Targeted chemoimmunotherapy using drug-loaded –dendrimer bioconjugates aptamer ⁎

Transcription

Targeted chemoimmunotherapy using drug-loaded –dendrimer bioconjugates aptamer ⁎
Contents lists available at ScienceDirect
Journal of Controlled Release
j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / j c o n r e l
Targeted chemoimmunotherapy using drug-loaded
aptamer–dendrimer bioconjugates
In-Hyun Lee, Sukyung An, Mi Kyung Yu, Ho-Keun Kwon, Sin-Hyeog Im, Sangyong Jon ⁎
Cell Dynamics Research Center, School of Life Sciences, Gwang ju Institute of Science and Technology, 261 Cheomdan-gwagiro, Buk-gu, Gwang ju 500-712, Republic of Korea
a r t i c l e
i n f o
Article history:
Received 22 February 2011
Accepted 22 May 2011
Available online 27 May 2011
Keywords:
PSMA aptamer
Dendrimers
Doxorubicin
Targeted chemoimmunotherapy
Cancer therapy
a b s t r a c t
We reported an innovative, targeted chemoimmuno drug-delivery system. Although chemoimmunotherapy,
as an alternative to or in combination with conventional therapeutic systems, has been in the forefront of
recent oncological research, as presently configured, such systems face several major obstacles for efficient
clinical application. Here, we establish a novel nano-platform for effective chemoimmunotherapy designed to
overcome the drawbacks of conventional cancer therapies, describing a delivery system based on a dendrimer
and a single-strand DNA-A9 PSMA (prostate-specific membrane antigen) RNA aptamer hybrid. Employing
these vehicles, we demonstrate the promising possibility of this chemoimmuno therapeutic system against
prostate cancer in in vivo and in vitro models.
© 2011 Elsevier B.V. All rights reserved.
1. Introduction
2. Materials and methods
Combination therapy using either two different types of chemical
drugs or a chemical drug in conjunction with a biologic, such as a
monoclonal antibody, has been a recent trend for maximizing
therapeutic efficacy [1–6]. Recently, combination of chemotherapy
with immunotherapy (i.e., chemoimmunotherapy) using unmethylated CpG oligonucleotides (ONTs) as immune-stimulants has also
shown promise [7–15], providing invaluable preclinical and clinical
outcomes. Because the synergism of combination therapy depends
greatly on appropriate timing and administration schedules [2,6,16], it
is desirable to develop carriers that incorporate and simultaneously
deliver both immune-stimulating and cytotoxic chemotherapeutic
agents [17]. To achieve that, we recently developed a combined
chemoimmunotherapy strategy using a plasmid–doxorubicin (Dox)
complex in which a plasmid bearing unmethylated CpG acts as both
an immune-stimulating agent and a carrier of the chemical drug, Dox
[17]. The plasmid–Dox complex showed greater antitumor efficacy
with much lower toxicity than the same dose of free Dox in in vivo
murine tumor models. However, our previous system was limited by
its lack of cancer-specific targeting ability. Thus, a combination of
cancer-specific targeting, chemotherapy, and immunotherapy might
be expected to yield a more efficacious cancer therapy. To this end, we
here report a targeted chemoimmunotherapy approach based on
aptamer–dendrimer conjugates.
2.1. Chemicals
⁎ Corresponding author. Tel.: + 82 62 970 2504; fax: + 82 62 970 2484.
E-mail address: syjon@gist.ac.kr (S. Jon).
0168-3659/$ – see front matter © 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.jconrel.2011.05.025
Purified Dox and a commercial Dox formulation (K.U. Dox HCl for
Injection) were purchased from Boryung Pharmaceutical (Seoul, Korea)
and Korea United Pharm (Seoul, Korea), respectively. PAMAMsuccinamic acid dendrimer, and most other chemicals were purchased
from Sigma-Aldrich (St. Louis, MO, USA). Analytical grade water and
acetonitrile were obtained from JT Baker (Phillipsburg, NJ, USA), and
oligonucleotide DNA was purchased from Bioneer (Daejon, Korea).
2.2. Mice
Male athymic and normal BALB/c mice were obtained from Orient
Bio (Seoul, Korea) and housed under pathogen-free conditions.
Animal care was provided in accordance with the guidelines of the
animal care facility at Gwangju Institute of Science and Technology.
2.3. Synthesis and characterization of sONT-DEN conjugates
PAMAM-succinamic acid dendrimers (Generation 4; 0.125 nmol),
N-hydroxy succinimide (NHS; 1 μmol), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC; 1 μmol), 4-(dimethylamino)pyridine (100 nmol), and amine-modified oligonucleotide
(ONT) (5′ NH2-AAA AAA AAA ATC GTC GTC GTC GTC GTC GTC G-3′;
8.8 nmol) were dissolved in DMSO. The mixture was stirred for 36 h at
room temperature, concentrated using a centrifugal vacuum system
(Hanil, Seoul, Korea), and loaded onto a Superdex 200 gel filtration
column. The purified dendrimer conjugate (sONT-DEN) was
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lyophilized, and the amount of DNA was quantified by measuring UV
absorption at 260 nm.
2.4. Synthesis of (CGA)7 extended A9 aptamer and (CGA)7 extended A9
scrambled aptamer
The sequence of the extended A9 aptamer [A9 (CGA)7 extended
aptamer] and extended A9 scrambled aptamer [A9 scramble (CGA)7
extended aptamer] was 5′-GGG AGG ACG AUG CGG ACC GAA AAA
GAC CUG ACU UCU AUA CUA AGU CUA CGU UCC CAG ACG ACU CGC
CCG ACG ACG ACG ACG ACG ACG ACG A-3′ and 5′-CAG GCA UGC CUA
GCU AAG CAG CCC AUG GCU UAU GCG CGG AAU AUU GGC UUC CGU
UCT CGA CGA CGA CGA CGA CGA CGA-3′, respectively (extended
sequence of the A9 aptamer underlined). The extended A9 aptamer
was synthesized by in vitro transcription (17 h at 37 °C) from a
double-stranded DNA template that included the T7 RNA promoter
using a MEGAscript kit (Ambion, Austin, TX, USA). Following
transcription, samples were treated with DNase for 15 min at 37 °C.
Aptamers were purified by LiCl precipitation.
2.5. Complexation and drug loading of Apt•dONT-DEN conjugates
sONT-DEN in distilled water was mixed with an equal concentration of the complementary sequence extended aptamer. The mixture
was heated at 90 °C for 10 min and allowed to cool slowly to room
temperature to obtain double-stranded Apt•dONT-DEN. A physical
complex between Apt•dONT-DEN or A9 (CGA)7 extended aptamer
(Apt-(CGA)7) and Dox was generated as described previously. Briefly,
increasing levels of dendrimer conjugate (in the picomolar range)
were added stepwise to a fixed concentration of Dox (1.5 μM) in
phosphate-buffered saline (PBS), and the fluorescence of Dox was
measured using a spectrofluorophotometer SH-2 (Sinko, Daejon,
Korea). Prior to in vivo injection, Dox-loaded aptamer dendrimer
bioconjugate (Dox@Apt•dONT-DEN) was freshly prepared by mixing
Apt•dONT-DEN with Dox in sterile saline.
2.6. Serum stability of Apt•dONT-DEN
2.9 mL Mouse serum (2.9 mL) was mixed with Apt•dONT-DEN
(0.1 mL) or A9 (CGA)7 extended aptamer (Apt-(CGA)7, 0.1 mL), as a
positive control, and incubated at 37 °C. Aliquots were withdrawn at
each time point, immediately frozen in liquid nitrogen, and subjected
to gel electrophoresis. Degradation was quantified by densitometry
using Image J software (National Institutes of Health, USA; http://rsb.
info.nih.gov/ij/).
2.7. In vitro immune response
The RAW264.7 murine monocytic cell line (American Type Culture
Collection [ATCC], Manassas, VA, USA) was cultured in Dulbecco's
modified Eagle medium (DMEM) containing 10% (v/v) heat-inactivated fetal bovine serum (FBS) (Invitrogen, Carlabad, CA, USA),
100 units/mL penicillin, and 100 mg/mL streptomycin (Invitrogen).
Changes in cytokine mRNA were measured in RAW264.7 cells treated
for 4 h with LPS (10 ng/mL), A10(TCG)7 ONT (10 μM), A10(TCG)7 ONT•
(CGA)7 extened RNA aptamer (Apt•dONT, 10 μM) or medium only
(negative control). After treatment, cells were collected and total RNA
was extracted using the Welprep reagent (Jeil Biotech Services,
Daegu, Korea). Total RNA (1 μg) was reverse-transcribed with
UmProm-II Reverse Transcriptase (Promega, Madison, WI, USA) and
amplified with an MJ Mini PCR system (Bio-Rad, Hercules, CA, USA).
Reverse transcription-polymerase chain reaction (PCR) analyses were
done using the following primers: β-actin, 5′-TCA TGA AGT GTG ACG
TTG ACA TC CGT-3′ (forward) and 5′-TTG CGG TGC ACG ATG GAG GGG
CCG GA-3′ (reverse); IL-12p40, 5′-GAA GTT CAA CAT CAA GAG CAG
TAG-3′ (forward) and 5′-AGG GAG AAG TAG GAA TGG GG-3′
(reverse); IL-1β, 5′-CCT GTG GCC TTG GGC CTC AA-3′ (forward) and
5′-GAG GTG CTG ATG TAC CAG TTG G-3′ (reverse); IL-6, 5′-ATG AAG
TTC CTC TGC AAG AGA CT-3′ (forward) and 5′-CAC TAG GTT TGC CGA
GTA GAT CTC-3′ (reverse); and TNF-α, 5′-AAA ATT CGA GTG ACA AGC
CTG TAG-3′ (forward) and 5′-CCC TTG AAG AGA ACC TGG GAG TAG-3′
(reverse).
In order to measure the TNF-α production in RAW264.7 cells was
measured after treatment with LPS (1 or 10 ng/mL), CpG 1668
(10 μM), A10(TCG)7 ONT (10 μM), A10(TCG)7 ONT•(CGA)7 extened
RNA aptamer (Apt•dONT, 10 μM) or medium only (negative control).
After 36 h, the medium was sampled and TNF-α was measured by
enzyme-linked immunosorbent assay (ELISA) using a Quantikine kit
(R&D Systems, Minneapolis, MN, USA).
2.8. Flow cytometric analysis
Cellular uptake of dendrimer conjugates was confirmed using flow
cytometry (EPICS XL Flow cytometry system, Beckman Coulter, Inc.,
Miami, FL). Briefly, LNCaP and 22RV1 (1 × 10 4 cells) cells were seeded
onto 6-well plates and then incubated for 24 h, after which
Dox@Apt•dONT-DEN or Dox@scApt•dONT-DEN (1.5 μM Dox) were
added and cells were incubated for an additional 0.5 h. After
incubation, cells were washed twice with Dulbecco's PBS (DPBS),
trypsinized, centrifuged at 300 g for 3 min, and then resuspended in
DPBS for FACS analysis. The data were processed with EXPO32
software.
2.9. Confocal microscopy
LNCaP and 22RV1 cells in RPMI-1640 medium were grown to 70%
confluence in Lab-Tek chamber slides (Nalgene Nunc, Naperville, IL)
followed by incubation with Dox@Apt•dONT-DEN or Dox@scApt•dONTDEN (1.5 μM Dox) for 30 min; after incubation, cell were washed
twice with DPBS, fixed by incubating with 3.5% HCHO for 10 min,
washed three times with DPBS, and mounted for fluorescence imaging.
Fluorescence images were obtained using an FV1000 laser-scanning
confocal microscope (Olympus, Tokyo, Japan) equipped with 100×
objectives.
2.10. In vitro anticancer effect of Dox@Apt•dONT-DEN
LNCaP, 22RV1, DU145, and PC3 cells were grown to 70% confluence
in 96-well plates. Before incubation with complex, cells were washed
with PBS and incubated with OPTI-MEM media for 30 min. The
medium in each well was then replaced with 100 μL of fresh medium
containing identical concentrations of Dox (5 μM) or Dox@Apt•dONTDEN (5 μM) for 1 h, washed, and further incubated in fresh medium
for 24 h. After washing cells twice with PBS, 100 μL of fresh culture
medium was added to each well, followed by the addition of 20 μL of
MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazoniumbromide) solution (2.5 mg/mL in PBS). After 4 h, 100 μL of DMSO was
added to each well, and absorbance was measured at 570 nm using a
Spectra Maxplus microplate reader (Molecular Devices, Sunnyvale,
CA, USA).
2.11. In vivo antitumor effects
The human 22RV1 prostate cancer cell line (ATCC) was grown as
recommended by the suppliers. Six-week-old male athymic BALB/c
mice were subcutaneously injected with 1 × 10 7 22RV1 cells delivered
into dorsal flanks. When tumors were at least 190–210 mm 3 in
volume, mice were randomly divided into groups of six (day 0). Mice
were then given four intravenous injections of 100 μL Dox (1 or 4 mg/
kg) or Dox@dONT-DEN (Dox 1 mg/kg) or Apt•dONT-DEN or Dox@Apt•dONT-DEN (Dox 1 mg/kg) in sterile saline. An untreated control
group was injected with sterile PBS, whereas the vehicle group
received Apt•dONT-DEN. For evaluate the effect of Dox@scApt•dONTDEN, when tumors were at least 80–120 mm 3 in volume, mice were
randomly divided into groups of four (day 0). Mice were then given
four intravenous injections of 100 μL Dox (1 mg/kg) or Dox@dONTDEN (Dox 1 mg/kg) or Dox@scApt•dONT-DEN (Dox 1 mg/kg) in
sterile saline. An untreated control group was injected with sterile
saline. At various times, mice tumors were measured using vernier
calipers. Tumor volume was calculated using the formula, (length ×
width × height)/2.
2.12. Statistical analysis
Data are shown as means±standard errors of the mean (SEMs).
Statistical significance was determined by ANOVA using SigmaStat 3.0
(Jandel Scientific, San Rafael, CA, USA). A P-valueb 0.005 was considered
to be statistically significant for differences between experimental groups.
3. Results and discussion
The concept underlying the approach and a synthetic scheme for the
conjugate is illustrated in Fig. 1. An RNA aptamer that specifically
Fig. 1. Schematic bioconjugation representation of the Dox@Apt•dONT-DEN for
targeted chemoimmunotherapy.
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recognizes a prostate-specific membrane antigen (PSMA) was used as a
prostate cancer-targeting ligand [18–31]. Dox was chosen as a chemotherapeutic agent because it is known to form stable complex with duplex
oligonucleotides (dONTs) by intercalating into base pairs, preferentially
into consecutive CG pairs [17,18,22,32]. As indicated in Fig. 1a, the
duplexed, unmethylated CpG ONT functions not only as a loading site for
Dox but also an immune-stimulating agent. Lastly, all of these components
were combined into a unique dendrimeric nanostructure that possesses
several favorable features for use in drug delivery, including a confined
nanometer size; ease of modification at peripheral sites; and high in vivo
stability [32–35]. The resulting aptamer–dendrimer bioconjugate would
be expected to provide specific targeting, drug delivery, and immune
stimulation to PSMA-overexpressing prostate cancers.
As shown in Fig. 1b, an oligodexoxynucleotide with an amine and
an adenine linker (5′-NH2-A10-(TCG)7-3′) was conjugated to a fourth
generation poly amido amine (PAMAM) dendrimer by reacting to
activated esters of periphery carboxylic acids in the dendrimer (64
copies per dendrimer), resulting in a single-stranded ONT-conjugated
dendrimer (sONT-DEN). The number of sONTs per dendrimer was
calculated through a general ONT quantification method by measuring UV absorption of oligonucleotide, revealing that ~41 sONTs were
conjugated to a dendrimer (see Supplementary Materials). In the
second step, an elongated version of the PSMA-specific A9 RNA
aptamer with additional sequence at the 3’-end (5′-Apt-(CGA)7-3′)
was hybridized with the sONT-DEN to create a double-stranded
Fig. 2. a) Gel electrophoresis data of size marker (lane 1), sONT-DEN (lane 2),
Apt•dONT-DEN (lane 3), and Apt-(CGA)7 (lane 4). The concentration of materials in all
lanes was identical. b) Fluorescence spectra of Dox (1.5 nmole) in the presence of 0,
150, 225, 375, and 575 pmol (top to bottom) Apt•dONT-DEN.
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Fig. 3. a) Confocal laser-scanning microscopy images of 22RV1 and LNCaP cells after treatments 1.5 μΜ of Dox@ Apt•dONT-DEN and of Dox@scApt•dONT-DEN for 0.5 h. b) Flow
cytometry histogram profiles of 22RV1 and LNCaP cells obtained after treatments with cell culture medium, Dox@scApt•dONT-DEN (1.5 μΜ of Dox) and Dox@Apt•dONT-DEN
(1.5 μΜ of Dox). c) Growth-inhibition assay for four prostate cancer cell lines LNCaP, 22RV1, DU145 and PC3 after 2 h of incubation with free Dox, Apt•dONT-DEN and
Dox@Apt•dONT-DEN and 24 h of subsequent incubation. All groups of Dox concentration is 5 μΜ.
Apt•dONT-DEN conjugate. In the last step, Dox was intercalated into
the double-stranded dONT site to yield Dox-loaded Apt•dONT-DEN
conjugates (Dox@Apt•dONT-DEN).
Fig. 2a shows agarose-gel electrophoresis data of each step. The
sONT-DEN band is fainter and shows retarded mobility compared to
the Apt-(CGA)7 band. After hybridization with the aptamer, however,
the ethidium bromide-stained band, which corresponds specifically to
double-stranded ONTs, became much brighter than sONT-DEN and
Apt-(CGA)7 bands at the same concentrations, indicating successful
formation of dONTs in the conjugate through hybridization. Intercalation of Dox into a base pair results in a decrease in its fluorescence
compared to unbound, free Dox [17,18,22,32]. To determine how
many Dox molecules are actually loaded onto the Apt•dONT-DEN
conjugate, we monitored quenching of Dox fluorescence with
increasing amounts of Apt•dONT-DEN conjugate. The fluorescence
spectrum of free Dox (1.5 nmol) was totally quenched by ~9.2 pmol of
Apt•dONT-DEN, indicating that ~163 Dox molecules were loaded into
an Apt•dONT-DEN that contains ~ 41 copies of Apt•dONT, which
corresponds to ~ 4 Dox per Apt•dONT unit (Fig. 2b).
Moreover, a Hill plot obtained from the fluorescence-quenching
curve yielded a dissociation constant (Kd) of 93.9 ± 10.9 nM for the
interaction between Dox and the Apt•dONT-DEN conjugate with a Hill
slope of 1.31 ± 0.59, indicating strong Dox binding to a base pair. In
contrast, no quenching effect of Dox was observed with the elongated
aptamer (Apt-(CGA)7) alone (Figure S1), which lacks the duplexed -CGcomponent that provides the intercalation sites necessary for Dox
Fig. 4. a) Macrophage cell line immune stimulation by LPS (10 ng/mL, lane 1), A10(TCG)7ONT (10 μM, lane 2), Apt•dONT (10 μM, lane 3), and cell culture medium treated
(lane 4) for 4 h. Total RNAs were extracted and each gene expression was determined
by RT-PCR analysis. b) TNF-α production in RAW 264.7 cell after LPS (1 or 10 ng/mL),
CpG1668 ONT (10 μM), A10-(TCG)7 (10 μM), and Apt•dONT (10 μM) for 24 h. TNF-α
was determined by ELISA.
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binding. Collectively, these data indicate that the Apt•dONT-DEN system
is able to carry large amounts of drug through a facile, intercalationbased drug-loading process, showing potential for in vivo use in
preclinical and clinical studies.
Serum stability of drug-delivery vehicles is also an essential
prerequisite for in vivo applications, particularly when nucleasecleavable, unmodified ONTs are used as components. Using gel
electrophoresis to examine the rate of Apt•dONT-DEN and elongated
PSMA-specific RNA aptamer (Apt-(CGA)7, negative control) degradation
in serum (Figure S2), we found that the Apt-(CGA)7 was totally degraded
within 3 h. In sharp contrast, approximately 62% of the Apt•dONT-DEN
conjugate remained undegraded, even after 24 h, clearly indicating that
the stability of the RNA aptamer in serum was dramatically enhanced
through attachment to the dendrimer. This enhanced stability of the
conjugate is likely attributable to the steric hindrance generated around
neighboring Apt•dONTs attached to the dendrimer core, which may
prevent access of nucleases to the cleavage site in the conjugate [32].
Increasing biostability through nanostructuring may prove to be an
effective strategy for designing RNA or DNA-based therapeutics.
To evaluate the feasibility of using Apt•dONT-DEN as a targeted drugdelivery vehicle, we carried out in vitro cellular uptake experiments with
22RV1 and LNCaP cells, both of which are prostate cancer cells
overexpressing PSMA protein on their plasma membranes. As shown
in Fig. 3a, uptake of Dox in both 22RV1 and LNCaP cells was much higher
with the Dox-loaded conjugate (Dox@Apt•dONT-DEN) than with the
scrambled aptamer-conjugated vehicle (Dox@scApt•dONT-DEN),
which is unable to recognize PSMA protein, as evidenced by strong
Dox fluorescence in the nuclei of most cells. Flow cytometry data
provided further support for the targeting specificity of Apt•dONT-DEN
(Fig. 3b), showing that Dox uptake was significantly enhanced in cells
incubated with Dox@Apt•dONT-DEN (~94% for 22RV1 and 57% for
LNCaP) compared to those incubated with Dox@scApt•dONT-DEN
(~35% for 22RV1 and 7% for LNCaP). Furthermore, the Apt•dONT-DEN
vehicle itself exhibited no appreciable cytotoxicity toward prostate
cancer cells (LNCaP, 22RV1, DU145, and PC3 cells) in a tetrazoliumbased MTT assay. Importantly, Dox@Apt•dONT-DEN exerted much
greater cytotoxicity toward PSMA-overexpressing LNCaP and 22RV1
target cells than toward PSMA-negative PC3 and DU145 cells (Fig. 3c).
Taken together, these results clearly indicate that Apt•dONT-DEN is
capable of delivering Dox to target cancer cells in a specific manner.
DNA•RNA chimeric hybrids, such as those used here, are known to
stimulate both TLR7 and TLR9 [10], whereas single-stranded DNA
oligonucleotides are only able to stimulate TLR9, suggesting that the
former may act as better immune-stimulating agents. To examine
whether the DNA•RNA chimeric hybrid unit in the Apt•dONT-DEN
conjugate can activate an immune response, we measured changes in
Fig. 5. Change of tumor sizes in 22RV1 xenograft after treated with four intravenous
injections of Dox 4 mg/kg (Δ) or 1 mg/kg (▼), Apt•dONT-DEN (○), Dox@dONT-DEN
(1 mg Dox/kg; ■), Dox@Apt•dONT-DEN (1 mg Dox/kg; □) or saline (●). The arrows
represent the injection schedule for all groups. All data are reported as means ± SEs;
n = 6, (*indicates P b 0.001 vs. control or Dox@dONT-DEN).
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expression levels of various immune-associated cytokines in macrophage cells (Raw264.7) using reverse transcription-polymerase
chain reaction (RT-PCR) and enzyme-linked immunosorbent assays
(ELISAs). As shown in Fig. 4a, RT-PCR revealed that levels of the
cytokines interleukin (IL)-1β, IL-12, IL-6, and tumor necrosis factor
(TNF)-α in cells treated with Apt•dONT were much higher than those
treated with either single-stranded ONT or cell culture medium.
Strikingly, Apt•dONT up-regulated these cytokines to an extent
similar to those obtained by treatment with the highly immunogenic
positive controls, lipopolysaccharide (LPS) and CpG1668. TNF-α
protein expression measured by ELISA is in good agreement with
RT-PCR data on mRNA levels (Fig. 4b), supporting the potential of the
Apt•dONT chimeric hybrid as a suitable immune adjuvant. Collectively, these data indicate a powerful adjuvant effect of Apt•dONT with the
potential to offset the local and systemic immune suppression caused
by cancer.
Finally, we evaluated the antitumor efficacy of Dox@Apt•dONT-DEN
using a 22RV1 xenograft tumor model. Fig. 5 shows the tumor growth
profiles following intravenous injection (four times treatments) of each
therapeutic modality. Measurement of tumor size on day 36 revealed
that Dox@Apt•dONT-DEN (dose: 1 mg Dox/kg) exhibited excellent
antitumor efficacy, reducing tumor volume by 78% compared to the
saline-treated control group (619 ± 52 mm3 vs. 2726 ± 411 mm3).
Moreover, the degree of tumor reduction in the Dox@Apt•dONT-DEN
group on day 44 was superior to that of the high-dose (4 mg/kg) freeDox group (830 ± 32 mm 3vs. 1939 ± 163 mm 3), even though the
former used a 4-fold lower dose of Dox. Interestingly, Dox@dONTDEN lacking the aptamer-targeting ligand exhibited an antitumor
efficacy comparable to that of the high-dose (4 mg/kg) free-Dox group
(46% vs. 45%). This result may be attributable to the enhanced
permeability and retention (EPR) effect of the dendrimer conjugate, as
has been demonstrated for nano-sized particles [32,33,35]. On the other
hand, the scrambled aptamer-conjugated group, Dox@scApt•dONTDEN, exhibited antitumor efficacy similar to Dox@dONT-DEN (Figure
S3), indicating that the dendrimer conjugate is unable to recognize
PSMA protein in the 22RV1 xenograft tumor. In contrast, the low-dose
free-Dox group (1 mg/kg; equivalent dose to Dox@Apt•dONT-DEN) and
the conjugate vehicle alone (without Dox loading) showed no
significant difference in tumor growth compared to the control group
treated with phosphate-buffered saline (PBS). Taken together,
these results indicate that the much greater antitumor efficacy of
Dox@Apt•dONT-DEN compared to the same dose of free-Dox or
aptamer-free dendrimer conjugate (Dox@dONT-DEN) was not attributable merely to prolonged circulation in the blood, but also reflected
targeted drug accumulation in the tumor.
In conclusion, we have developed a novel targeted chemoimmunotherapy system based on an aptamer–dendrimer bioconjugate that
possesses cancer-targeting ability, immune-stimulating function, and
drug delivery for chemotherapy. The drug-loaded conjugate showed
excellent antitumor efficacy and target specificity in an in vivo
prostate tumor model. The high drug-loading capacity and enhanced
in vivo stability of oligonucleotides made possible by the dendrimer
nanostructure can also be adapted to the development of RNA- or
DNA-based drug-delivery systems. This proof-of-concept demonstrates the potential of this nanostructure system as a new
combination approach for improving cancer treatments.
Acknowledgement
This study was supported by a grant of the Korea Healthcare
technology R&D Project, Ministry for Health, Welfare & Family Affairs,
Republic of Korea (grant number: A084764-0902-0000100) and by
the Converging Research Center Program through the National
Research Foundation of Korea (NRF) funded by the Ministry of
Education, Science and Technology (grant number. 20100028753).
Appendix A. Supplementary data
Supplementary data to this article can be found online at doi:10.
1016/j.jconrel.2011.05.025.
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