FUdR-DNA strands were synthesized for
formation of DNA tetrahedron according to the principle
of base pairing. Considering the influence of the addition
of FUdR on DNA tetrahedron formation, the DNA strand
was screened and optimized. Finally, four 41-mer DNA
strands (A13, B13, C13, and D13) were selected to con-
struct DNA tetrahedron with 13 bp in each edge. The
DNA sequences information was listed in Table S1. To
realize the construction of FUdR-DNA strand, FUdR was
derivatized to its phosphoramidite form (Figures S2 and
S3), and then linked to the 5ʹ end of each 41mer DNA
strand one by one by solid-phase synthesis. The new four
FUdR-DNA strands (A13F, B13F, C13F, and D13F) were
characterized by mass spectrometry. The results showed
that the measured molecular weights were consistent with
the theoretical values (Figure S4, Table S2). It was also
confirmed by denatured polyacrylamide gel electrophor-
esis (PAGE) that FUdR-containing DNA strands were
apparently less mobile than unmodified DNA strands
(Figure S5). It was also a proof to illustrate FUdRs were
successfully inserted into the DNA strands. Furthermore,
drug loading with covalent conjugation between FUdRs
and DNA strands is more stable and controllable by accu-
rate calculation and solid-phase synthesis process.
Preparation and Characterization of
Self-Assembled Affi/F-TDNs
The obtained FUdR-DNA-affibody chimera (A13F-
affibody) was mixed with other three single strands
(B13F, C13F, and D13F) in an equimolar amount, and
self-assembled into affi-F/TDNs (Scheme 1A). Each edge
of the tetrahedron consists of 13 base pairs and each vertex
connects 10 FUdR molecules. An affibody molecule is
linked to one vertex of the tetrahedron through polymeric
FUdR. As a comparison, TDNs were constructed using the
above method. The obtained nanoparticles were character-
ized by 10% native PAGE. As shown in Figure 2A,
a prominent band appeared in each DNA nanoparticle,
and their mobility apparently decreased along with the
drug modification and bioconjugation with affibody. The
hydrodynamic size of affi-F/TDNs was 23.27 0.85 nm as
determined by dynamic light scattering (DLS) (Figure 2B).
Furthermore, affi/F-TDNs were characterized by atomic
force microscopy (AFM) and showed about 20 nm length
and 3.76 ( 0.39) nm height at the dried state, which was
larger than F/TDNs (about 15 nm length) (Figures 2C and
S9). These results demonstrated that affi-F/TDNs were
constructed by self-assembly without influence of poly-
meric FUdR tails and affibody. In addition, 19.6% of
FUdR in molar ratio was loaded in affi/F-TDNs, which
was higher than that of the DNA polyhedron (18.3%).21
Moreover, as targeted nanodrug, each affi-F/TDNs was
conjugated with 40 drug molecules, far more than the
antibody-drug-conjugate (ADC) drug-loading capacity
(typically an average of 3–4 payloads per antibody).29
The test of stability of affi-F/TDNs was also conducted
under physiological conditions. The nanodrugs were incu-
Synthesis and Characterization of
FUdR-DNA-Affibody Chimera
In order to establish the targeting of DNA nanoparticles, we
adopted affibody molecule and connected it to the end of
FUdR-DNA strand. The affibody molecule ZHER2:342, which
was derived from the immunoglobulin G protein Z-domain
scaffold, is a small protein with the molecular weight of ~7.0
kDa, and specifically recognizes and binds HER2 receptor on
the cancer cell surface (Figure 1A).27,28 ZhcHER2:342 was gen-
erated by introducing a cysteine at the C-terminus of
ZHER2: 342 to provide a thiol group and by fusing with his-
tag at its N-terminus for purification by affinity chromatogra-
phy (Figure 1B and S6). The modified affibody molecule was
linked to FUdR at 5ʹ end of A13F-NH2 through a linker EMCS
according to Scheme S2. The A13F-affibody were obtained by
two-step purification (Capto DEAE column chromatography
and HisTrap HP column chromatography) to remove the bated with 50% non-inactivated fetal bovine serum (FBS).
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Figure 1 (A) 3D structure of affibody molecule ZHER2:342-HER2 complex. Affibody is composed of three α-helices and bind to HER2 receptor through hydrogen bond/salt
bridge. (B) The amino acid sequence of ZHER2:342 and ZhcHER2:342. The hexahistidine tag of ZhcHER2:342 was colored blue, and cysteine residue of ZhcHER2:342 was colored red.
(C) Characterization of purified A13F-affibody via UV-vis spectroscopy. (D) Characterization of purified A13F-affibody by SDS-PAGE. The gel was stained by Coomassie
Brilliant Blue R-250. Lane M, protein Marker; Lane 1, A13F-affibody; lane 2, DNA strand A13F; lane 3, affibody; The gel stained with GelStain. Lane 4, A13F-affibody; lane 5,
DNA strand A13F; lane 6, affibody.
When they were incubated at 37°C for 4 hrs, no obvious employed for cell uptake test by confocal microscopy and
change was observed by gel electrophoresis (Figure S10), flow cytometry after incubated with FAM-labeled DNA
indicating that they were stable in FBS. In contrast, single- nanoparticles. F/TDNs and FUdR-DNA strands were used
stranded DNA was almost completely degraded by the as the controls for analysis of cell uptake. In BT474 cells,
nuclease in FBS when incubated for only 2 hrs. the different uptake capability was observed. A strong
Therefore, these results confirmed that the affi-F/TDNs green fluorescence appeared after BT474 cells were trea-
can perfectly work as a nanodrug, which retains intact ted with affi-F/TDNs, whereas the weak fluorescence was
nanoparticles in plasma until it enters cancer cells.
captured in BT474 cells treated with F/TDNs and FUdR-
DNA strands (Figure 3A). There was a difference of more
than 2.0-fold intensity between the strong and weak fluor-
Specific Cellular Uptake of Affi-F/TDNs
To investigate the targeting efficiency of affi-F/TDNs, escence (Figure 3B). It was inferred that affi-F/TDNs had
breast cancer cells, BT474 (HER2 overexpressing cell significant targeting ability to HER2-overexpressing can-
line) and MCF-7 (HER2 low expressing cell line)30 were cer cells. This was mainly attributed to the specific binding
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Figure 2 Characterization of DNA nanoparticles. (A) Electrophoresis analysis of the self-assembled DNA nanoparticles. Lane M, DNA Marker-B; lane 1, DNA single strand
A13F-NH2; lane 2, A13F-affibody; lane 3, TDNs; lane 4, F/TDNs; lane 5, affi-F/TDNs. (B) DLS data of affi-F/TDNs. (C) AFM image of affi/F-TDNs.
of the affibody to the HER2 receptor through eight hydro- (HER2 low expressing cell line) instead of BT474 cells,
gen bonds and six salt bridges (Figure 1A). As illustrated a relatively weak fluorescence was detected, which was
in Scheme 1B, the affi-F/TDNs-HER2 receptor complex suggested that the ability of affi-F/TDNs to bind to HER2
was formed on the surface of BT474 cells, which was low expressing cancer cells was inferior to that of HER2-
shown in its fluorescence image. Then, the HER2 receptor- overexpressing cancer cells. The flow cytometry results
mediated endocytosis enabled cells to absorb extracellular further demonstrated that affi-F/TDNs had excellent tar-
substances. Subsequently, the affi-F/TDNs-HER2 receptor geting ability and high specificity for BT474 cells (Figure
complex was internalized into endosomes and further pro- 3C). This targeted nanodrug is of potential for future
cessed into late endosomes/multivesicular bodies and lyso- application in preventing rapid metabolism of FUdR in
somes. In this process, affi-F/TDNs were degraded in plasma and reducing toxicity to healthy tissue.
lysosomes by intracellular nucleases and protease to
release FUdR (Figure S11) as the HER2 receptors were In vitro Cytotoxicity of Affi-F/TDNs
recycled to the cell membrane.31 On the contrary, F/TDNs MTT assay was taken to assess the potential cytotoxicity
and FUdR-DNA strands were not efficiently absorbed by of affi-F/TDNs. As a blank test, affibody molecule and
BT474 cells due to the lack of affibody in their TDNs did not show any cytotoxicity in normal (MCF-
structures.32,33 When affi-F/TDNs acted on MCF-7 10A) and tumor (BT474 and MCF-7) cells with a wide
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Figure 3 The cellular uptakes of DNA nanoparticles. (A) Confocal laser scanning microscopy of FAM-B13F, F/TDNs, and affi-F/TDNs in BT474 and MCF-7 cells. Scale bar:
20 µm. (B) Semi-quantitative analysis of fluorescence intensity. DATA are presented as mean SD (n=3). Statistical analysis: ***p<0.001. (C) Flow cytometry analysis of
cellular uptake of DNA nanoparticles to BT474 and MCF-7 cells.
range of concentrations (50 nM to 1000 nM) (Figure 4A). HER2-overexpressing cancer cells. Due to the higher cyto-
Based on these findings, it can be assumed that these toxicity of the polymerized form of FUdR,34 F/TDNs also
protein and DNA materials present an adequate biocom- displayed a higher inhibitory effect than free FUdR. The
patibility and safety for drug delivery. Then, the potential lower cytotoxicity of free FUdR may be attributed to the
toxicity of affi-F/TDNs as a nanodrug to normal cells was inadequacy of specialized drug transporter proteins and the
tested, and Figure 4B shows that affi-F/TDNs displayed efflux of drug molecules by P-glycoprotein (P-gp)
lower cytotoxicity in MCF-10A than free FUdR and F/ pumps.35 Next, the cytotoxicity of affi-F/TDNs to MCF-7
TDNs, which may be effective in reducing the side effects cells was also evaluated by the same method described
of FUdR-based chemotherapy.
above. Compared with BT474 cells, affi-F/TDNs caused
Next, Figure 4C exhibits the in vitro cytotoxic effect of lower cytotoxicity against MCF-7 cells, which may be due
affi-F/TDNs, F/TDNs, and free FUdR on BT474 and to the decreased drug uptake into the cells with low HER2
MCF-7 cells, and it was found that they inhibited cell expression levels. Based on the above results, it was
proliferation in a dose-dependent manner. For BT474 demonstrated that affi-F/TDNs were effectively trans-
cells, affi-F/TDNs presented the highest cytotoxicity and ported into HER2-overexpressing cancer cells because of
the cell viability decreased to 18.8%, whereas in the exis- the excellent targeting ability to HER2, resulting in
tence of free FUdR and F/TDNs, their cell viability was a significantly enhanced antitumor activity.
61.8% and 39.8%, respectively (Figure 4C). The highest
inhibition of affi-F/TDNs may be associated with an Cell Apoptosis Assays
enhanced cellular uptake via HER2-mediated endocytosis, As a chemotherapeutic drug, FUdR has been reported cap-
thus leading to a significantly enhanced effect against the able of inducing cell apoptosis.36 So, the Annexin V-FITC/
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Figure 4 In vitro cytotoxicity assay. (A) Cell viability of BT474, MCF-7, and MCF-10A cells incubated with increasing concentrations of affibody molecule and TDNs for 72
hrs. (B) Cell viability of MCF-10A cells incubated with FUdR, F/TDNs, and affi-F/TDNs for 72 hrs. (C) Cell viability of BT474 and MCF-7 cells incubated with FUdR, F/TDNs,
and affi-F/TDNs for 72 hrs. The results are expressed as a percentage of the control group, Data are presented as mean SD (n=5). Statistical analysis: ***p<0.001, No
significant difference (NS).
propidium iodide (PI) stain was used to measure the affi-F/ apoptosis (7.4 1.2%) in MCF-10A cells than that of free
FUdR and F/TDNs chemotherapy, thus supporting the lower
TDNs induced total apoptosis, including early (Annexin
V-FITC+/PI−) and late apoptosis (Annexin V-FITC+/PI+).
The breast cancer cells (BT474 and MCF-7) and normal
breast cells (MCF-10A) were pretreated with free FUdR,
F/TDNs, and affi-F/TDNs for 12 hrs. As shown in flow
cytometry (Figure 5A and B), free FUdR and F/TDNs
induced a low apoptosis rate in the BT474 cells, whereas
a significant increase in the percentage of apoptotic cells was
observed in cells treated with affi-F/TDNs (31.9 0.6%),
indicating that affi-F/TDNs enhanced apoptosis induction of
HER2-overexpressing cancer cells through targeted che-
motherapy. By contrast, the apoptosis rate of the affi-F/
TDNs treated MCF-7 cells were 14.7 1.5%, which was
similar to that of MCF-7 cells treated with F/TDNs. This
may be due to affi-F/TDNs have no targeted therapeutic
effect on cancer cells with low HER2 expression. It was
toxicity of affi-F/TDNs against normal cells.
Targeting of Affi-F/TDNs to Cancer
Xenograft in vivo
In order to verify whether affi-F/TDNs still had the
targeting ability in vivo, the Cy5.0-labeled nanodrugs
were injected into tumor-bearing mice via tail vein and
the fluorescence distribution was monitored using an
IVIS Lumina Series III. It can be clearly observed
from Figure 6A that both affi-F/TDNs and F/TDNs
rapidly covered the whole body of the mice at one-
hour post-injection. As the blood circulation continued,
the distribution of the two nanodrugs in mice showed
significant differences. At 4 hrs after injection, the affi-
F/TDNs-treated mice had a stronger fluorescent signal at
worth mentioning that affi-F/TDNs induced a lower rate of the tumor region compared with F/TDNs-treated mice,
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Figure 5 Cell apoptosis assay using flow cytometry. (A) Flow cytometry scatterplots of apoptosis rate in BT474, MCF-7 and MCF-10A cells. (B) Quantitative analysis of the
total apoptosis rate in above cells. Data are presented as mean SD (n=3). Statistical analysis: ***p<0.001, no significant difference (NS).
indicating that affi-F/TDNs could largely accumulate in also found from Figure 6B that affi-F/TDNs showed low
the tumor region. Such differential result might be accumulation in all normal tissues and were rapidly
attributed to the affibody ligand in affi-F/TDNs to metabolized in a short period of time (about 8 hrs),
potentially inducing specific active transport via the which might contribute to the low systematic toxicity.
HER2-mediated endocytosis.30 Furthermore, to investi- It was also noteworthy that affi-F/TDNs were mainly
gate the localization of affi-F/TDNs within specific tis- accumulated in tumor tissues and last for more than 12
sue compartments, various organs and tumors were hrs, which might be helpful to improve the therapeutic
dissected and collected at 1, 2, 4, 8, and 12 hrs. It is effect of FUdR through sustained release.
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Figure 6 In vivo biodistribution and antitumor effects of affi-F/TDNs on female NOD/SCID (Mus Musculus) mice with BT474 tumor xenograft. (A) In vivo fluorescent image
of tumor-bearing mice after 12 hrs of intravenous injection of the Cy5.0 labelled F/TDNs and affi-F/TDNs. (B) The fluorescence imaging of various tissues (tumors, lungs,
hearts, spleens, kidneys, and livers) at 1, 2, 4, 8, and 12 hrs after of intravenous injection of the Cy5.0 labelled affi-F/TDNs.
antitumor effect might be due to the specific binding and
In vivo Tumor Inhibition by Affi-F/TDNs
internalization of tumor cells mediated by the overex-
pressed HER2. In addition, the body weights of mice
were monitored to evaluate the systemic toxicities of
the treatments. The monitoring results (Figure 7D)
showed that the bodyweight of mice in the F/TDNs and
affi-F/TDNs treatment groups increased slightly during
the treatment process, implying their low systemic toxi-
cities. However, the weight of free FUdR treatment group
was significantly reduced, which may be related to the
strong toxic and side effects of FUdR. The data of tumor/
body weight (Figure 7E) also confirmed the above
results. Finally, H&E staining was used for pathological
examination of tumors and various tissues and organs. As
Based on the outstanding performance of affi-F/TDNs
in vitro, the in vivo antitumor efficacy was evaluated on
a female NOD/SCID (Mus Musculus) mice model bear-
ing BT474 tumor xenografts. After three consecutive
intravenous injections of the nanodrugs, the tumor
growth and body weight were recorded every 3 days
during the treatment. As shown in Figure 7A–C, com-
pared with the free FUdR treatment group, both F/TDNs
and affi-F/TDNs groups exhibited more significant
growth inhibitory effects. These results once again
proved that DNA nanoparticles integrated with FUdRs
could achieve the enhanced antitumor efficacy.
Interestingly, affi-F/TDNs were proved to be a more effi-
cient nanodrug with an inhibition ratio (IR) of 58.1% shown in Figure 7F, the tumor sections in the group
compared with F/TDNs (IR, 41.2%), and this excellent treated with affi-F/TDNs exhibited more severe cancer
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Figure 7 In vivo antitumor effects of affi-F/TDNs on female NOD/SCID (Mus Musculus) mice with BT474 tumor xenograft. (A) Images of resected tumors. (B) Tumor
volumes of each group during the whole treatment. (C) Tumor growth inhibition ratio (IR). (D) Bodyweight changes (E) Tumor/bodyweight of mice after the end of study.
Data represent as mean SD (n=6). Statistical analysis: *p<0.05, **p<0.01, ***p<0.001, no significant difference (NS). (F) Histological images of BT474 tumor slices treated
with PBS, FUdR, F/TDNs, and affi-F/TDNs after staining with H&E (magnification 200×).
necrosis and a lower nuclear-to-cytoplasmic ratio than the and affi-F/TDNs (Figure S12). Therefore, these results
other groups. Meanwhile, no obvious abnormality was demonstrated that the DNA-based nanodrugs had very
observed in the major organs of mice treated with PBS good treatment effect and low systemic toxicity.
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Zhang et al
2. Slamon DJ, Godolphin W, Jones LA, et al. Studies of the HER-2/neu
proto-oncogene in human breast and ovarian cancer. Science.
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In summary, a new affibody-DNA tetrahedron template
was successfully constructed for loading nucleoside
analogue FUdR. This DNA nano-structure had a very
high drug-loading ratio, and the amount of FUdR can
be accurately controlled. In addition, the affi-F/TDNs
exhibited good stability and entered cancer cells with
intact nanoparticles. Then, FUdRs were sustained-
released by degradation of nuclease in cancer cells.
The in vitro and in vivo results demonstrated that the
affi-F/TDNs had a capability of specifically targeting
HER2-overexpressing breast cancer cells and displayed
excellent anticancer efficacy, which promoted the pre-
clinical application of the nanodrug. Furthermore, the
affibody-DNA tetrahedrons as a platform facilitates
efficient loading of many other nucleoside analogues,
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Ethics Approval and Consent to
Participate
All animal experiments were carried out in compliance
with the Animal Management Rules of the Ministry of
Health of the People’s Republic of China (Document No.
552001) and the guidelines for Care and Use of
Laboratory Animals of Hebei University.
Acknowledgments
The authors acknowledge the financial support from the
Natural Science Foundation of Hebei Province (grant
number B2016201031), Hebei Province Science
Foundation for High-level Personnel (grant number
GCC2014013), Hebei University Science Foundation
(grant number 3333112), and the Post-graduate’s
Innovation Fund Project of Hebei University (grant num-
ber hbu2018bs03).
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acterization of nanoliposomal 5-fluorouracil for cancer nanotherapy.
J Liposome Res. 2014;24:1–9. doi:10.3109/08982104.2013.810644
18. Roth SK, Epley CC, Novak JJ, Mcandrew ML, Jie Z, Dawn CH.
Photo-triggered release of 5-fluorouracil from a MOF drug delivery
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19. Shayan K, Nowroozi A. Boron nitride nanotubes for delivery of
5-fluorouracil as anticancer drug: a theoretical study. Appl Surf Sci.
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Disclosure
The authors report no conflicts of interest in this work.
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