An antisense oligodeoxynucleotide-doxorubicin conjugate: Preparation and its reversal multidrug resistance of human carcinoma cell line in vitro

Article abstract of DOI:10.1081/NCN-200031419

An antisense oligodeoxynucleotide-doxorubicin conjugate was synthesized by an aminocaproic acid linker. The synthetic conjugate was identified by HPLC analysis and UV-vis spectra. Properties of the conjugate in vitro conditions were investigated. The results demonstrated that the conjugate was remarkably stabilized by doxorubicin. When incubated in Dulbecco Phosphate-Buflered Saline (pH 7.4) at 37°C, the conjugate was more stable than doxorubicin or the mixture of doxorubicin and antisense oligodeoxynucleotide. When incubated in 10% fetal serum at 37°C, the conjugate showed a remarkable stabilization as compared to the unmodified oligodeoxynucleotide. Melting experiments demonstrated that the covalent attachment of doxorubicin strongly stabilized the binding of the oligodeoxynucleotide to its complementary sequence. In addition, in vitro reversion of multidrug resistance by the conjugate was assayed in a human carcinoma cell line (KB-A-1) resisting to doxorubicin. The result showed that the conjugate displayed very high reversal multdrug resistance activity in KB-A-1 cells in vitro. The conjugate lowered the IC50 value from 21.5 pM to 2.2 μM with a fold-reversal factor of 10. In contrast, a slight decrease of the IC50 value was observed when they combined with the "free" antisense oligodeoxynucleotide: the IC50 value was down from 21.5 pM to 16.8 pM. This study suggested that antisense oligodeoxynucleotide-doxorubicin conjugate might be helpful in multidrug resistance reversal.

Full text of DOI:10.1081/NCN-200031419

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An Antisense Oligodeoxynucleotide‐Doxorubicin
Conjugate: Preparation and Its Reversal Multidrug
Resistance of Human Carcinoma Cell Line In Vitro
Yuhong Ren a b , Dongzhi Wei a , Jianwen Liu a & Wu Su a
a State Key Laboratory of Bioreactor Engineering , Institute of Biochemistry , East China
University of Science and Technology , Shanghai, 200237, China
b Jiangxi‐OAI Gemeinsames Institute , Nanchang University , Nanchang, 330047, China
Published online: 01 Jun 2007.
To cite this article: Yuhong Ren , Dongzhi Wei , Jianwen Liu & Wu Su (2004) An Antisense Oligodeoxynucleotide‐Doxorubicin
Conjugate: Preparation and Its Reversal Multidrug Resistance of Human Carcinoma Cell Line In Vitro, Nucleosides, Nucleotides
and Nucleic Acids, 23:10, 1595-1607, DOI: 10.1081/NCN-200031419
To link to this article: http://dx.doi.org/10.1081/NCN-200031419
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NUCLEOSIDES, NUCLEOTIDES & NUCLEIC ACIDS
Vol. 23, No. 10, pp. 1595–1607, 2004
An Antisense Oligodeoxynucleotide-Doxorubicin
Conjugate: Preparation and Its Reversal
Multidrug Resistance of Human
Carcinoma Cell Line In Vitro
1
1
Yuhong Ren,1,2 Dongzhi Wei,1, Jianwen Liu, and Wu Su
*
1State Key Laboratory of Bioreactor Engineering, Institute of Biochemistry,
East China University of Science and Technology, Shanghai, China
2Jiangxi-OAI Gemeinsames Institute, Nanchang University, Nanchang, China
ABSTRACT
An antisense oligodeoxynucleotide—doxorubicin conjugate was synthesized by an
aminocaproic acid linker. The synthetic conjugate was identified by HPLC analysis
and UV-vis spectra. Properties of the conjugate in vitro conditions were investigated.
The results demonstrated that the conjugate was remarkably stabilized by doxorubicin.
When incubated in Dulbecco Phosphate-Buflered Saline (pH 7.4) at 37°C, the
conjugate was more stable than doxorubicin or the mixture of doxorubicin and
antisense oligodeoxynucleotide. When incubated in 10% fetal serum at 37°C, the
conjugate showed a remarkable stabilization as compared to the unmodified oligo-
deoxynucleotide. Melting experiments demonstrated that the covalent attachment of
doxorubicin strongly stabilized the binding of the oligodeoxynucleotide to its com-
plementary sequence. In addition, in vitro reversion of multidrug resistance by the
conjugate was assayed in a human carcinoma cell line (KB-A-1) resisting to doxo-
rubicin. The result showed that the conjugate displayed very high reversal multdrug
resistance activity in KB-A-1 cells in vitro. The conjugate lowered the IC50 value
*
Correspondence: Dongzhi Wei, State Key Laboratory of Bioreactor Engineering, Institute of
Biochemistry, East China University of Science and Technology, Shanghai 200237, China;
Fax: +86-21-64250068; E-mail: dzhwei@ecust.edu.cn.
1595
DOI: 10.1081/NCN-200031419
1525-7770 (Print); 1532-2335 (Online)
www.dekker.com
Copyright D 2004 by Marcel Dekker, Inc.

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Ren et al.
from 21.5 mM to 2.2 mM with a fold-reversal factor of 10. In contrast, a slight decrease
of the IC50 value was observed when they combined with the ‘‘free’’ antisense
oligodeoxynucleotide: the IC50 value was down from 21.5 mM to 16.8 mM. This study
suggested that antisense oligodeoxynucleotide-doxorubicin conjugate might be help-
ful in multidrug resistance reversal.
Key Words: Antisense oligodeoxynucleotide; Doxorubicin; Antisense oligodeoxy-
nucleotide-doxorubicin conjugate; Synthesis, multdrug resistance reversal; KB-A-1
cell.
INTRODUCTION
Antisense strategy is a simple approach to down-regulate the gene expression by
inhibiting transcription or translation via sequence-specific blinding of either DNA or
RNA, respectively.[1,2] But the efficiency of the synthetic oligodeoxynucleotide (ODN) in
regulating gene express in living cell depends on its thermodynamic stability, resistance
toward nuclease and cellular uptake. Much research has indicated that a synthetic ODN
coupled with DNA intercalators such as acridine,[3] adriamycin,[4] naphthyl imide,[5]
psoralen[6] and pyrene[7] might increase its stability.
Doxorubicin (DOX), one of anthracycline antibiotics that induces genetic damage
leading to cell death, is one of the most useful drugs in cancer chemotherapy. However, its
clinical efficiency is limited by its cardiotoxicity and suppression of bone marrow
function.[8,9] Moreover, DOX often causes a multidrug resistance (MDR) phenomenon,
which reduces the intracellular drug content and results in therapy failure.[10,11] Many
DOX analogs are synthesized in order to mitigate the toxicity and the MDR phenomenon,
but no clearly improved drug has been found.[12] In the past few years, a prevailing
approach to overcome MDR is inhibiting MDR gene expression by AS ODN.[13–15]
Recently, Chi et al.[16] reported that combining AS ODN (G3139) with free
DOX and liposomal DOX increased their antitumor activity. Their results indicated
that there exists a synergistic action of AS ODN and DOX. Similar results were also
reported by other studies.[17,18] In our laboratory, we have found that an AS ODN,
5 TCCTCCATTGCGGTCCCCTT-3 the 30–11 region of mdr1 gene which associated
with MDR in a human epidemic carcinomata cell line, could control expression of the
mdr1 gene.[5] In this study, we have synthesized the AS ODN-DOX conjugate and
investigated its properties in Dulbecco Phosphate-Buffered Saline (PBS) or in cell
culture. The activity of the conjugate to reverse MDR of KB-A-1 cells in vitro has also
been investigated.
MATERIALS AND METHODS
Materials
DOX was a gift from Zhejiang Hisun Pharmacentical Co. Ltd. (Zhejiang China).
Other chemicals were obtained from commercial sources. All the solvents were dried
and distilled before used. The AS ODN with phosphate group at the 3 end and its

Oligodeoxynucleotide-Doxorubicin Conjugate
1597
complementary sequence RNA were purchased from Shanghai Bioasia Biotech Co.
Ltd.(Shanghai China).
Cell Line and Culture Conditions
A human oral epidermoid carcinoma, DOX-resistance cell line (KB-A-1) was
kindly provided by Dr. Ira Pastan and Micheal M. Gottesman (National Institutes of
Health, USA). KB-A-1 was cultured in Dulbecco Modified Eagle Medium (DMEM)
(Life Technologies, Inc.MA, USA) supplemented with 10% (v/v) fetal bovine serum
(Gibco, USA), 100 U/ml penicillin G, 100 mg/ml streptomycin sulfate, and 1 mg/ml
DOX. The cells were incubated at 37°C in a humidified atmosphere of 95% air and
5% CO2.
Synthesis of Boc-Caproic Acid (Compound 1)
A mixture of 6-aminocaproic acid (0.66 g, 5 mmol) and di-tert-butyl dicarbonate
(Boc2O) (1.11 g, 5.1 mmol) in tert-butyl alcohol/H2O (1:1, V:V) solution (10 ml)
(pH8.5 with a modified by 2 N NaOH) was stirred at room temperature. The reaction
was monitored by TLC analysis on silica gel plates using methanol (MeOH)/ethyl
acetate (EtOAc) (90:10, V:V) as eluent (Rf 6-aminocaproic acid = 0.18, Rf 6-Boc-
aminocaproic acid = 0.64). When the reaction was completed, the solution was concen-
trated under reduced pressure, the residue was partitioned between EtOAc and H2O.
After being dried by MgSO4, the organic solution was evaporated and obtained com-
pound 1 (0.9 g, 3.9 mmol) yield 78%. 1H-NMR (CDCl3): d1.39 (s, 9 H, –CH3), 1.28–1.52
(m, 6 H, J 7.1, 7.2, 7.3, –CH2–), 2.15 (m, 2 H, J 7.24, –CH2COO–), 3.01 (m, 2 H, J 7.0,
NCH2–). MS (TOF-ES+) m/z: 254.1 (M + 23).
Synthesis of Aminoalkyl Derivate of DOX (Compound 3)
The mixture of compound 1 (14 mg, 0.06 mmol), N-ethyl-N- (3-dimethylamino-
propyl) (EDC) (12 mg, 0.06 mmol), N-hydroxybenzotriazole (8 mg, 0.06 mmol) in dried
N, N-dimethylformamide (DMF)/CH2Cl2 (1:1, V:V) (4 ml) was stirred for 2 h at 4°C. A
solution of Doxorubiciin (35 mg, 0.06 mmol) and N-methylmorpholine (6 mg, 0.06 mmol)
in anhydrous DMF (2 ml) was poured into the mixture and the whole was stirred at 4°C.
The reaction was monitored by TLC using CH2Cl2/MeOH (90:10, V:V) as eluent (Rf
DOX = 0.05, Rf aminoalkyl derivate of DOX = 0.54 ) when the reaction was completed,
one drop of AcOH was added to decompose the redundant EDC, then the precipitate was
removed by filtration and the filtrate was concentrated under pressure. The residue was
kept in CHCl3 (20 ml) and the organic layer washed with cold water (2 ml). After being
dried over MgSO4 the organic solution was concentrated, and the residue was purified by
column chromatography using silica gel with CH2Cl2/MeOH (90:10, V:V) gave com-
pound 2 (30 mg, 0.04 mmol ), yield 65%. 1H-NMR (CDCl3): d1.20 (3 H, d, –CH3), 1.38
(9 H, s,–(CH3)3), 1.25–1.8 (8 H, m,–CH2–), 2.15 (2 H, m, –CH2COO–), 3.0–3.4 (4 H,
m, NCH2–, –NHCO–, –NHOCO–), 3.6 (2 H, d, –CH2OH), 3.8 (1 H, m, –CHN–), 4.1
(3 H, s, –OCH3), 7.4–8.0 (3 H, d, t, d, aromatic), 13.2–13.8 (2 H, s, s, Ph-OH). MS (TOF-
ES+) m/z: 779.3 (M + 23).

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Ren et al.
Compound 2 (30 mg, 0.04 mmol) was dissolved in 3 M HCl EtOAc (0.5 ml) and
stirred 30 min at room temperature, then the solution was removed in vacuum and the
residue was purified on preparative silica gel plates using CH2Cl2/MeOH (95:5, V:V)
1
as eluents. The compound 3 was obtained (21 mg, 0.015 mmol) yield 80%. H-NMR
(CDCl3): d1.20 (3 H, d, –CH3), 1.25–1.86 (8 H, m, –CH2–), 2.15 (2 H, m, –CH2COO–),
3.1–3.2 (5 H, m, NCH2–, –NHCO–, –NH2), 3.6 (2 H, d, –CH2OH), 3.8 (1 H, m, –CHN–),
4.1 (3 H, s, –OCH3), 7.4–8.0 (3 H, d, t, d, aromatic), 13.2–13.8 (2 H, s, s, Ph-OH). MS
(TOF-ES+) m/z: 679.1 (M + 23).
Synthesis of AS ODN-DOX Conjugate
10 A260 O.D.(50 nmol ) of AS ODN with a phosphate group at 3 end in water
(100 )was completely precipitated with 8% N-cetyl-N,N,N-trimethylammonium bromide.
The sediment was separated by centrifuged and dried in vacuum over P2O5. A mixture of
this dried N-cetyl-N,N,N-trimethylammonium salt of oligodeoxynucleotide, triphenyl-
phosphine (20 mg, 0.77 mmol), dipyridyldisulfide (20 mg, 1.0 mmol), 1-methylmidazole
(8 ml) and anhydrous DMF (50 ml)was subjected to several (3ꢀ5) brief (1 min) heating at
50°C followed by intensive vortexing and incubation at room temperature for 15 min.
Then 2 mg of the aminoalkyl derivative of DOX and 2 Fl of anhydrous N, N-diisopropyl-
ethylamine were added. After stirred for 1 hour at room temperature, the solution was
precipitated with 1.5 ml of 2% LiClO4 in dry acetone. The sediment was re-precipitated
by 2% LiClO4 in dry acetone from 3 M LiClO4 water solution and washed by acetone.
The residue was dissolved in 1 ml of water. Purified by reverse-phase HPLC using
Lichrospher RP-18, 10 mm, 10 Â 250 mm column (Merk), Agilent 1100 chromatograph
system and Agilent diode-array absorbance detector (Germany). Linear (0 to 60%)
gradient of acetonitrile in 0.1 M aqueous triethylammonium acetate, pH 7.0, (flow rate of
4 ml/min) was used. And then was lyophilized and storage at À20°C. The conjugate was
characterized by combination of denaturation polyacrylamide gel electrophoresis,
reverse-phase HPLC and UV-vis spectroscopy.
In Vitro Stability in Dulbecco’s Phosphate-Buflered Saline (PBS)
The solution of DOX, a mixture of AS ODN and DOX, and the conjugate were
incubated in PBS (pH 7.4) at 37°C, respectively. The absorbance at 480 nm was detected
at each 8 hours within 72 hours with a Model 752 UV-vis spectrophotometer (China).
In Vitro Stability in Calf Serum
The conjugate (8 ml, 1 mg/ml) and AS ODN (8 ml 1 mg/ml) were incubated at 37°C
in 10% activated fetal calf serum (32 ml), respectively. After incubation for various
regular times, the mixture was heated at 70°C for 5 min, and then 20 ml mixture was
taken out for denaturation polyacrylamide gel electrophoresis analysis and 2 ml for
HPLC analysis.
Thermal Denaturation Experiments
Thermal denaturation of ODN complexes was carried out in cacodylate buffer pH
7.4, containing 140 mM KCl, 2.0 mM sodium cacodylate, 1.5 mM MgCl2, and 0.8 mM

Oligodeoxynucleotide-Doxorubicin Conjugate
1599
spermine, 0.5 mM EDTA. The concentration of each component was 1.0 mm. The
melting curves (absorbance versus temperature) were obtained with the use of a Model
UV-2501PC spectrometer (Shimadzu, Japan) at 260 nm. The temperature of the cell
holder was regulated by circulating liquid used a Model TB-85 thermo bath (Shimadzu,
Japan). A buffer solution of appropriate ionic strength was placed in a reference curette
occupying one of the cell hold. Each ODN mixture was contained in a quartz curette
with a Teflon cap. The temperature was initiated at 10°C and was increased at 1°C/min
increment with a 1 min waiting period for stabilization at each temperature. The duplex
melting temperatures (Tm) were evaluated as the maximum of the first derivative of the
melting profiles.
Treatment Cells with ODN-DOX Conjugate or
Combined with DOX
To assess the reversal of MDR ability of the conjugate to the cells, KB-A-1 cells
were seeded in 96-well plates (Costar Corp., Combridge, MA) in 200 ml complete
DMEM at the concentration of 1 Â 104 cells/well. After incubated at 37°C for 20–22 h
to allow the cells to attach, the medium was removed. The cells were washed twice
with serum-free medium, and were incubated in a serum-free DMEM medium sup-
plemented with 0.5 mM of the conjugate or a mixture of 0.5 mM of the AS ODN and
0.5 mM of DOX. In control wells, only serum-free medium was added. After incubated
for 24 h, the cells were washed once with complete medium, and then exposed to DOX
at doses from 0.1 mM to 25 mM for 48 h. At the end of the combination treatment,
samples from each group were harvested, counted and assayed for viability (trypan blue
dye exclusion).
Statistical Analysis
Each experimental value is expressed as the mean standard deviation (SD).
Students’ paired t test was used to evaluate the significance of differences between
groups, and the criterion of statistical significance was taken as p < 0.05 or p < 0.01.
RESULTS
Synthesis
The conjugate was synthesized as shown in Scheme 1. Before being reacted with
DOX, the amino group of 6-aminocaproic acid should be protected. In general, many
protective groups have been used for the amino group. In our studies, Boc group was used
because it can be easily cleaved at room temperature. The reaction of 6-aminocaproic
acid was completely converted into the Boc group. Isolation yield of the desired
compound 1 (N-tert-butyloxycarbonyl- 6-amino-caproic acid) was approximately 82%.
The compound 1 was then reacted with DOX in the presence of tertiary base to yield
compound 2. The reaction was catalyzed by EDC in 65% yield. Finally, the Boc group
was cleaved with 3 M HCl in ethyl acetate to give in nearly quantitative yield of the
compound 3.

1600
Ren et al.
Scheme 1. Synthesis route of the AS ODN-DOX conjugate.
Synthesis of the AS ODN-DOX conjugate was carried out according to Aitssando
Balbi et al.[19] and Kostenko et al.[20] with slight modifications. In the presence of 1-
methylimdazole and N, N-diisopropylethylamine, the conjugate was synthesized with
85% yield. Reverse-phase HPLC analysis of the crude mixture obtained by conjugating
the AS ODN with compound 3 showed a main peak corresponding to the 3 conjugated
oligomer which had a higher retention time (Rt = 14 min 21 sec) than that of 3 phos-
phorate (Rt = 8 min 10 sec) (Fig. 1). The UV-vis absorption spectrum of the conjugate
and DOX were shown as Fig. 2. The denaturation polyacrylamide gel electrophoresis
analysis was shown as Fig. 3. It is shown that the conjugate band was slightly above
the ODN band. This could be not only due to the increasing molecular weight of the
ODN, but also due to changing the ODN charge.

Oligodeoxynucleotide-Doxorubicin Conjugate
1601
Figure 1. Reverse phase analysis on a Lichrospher100RP18 (5 mm) colum (125 mmÂ4 mm)
(using a linear gradient of CH3CN: from eluent A to eluent B within 30 min, with a flow rate of
1 ml/min) of the crude mixture obtained after coupling of p-ODN with DOX aminoalkyl derivative.
The retention time is: the AS ODN-DOX conjugate: Rt = 14 min 21 sec, the AS ODN: Rt = 8 min
10 sec, DOX aminoalkyl derivative: Rt = 24 min 38 sec.
Stability of the Conjugate In Vitro
In order to examine their stability, DOX, the mixture of the AS ODN and DOX,
and the conjugate in PBS (pH 7.4) at 37°C were monitored at 480 nm (Fig. 4). It was
demonstrated that the absorbance of DOX fell to 76.7% of the initial level after
incubation for 72 hours. The mixture of the AS ODN and DOX showed slightly better
stability. However, the conjugate retained more than 84.7% of the initial absorbance
after 72 hours incubation.
Figure 2. UV-vis spectrum of AS ODN and AS ODN-DOX conjugate in water (-): AS ODN
(5.1 mM). (....): AS ODN-DOX (5.6 mM).

1602
Ren et al.
Figure 3. Polyacrylamide gel electrophoresis analysis the stability of AS ODN and AS ODN-
DOX conjugate. Lane 1ꢀ3: AS ODN-DOX (40 mM). Lane 4ꢀ6: AS ODN (46 mM).
The stability of the conjugate in fetal calf serum was analyzed by reverse-phase
HPLC. As shown in Fig. 5, 15.8% of the conjugate was degraded after 24 hours
incubation. And 97.2% of the control AS ODN was degraded under the same condition.
These results indicated that attachment of the DOX at the 3 end of the AS ODN could
protect it against degradation by nuclease.
Denaturation of Complexes
The cacodylate buffer was chosen due to the limited dependence of pH on
temperature. The UV melting curves at 260 nm for the 1:1 (molar ratio) mixtures of the
ODN or the ODN-DOX conjugate with the complementary RNA (5 AAGGGGACCG-
CAATGGAGGATGTACA) are shown in Fig. 6. Both profiles exhibit a clear melting
transition, giving the Tm of 62°C (hypochromicity: 9.6%) for duplex of AS ODN and
of 74°C (hypochromicity: 11.4%) for AS ODN-DOX conjugate. This result indicated
that the presence of DOX covalently attached to the ODN remarkably increase the
stability of the complexes.
Figure 4. Stability of DOX, DOX plus AS ODN and AS ODN-DOX conjugate in PBS at 37°C in
&
the dark. Estimated from the absorbance at 480 nm. ( ) DOX (21 mM). ( ) AS ODN (31 mM) plus
.
~
DOX (21 mM). ( ) AS ODN-DOX conjugate (26 mM). Each value represents the mean standard
deviation (SD) of three independent experiments. Significant differences from untreated control are
indicated by p < 0.05 (*) and p < 0.01 (**).

Oligodeoxynucleotide-Doxorubicin Conjugate
1603
Figure 5. Stability of AS ODN and AS ODN-DOX conjugate in 10% fetal calf serum at 37°C in
the dark. Determination by HPLC at 254 nm using a linear gradient of CH3CN in 0.1 M aqueous
~
triethylammonium acetate, ph 7.0, with a flow rate of 1 ml/min. ( ): AS ODN. ( ): AS ODN-DOX
.
conjugate. Each value represents the mean
standard deviation (SD) of four independent
experiments. Significant differences from untreated control are indicated by p < 0.05 (*) and
p < 0.01 (**).
In Vitro Reversal of MDR in KB-A-1 Cells
Treated with the Conjugate
In order to assess the ability of the conjugate to increase DOX cytotoxicity in KB-
A-1 cells and thus to reverse MDR, KB-A-1 cells were first exposed to the conjugate
and then to DOX. The results were shown in Fig. 7. When KB-A-1 cells were treated
with DOX alone, only at the higher dose (15 mM) could the inhibition of cells growth
observed (about ꢀ 28% inhibition compared to the control). However, when treated
Figure 6. Melting curves for the mixture of AS ODN and AS ODN-DOX conjugate with the
complementary ODN. The pH was 7.4 with the cacodylate buffer. The ODN concentration was 1.0
~
mM in all cases. ( ): AS ODN. ( ):AS ODN-DOX conjugate.
.

1604
Ren et al.
Figure 7. Reveral effect of the conjugate or the AS ODNs + DOX on the MDR in KB-A-1 cell
~
growth. Cells were treated with 0.5 mM the conjugate ( ), the mixture of the 0.5 mM AS ODN
&
AND 0.5 mM DOX ( ). DOX alone ( ). Cell viability was measured by trypan blue dye
.
exclusion assay. Relative cell viability was calculated by dividing the absolute survival of the
treated samples by that of the control samples. Each value represents the mean standard
deviation (SD) of four independent experiments. Significant differences from untreated control
are indicated by p < 0.05 (*) and p < 0.01 (**).
with the conjugate, a significant increase of DOX cytotoxicity was observed: the
inhibition of the cells growth was about 47% at the lower DOX dose (2.0 mM). In
contrast, a slight effect was observed when the cells were treated with the mixture of the
‘‘free’’ AS ODN (0.5 mM) and DOX (0.5 mM). The DOX IC50 values (dose of DOX that
caused 50% of cells growth inhibition) were calculated from the percentage of growth
inhibition caused by the different treatments on KB-A-1 cells. The DOX IC50 value for
the conjugate was 2.2 mM. In contrast, the DOX IC50 value for the AS ODN mixed with
DOX was 16.8 mM, and for DOX alone the value was 21.5 mM. The reversal index of the
conjugate (= the DOX IC50 value for DOX alone/the DOX IC50 value for the conjugate)
was nearly 10. However, the reversal index of the AS ODN was 1.3. These data
demonstrated that the conjugate displayed an evident reversal effect on MDR cells.
DISCUSSION
It has been reported that the contribution of a linker group is very important to the
properties of the conjugate.[21,22] Previous studies showed that the –(CH2)5– linker of
ODN-acidine conjugates led to more stable complexes than other linkers.[21] In our
case, we chose 6-aminocaproic acid as the linker. The UV-vis absorption spectrum of
the conjugate, which exhibited the expected absorbance radio at l = 260 nm and
l = 480 nm according with the published lmax of DOX (Fig. 2). A slight red-shift of
the absorption maximum of DOX was observed similar to the oligonucleotide-
daunomycin conjugates.[6] The denaturation polyacrylamide gel electrophoresis analysis
showed that the conjugate migrated slightly slower compared to the ODN (Fig. 3). The
result suggested that the conjugate had similar character of the ODN and its molecular
weight was slightly bigger than the ODN. These results confirmed that DOX was
linked to the AS ODN.

Oligodeoxynucleotide-Doxorubicin Conjugate
1605
The results presented in this study showed that the presence of DOX covalently
linked to the AS ODN on the 3 phosphate group significantly increased the stability of
DOX and the AS ODN either in PBS or in cell culture. In PBS, after incubated for 72 h
at 480 nm, the absorbance of the conjugate decreased less than that of free DOX
(Fig. 4). The observed stabilization of the conjugate in PBS was due to the amino group of
DOX forming more stable amide derivate. The results also showed that the AS ODN
linked to DOX had remarkably improved its resistance to endonuclease in cell culture. The
inhibition to endonuclease degradation of the conjugate is nearly 40-fold as compared to
that of the ree AS ODN in cell culture condition (Fig. 5). It is known that 3 exonuclease
activity is mainly responsible for the degradation of ODNs in calf serum.[23] This 3
exonuclease activity is similar to that of snake venom phosphodiesterase, which degrades
ODNs with a free 3 hydroxyl group from the 3 end. Therefore, the conjugate with DOX
attached to the 3 phosphate may prevent the attack from other 3 exonucleases.
In our study, the results also suggested that the conjugate strongly stabilized the
complex formed from the AS ODN and its complementary sequence. A considerable
increase of Tm of the intramolecular structures of the conjugate is observed: D Tm for
the conjugate is +12°C compared to the AS ODN (Fig. 6). The result is in agreement
with the ODN-acridine conjugates 3. The possible reason is that the presence of an
additional positively charged substituent on the 3’-phosphate group affects the stability
of the complexes. It is considered that substitution of the 3 phosphate group by the
positively charged group not only adds a positive charge to the ODN but also removes
one of the negative charges originally borne by the terminal phosphate group.[4]
In the present study, we also investigated the reversal of MDR in KB-A-1 cells
treated with the conjugate combination with DOX in vitro. The KB-A-1 cell line is a
strong DOX resistant cell line (the IC50 value for DOX is up to 21.5 mM). The ability
of the conjugate to elevate the DOX cytotoxicity in KB-A-1 cells and thus to reverse
MDR resistance was assessed in combination experiments in which KB-A-1 cells were
first exposed to the conjugate and then to DOX. The results demonstrated that a strong
decrease of the IC50 value was observed in KB-A-1 cells treated with the combination
of the conjugate and DOX. The conjugate lowered the IC50 value from 21.5 mM to
2.2 mM with a fold-reversal factor of 10. In contrast, a slight decrease of the IC50 value
was observed when DOX was combined with the ‘‘free’’ AS ODN: the IC50 value was
down from 21.5 mM to 16.8 mM (Fig. 7).
Antisense strategy is an efficacious approach to overcome MDR.[12,13] Our
previous studies showed that the AS ODN could inhibit the expression of the mdr1
gene but not strongly as expected.[24] This might be due to the poor absorbability of the
AS ODN, the rapid degradation of the AS ODN, or the poor binding affinity to the
target nucleic acid. In this case, however, our data suggested that coupling DOX with
the AS ODN efficiently elevated the antisense activity against DOX resistant KB-A-1
cells. The conjugate led to an IC50 that was about 8-fold less than that of the ‘‘free’’
AS ODN (16.8 mM verse 2.2 mM). Although the mechanism by which the conjugate
inhibits the KB-A-1 cell growth is still unknown, this might be explained in that the
conjugate increases cellular uptake, inhibits degradation by exonucleases, and improves
binding affinity to the complementary sequence. These results suggested the possibility
of utilizing this conjugate to overcome MDR. Further research is needed to explore the
antitumor potential of the conjugate in vitro and in vivo.
In conclusion, we synthesized the AS ODN-DOX conjugate and found that the
conjugate was very stable either in PBS or in cell culture. This study also showed that the

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Ren et al.
conjugate displayed very high reversal MDR activity in KB-A-1 cells in vitro. Further
studies such as the antitumor activity of the conjugate in vivo using severely combined
inmunodeficient mice bearing human resistant tumour xenografts are being explored.
ACKNOWLEDGMENTS
This work was supported by The National Natural Foundation of China (No.
30171088), Shanghai Key Disciplinary Foundation and 63 Project of China (No.
2001AA215261).
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Received April 4, 2003
Accepted May 4, 2004