Actively Targeted Delivery of Doxorubicin to Bone Metastases by a pH-Sensitive Conjugation

Article abstract of DOI:10.1002/jps.24476

Alendronate-monoethyl adipate-(hydrazone)-doxorubicin conjugate (ALN-MA-hyd-DOX) was synthesized to specifically deliver doxorubicin (DOX) to bone tumor tissue. The binding kinetics of ALN-MA-hyd-DOX with hydroxyapatite (HA) and natural bone were detected by using spectrophotometer. Cytotoxicity of ALN-MA-hyd-DOX on tumor cells was determined by MTT [3-(4,5-dimethylthiaol-2-yl)-2,5-diphenyl-tetrazolium bromide] method. The cellular uptake of ALN-MA-hyd-DOX was observed by using fluorescence microscopy. The in vivo antitumor activity of ALN-MA-hyd-DOX was investigated by using tumor-bearing nude mice model. The results indicated that ALN-MA-hyd-DOX was able to quickly bind with HA and natural bone. ALN-MA-hyd-DOX immobilized on the natural bone released more DOX in pH 5.0 medium than that in pH 6.0 or 7.4 medium. The cytotoxicity of ALN-MA-hyd-DOX toward A549 cells and MDA-MB-231/ADR cells was greater than DOX. ALN-MA-hyd-DOX was rapidly uptaken by A549 cells and MDA-MB-231/ADR cells. Compared with the same dose of free DOX, ALN-MA-hyd-DOX significantly decreased tumor volume of tumor-bearing nude mice. DOX mainly distributed in bone tumor tissue after ALN-MA-hyd-DOX was intravenously administered to tumor-bearing nude mice, whereas DOX distributed through the whole body after DOX was intravenously administered to tumor-bearing nude mice. These findings implied that the ALN-MA-hyd-DOX was a promising bone-targeted conjugate for treating bone neoplasms.

Full text of DOI:10.1002/jps.24476

RESEARCH ARTICLE – Pharmaceutics, Drug Delivery and Pharmaceutical Technology
Actively Targeted Delivery of Doxorubicin to Bone Metastases by a
pH-Sensitive Conjugation
WEI-LIANG YE,1 YI-PU ZHAO,1 REN NA,2 FEI LI,1 QI-BING MEI,3 MING-GAO ZHAO,3 SI-YUAN ZHOU1
1Department of Pharmaceutics, School of Pharmacy, Fourth Military Medical University, Xi’an 710032, China
2West Changle Sanatorium for Xi’an Army Retired Cadres of Fourth Military Medical University, Xi’an 710032, China
3Department of Pharmacology, School of Pharmacy, Fourth Military Medical University, Xi’an 710032, China
Received 19 December 2014; revised 24 March 2015; accepted 16 April 2015
Published online 15 May 2015 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/jps.24476
ABSTARCT: Alendronate-monoethyl adipate-(hydrazone)-doxorubicin conjugate (ALN-MA-hyd-DOX) was synthesized to specifically de-
liver doxorubicin (DOX) to bone tumor tissue. The binding kinetics of ALN-MA-hyd-DOX with hydroxyapatite (HA) and natural bone were
detected by using spectrophotometer. Cytotoxicity of ALN-MA-hyd-DOX on tumor cells was determined by MTT [3-(4,5-dimethylthiaol-2-
yl)-2,5-diphenyl-tetrazolium bromide] method. The cellular uptake of ALN-MA-hyd-DOX was observed by using fluorescence microscopy.
The in vivo antitumor activity of ALN-MA-hyd-DOX was investigated by using tumor-bearing nude mice model. The results indicated
that ALN-MA-hyd-DOX was able to quickly bind with HA and natural bone. ALN-MA-hyd-DOX immobilized on the natural bone re-
leased more DOX in pH 5.0 medium than that in pH 6.0 or 7.4 medium. The cytotoxicity of ALN-MA-hyd-DOX toward A549 cells and
MDA-MB-231/ADR cells was greater than DOX. ALN-MA-hyd-DOX was rapidly uptaken by A549 cells and MDA-MB-231/ADR cells.
Compared with the same dose of free DOX, ALN-MA-hyd-DOX significantly decreased tumor volume of tumor-bearing nude mice. DOX
mainly distributed in bone tumor tissue after ALN-MA-hyd-DOX was intravenously administered to tumor-bearing nude mice, whereas
DOX distributed through the whole body after DOX was intravenously administered to tumor-bearing nude mice. These findings implied
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that the ALN-MA-hyd-DOX was a promising bone-targeted conjugate for treating bone neoplasms. 2015 Wiley Periodicals, Inc. and the
American Pharmacists Association J Pharm Sci 104:2293–2303, 2015
Keywords: bone-targeted drug delivery; doxorubicin; controlled release; alendronate; conjugaton; p-glycoprotein; cytotoxicity; imaging
methods; cancer
the clinic.11 Additionally, BPs can inhibit the angiogenesis of
tumor tissue.12,13 BPs exhibit strong binding affinity with bone
mineral.14 Therefore, BPs was used as excellent ligands for
bone-targeted therapy.15,16 Alendronate (ALN), one of BPs, was
approved by the US FDA (Food and Drug Administration) to
treat osteoporosis, tumor-associated hypercalcemia, and sev-
eral other bone-related diseases.17,18 Besides, ALN was proved
to have antitumor effect in several tumor models.19–21 DOX has
remained a widely used antitumor drug in the last decades,
although it can cause serious systemic side effect, such as car-
diotoxicity. In theory, if DOX is specifically delivered to the
tumor bone metastases, its antitumor activity will be greatly
enhanced and its systemic side effects will be significantly de-
creased.
In this paper, a new conjugate alendronate-monoethyl
adipate-(hydrazone)-doxorubicin conjugate (ALN-MA-hyd-
DOX) was synthesized to deliver DOX to the bone metastases
by using monoethyl adipate (MA) as a linker. MA was conju-
gated with ALN and DOX by amide bond and hydrazone bond,
respectively. The binding kinetics of ALN-MA-hyd-DOX with
hydroxyapatite (HA) and natural bone was determined. The
in vitro and in vivo antitumor activities of ALN-MA-hyd-DOX
were investigated on the tumor-bearing nude mice model.
INTRODUCTION
Bone is a major organ for tumor metastasis, particularly for
prostate and breast tumor. Although the tumor bone metastasis
is not the major reason for causing death, symptoms associated
with tumor bone metastasis such as bone pain, life-threatening
hypercalcemia, nerve compression syndromes, and pathologic
fractures significantly lowered the life quality of patients.1–3
Currently, there is a lack of effective treatments for patients
with tumor bone metastasis. Therefore, it is imperative to de-
velop effective methods to treat tumor bone metastasis.
It was reported that when tumor bone metastasis occurred,
the acid-base balance in tumor bone metastasis tissue was bro-
ken, and osteoclasts secreted protons and acidic hydrolases into
the bone resorption compartment, which led to the digestion of
the mineral and organic phase of bone matrix.4,5 The pH value
decreased to 4.5 in bone resorption microenvironment.6 Thus,
a pH-sensitive bone-specific drug delivery system, the drug re-
lease of which is dependent on the environmental pH value, has
attracted much attention.7,8 In our laboratory, hydrazone bond
was used to prepare pH-sensitive site-specific drug delivery sys-
tems. The result showed that drug release was accelerated in
acidic environment.9,10
Bisphosphonates (BPs) are used to treat myeloma, osteo-
porosis, bone metastases, and other bone-related diseases in
MATERIALS AND METHODS
Materials
Correspondence to: Si-Yuan Zhou (Telephone: +86-29-84776783; Fax: +86-29-
84776783; E-mail: zhousy@fmmu.edu.cn)
This article contains supplementary material available from the authors upon
request or via the Internet at http://wileylibrary.com.
Wei-Liang Ye and Ren Na contributed equally to this work.
Alendronate, HA, N-hydroxysuccinimide (NHS), and 1-ethyl-3-
(3-dimethyllaminopropyl) carbodiimide (EDCI) were purchased
from J&K CHEMICA (Beijing, China). DOX was purchased
Journal of Pharmaceutical Sciences, Vol. 104, 2293–2303 (2015)
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from Hisun Pharmaceutical Company (Zhejiang, China). MA Binding Kinetics of DOX Conjugate with HA
was purchased from Aladdin reagent Company (Shanghai,
The binding kinetics of ALN-MA-hyd-DOX, ALN-MA-ami-
China). All other chemicals were of analytical grade and ob-
tained from commercial suppliers without further purification.
RPMI1640 medium, 4ꢁ,6-diamidino-2-phenylindole, and 3-(4,5-
dimethylthiaol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT)
were bought from Invitrogen Technologies Company (Carls-
bad, California). A549 cells were purchased from Institute
of Biochemistry and Cell Biology, Chinese Academy of Sci-
ence (Shanghai, China). MDA-MB-231/ADR cells were DOX-
resistant human breast cancer cells, which were induced in our
laboratory.
DOX, and DOX with HA were assessed according to the pre-
vious literature.23,24 Briefly, 1 mg conjugate was dissolved in
10 mL phosphate-buffered saline (PBS) in a falcon tube, and
100 mg HA was added. The mixture was gently shaken at
37°C in water bath. Another solution of conjugate without HA
was used as control. After 0, 5, 10, 15, 20, 30, 40, 50, 60, 70,
80, and 90 min, the mixture solution was centrifuged (4000g,
5 min) and the absorbance of the supernatant was measured
by Beckman DU-800 spectrophotometer at 233 nm. The bind-
ing percentage of conjugate with HA was calculated as by the
formula: [(ODwithout HA−ODwith HA)/(ODwithout HA)]×100%.
Methods
Synthesis of ALN-MA-hyd-DOX and ALN-MA-ami-DOX
Binding Kinetics of DOX Conjugate with Natural Bone
The synthetic route of ALN-MA-hyd-DOX is shown schemat-
ically in Figure 1 according to the literature.22 MA (500 mg,
2.8 mmol) was dissolved in 10 mL ethanol, and then 4 mL hy-
drazine hydrate (6.9 mmol) solution was drop-wise added. The
reaction mixture was refluxed for 12 h. Then the ethanol was
removed from the reaction solution. The residue was extracted
with diethyl ether for three times. The residue was spin-dried
to get MA hydrazide. The product was purified by silica ge1
column.
Monoethyl adipate hydrazide (80 mg, 0.45 mmol) was dis-
solved in 10 mL methanol, and then DOX (140 mg, 0.25 mmol)
and 60 :L of trifluoroacetic acid were added. The reaction mix-
ture was stirred for 12 h in the darkness at room temperature.
After ethanol was removed from the reaction mixture, the MA-
hyd-DOX was purified by silica gel column.
Bone fragments, isolated from the backbone of pig, were cut
into small pieces. The bone pieces were washed with wa-
ter and ethanol and then dried in drying oven. One mil-
ligram DOX (or ALN-MA-ami-DOX, ALN-MA-hyd-DOX) was
dissolved in 10 mL PBS in a falcon tube, after which 100 mg
bone pieces were added. The mixture was gently shaken at
37°C in water bath. Another solution of conjugate without
bone pieces was used as control group. After 0, 5, 10, 15,
20, 30, 40, 50, 60, 70, 80, and 90 min, the mixture solu-
tion was centrifuged (4000g, 5 min) and the absorbance of
the supernatant was measured by Beckman DU-800 spec-
trophotometer at 233 nm. The binding percentage of conjugate
with HA was calculated by the formula: [(ODwithout nature bone
ODwith nature bone)/(ODwithout nature bone)] × 100%.
−
Monoethyl adipate-(hydrazone)-doxorubicin (230 mg,
0.33 mmol) was dissolved in 8 mL dimethyl sulfoxide (DMSO)
DOX Release from DOX Conjugate at Different pH Medium
and was activated by both 100 mg EDCI (0.515 mmol) and The mixture solution contained nature bone and ALN-MA-ami-
80 mg NHS (0.695 mmol) for 12 h at room temperature. ALN DOX or ALN-MA-hyd-DOX was gently shaken in water bath at
(120 mg, 0.44 mmol) dissolved in 10 mL H2O was drop-wise 37°C for 2 h. Then, it was centrifuged at 4000g for 5 min, and
added to the reaction solution. Then, triethylamine was added the supernatant was discarded. The precipitation was washed
to adjust pH of the reaction solution to 8–9. The reaction three times with 2.0 mL water. The precipitation was dispersed
mixture was stirred for another 12 h at room temperature. in 10 mL PBS (pH 5.0, 6.0, and 7.4) in falcon tube. The falcon
Ethyl acetate was added to the reaction solution, and the tubes were then gently shaken in water bath at 37°C. After
resulting precipitate was filtered and rinsed three times with a predetermined time period, the mixture solution was cen-
ethyl acetate, then purified by reversed-phase column chro- trifuged (4000g, 5 min) and the absorbance of the supernatant
matography. The yield was 173.6 mg (0.185 mmol, 49.4%). The was measured by Beckman DU-800 spectrophotometer at
purification of ALN-MA-hyd-DOX was analyzed according to 233 nm to calculate the amount of DOX released from the na-
previously reported method.10 Lipo-hydro partition coefficient ture bone.
(Log P) of DOX and ALN-MA-hyd-DOX was determined by
using n-octanol/water method.10 The concentration of DOX (or
Cell Culture Condition
ALN-MA-hyd-DOX) in both phases was measured by using
A549 cells, also called human lung cancer cell lines, are easy
Beckman DU-800 spectrophotometer.
to metastasize to the bone.25 A549 cells were maintained in
The synthetic route of ALN-MA-ami-DOX is showed
a RPMI 1640 medium. MDA-MB-231/ADR cells, also called
schematically in Figure 2. MA (80 mg, 0.45 mmol) was dissolved
human DOX-resistant breast cancer cell line, are easy to
in 15 mL methanol, and then 104 mg NHS (0.91 mmol), 210 mg
EDCI (1.08 mmol), and 70 :L triethylamine were added. The
metastasize to the bone.26 MDA-MB-231/ADR cells were main-
tained in L-5 medium. All cell lines were supplemented with
reaction mixture was stirred at room temperature for 6 h, and
100 units/mL penicillin, 100 units/mL streptomycin, 10% fetal
then 160 mg DOX (0.29 mmol) was added. The reaction mix-
bovine serum, and cultured in a humidified atmosphere con-
ture was stirred for another 12 h. Finally, the ALN was conju-
taining 5% CO2 at 37°C.
gated with the MA-ami-DOX. The ALN-MA-ami-DOX was pu-
rified by reversed-phase column chromatography. The yield was
Cytotoxicity of DOX Conjugates
87.3 mg (0.094 mmol, 51.9%). The purification of ALN-MA-ami-
DOX was analyzed according to a previously reported method.10 The A549 and MDA-MB-231/ADR cells were seeded in 96-well
Log P of ALN-MA-ami-DOX was determined by using afore- plates at a density of 1 × 104 cells per well and incubated for
mentioned method.
overnight to allow cells attachment. Then, cells were incubated
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Figure 1. The synthetic route of ALN-MA-hyd-DOX.
with fresh medium containing various concentration of free Cellular Uptake of DOX Conjugates
DOX, ALN-MA-ami-DOX, or ALN-MA-hyd-DOX (0.08, 0.8, 8,
A549 cells and MDA-MB-231/ADR cells were seeded in cover-
glass containing 24-well plates at a density of 1 × 105 cells per
well and incubated for 24 h at 37°C. Then, free DOX (ALN-
MA-ami-DOX or ALN-MA-hyd-DOX, 10 :mol/L) was added
and incubated for 4 h at 37°C. The cells were washed three
times with PBS and treated with 4ꢁ,6-diamidino-2-phenylindole
(10 :g/mL) for 15 min for nucleus staining. Then, the cells
were washed with PBS for three times and fixed with 1.5%
formaldehyde. Cover slips were placed onto microscope slides,
and DOX uptake was observed by using 80i fluorescence micro-
scope (Nikon Corporation, Tokyo, Japan).
and 40 :mol/L DOX) for 24 or 48 h. MTT (5 mg/mL, 50 :L)
was added and incubated for 4 h. After removing the medium,
150 :L of DMSO were added to dissolve formazan crystals. The
absorbance was measured at 570 nm using a CODA Automated
EIA Analyzer (Bio-Rad Laboratories, Hercules, California).
The Effect of DOX Conjugates on Cell Apoptosis
Apoptosis of tumor cells, induced by DOX conjugates, was ob-
served by measuring the intracellular caspase-3 activities. The
A549 cells or MDA-MB-231/ADR cells were treated with free
DOX (ALN-MA-ami-DOX or ALN-MA-hyd-DOX) at the concen-
Antitumor Activity of ALN-MA-hyd-DOX In Vivo
tration of 10 :mol/L DOX for 24 or 48 h. The cells were collected Female athymic nude mice (body weight = 20–23 g) were in-
and lysed by lysate buffer. Then, the cells lysate were treated jected via intratibia with A549 cells (1 × 107 cells/animal).
with 50 :mol/L Ac-DEVD-AFC at room temperature for 60 min There were four mice in each group. Treatment was initiated
in darkness. The caspase-3 activity was determined by measur- on the 10th day after tumor cell inoculation. Normal saline,
ing the absorbance at 405 nm.
free DOX (10 :mol/kg) and ALN-MA-hyd-DOX (10 :mol/kg),
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Figure 2. The synthetic route of ALN-MA-ami-DOX.
was injected to tumor-bearing nude mice via the tail vein for
every 7th day (day 1, 7, and 14). Mice were observed every
3 days. The body weight was recorded and the tumor growth
was monitored by using caliper. The tumor volume was cal-
culated by using the formula: volume = LW2/2 (L is the long
diameter and W is the short diameter of a tumor). DOX usu-
ally leads to the damage of heart and kidney. Thus, at the end
of the treatment, the heart and kidney samples were removed
and stained by hematoxylin and eosin (H&E) to observe tissue
injury.
Biodistribution Study
Female athymic nude mice (body weight = 20–23 g) were in-
jected intratibia with A549 cells (1 × 107 cells/animal). When
the tumor volume reached about 300 mm3, free DOX (10
:mol/kg) and ALN-MA-hyd-DOX (10 :mol/kg) were adminis-
trated to tumor-bearing nude mice by tail vein injection. Twelve
hours after the injection, major organs including heart, liver,
spleen, kidney, lung, and bone in tumor tissue were removed.
The fluorescence intensity in organs and bone tumor tissues
was observed and semiquantitative by the Caliper IVIS Lu-
mina in vivo image (Caliper Life Science, Boston, MA, USA).
Figure 3. The ultraviolet spectrum of DOX, ALN-MA-ami-DOX, and
ALN-MA-hyd-DOX.
RESULTS
Characterization of ALN-MA-hyd-DOX and ALN-MA-ami-DOX
The typical HPLC chromatogram of ALN-MA-hyd-DOX is
showed in Supplementary Figure 1a. The purity of ALN-MA-
hyd-DOX was 98.7%. The ultraviolet spectrum of ALN-MA-hyd-
DOX is shown in Figure 3. The results indicated that there were
Statistical Methods
significant difference in ultraviolet spectrum between DOX and
1
Experiments were performed in triplicates. The results are ex- ALN-MA-hyd-DOX. The mass spectrum, IR spectrum, and H
pressed as mean
SD. Statistical analyses were performed NMR spectrum of ALN-MA-hyd-DOX are shown in Supple-
with Graph Pad Prism 5.0.
mentary Figures 1b–1d, respectively. The molecular ion peak
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Table 1. Molecular Properties of DOX Conjugates (n= 5)
Molecular
Number of Oxygen
and Nitrogen Atoms
Number of –OH
and –NH
Violations of
Rule of Five
Number of
Rotatable Bonds
Compound
Log P
Weight
DOX
ALN-MA-ami-DOX
ALN-MA-hyd-DOX
0.33
−0.163
−0.424
0.08
0.04
0.11
543
924
938
12
22
23
6
11
12
3
3
3
5
12
12
([M+H]+) of ALN-MA-hyd-DOX was 938. In IR spectrum of
ALN-MA-hyd-DOX, peak at 3300–3500 was because of amino
bond stretching bands; peak at 1634 was because of the C=C
stretching vibration band; peak at 1608 was because of the hy-
drazone bond stretching vibration band; peaks at 451 and 540
were because of the O-P-O stretching bands; peak at 1362 was
because of the P=O vibration band.27,28 The 1H NMR spectrum
of ALN-MA-hyd-DOX confirmed the presence of the MA moiety
(signal at 1.2, 2.5, and 3.4 ppm) and the ALN moiety (signal at
4.6, 3.9, 3.6, 3.4, and 2.9 ppm). Conjugation of DOX was con-
firmed by the presence of signal at 8.0, 7.8, 7.5, 5.4, 2.1, and
1.9 ppm.
The typical HPLC chromatogram of ALN-MA-ami-DOX is
shown in Supplementary Figure 2a. The purity of ALN-MA-
ami-DOX was 97.9%. The ultraviolet spectrum of ALN-MA-
hyd-DOX is shown in Figure 3. The mass spectrum, IR spec-
trum, and 1H NMR spectrum of ALN-MA-ami-DOX are respec-
tively shown in Supplementary Figure 2b–2d. The molecular
ion peak ([M+H]+) of ALN-MA-ami-DOX was 924. In IR spec-
trum of ALN-MA-ami-DOX, peak at 3300–3500 was because
of amino bond stretching bands; peak at 1634 was because of
the C=C stretching vibration band; peaks at 456 and 553 were
because of the O-P-O stretching bands; peak at 1377 was be-
cause of the P=O vibration band.27,28 The 1H NMR spectrum of
ALN-MA-ami-DOX confirmed the presence of the MA moiety
(signal at 1.3, 2.5, and 3.5 ppm) and the ALN moiety (signal at
4.2, 3.5, 3.4, and 2.6 ppm). Conjugation of DOX was confirmed
by the presence of signal at 7.9, 7.6, 7.1, 5.2, 2.1, and 1.8 ppm.
Rule of five is a rule of thumb to evaluate whether a chemical
compound with a certain pharmacological activity has proper-
ties to be an orally active drug in humans. Table 1 showed the
molecular description of DOX, ALN-MA-ami-DOX, and ALN-
MA-hyd-DOX, and there were three parameters that violated
rule of five in DOX conjugates. Compared with free DOX, the
water solubility of ALN-MA-ami-DOX and ALN-MA-hyd-DOX
increased. This was an expected result caused by the sub-
stituent of ALN. There was no significant difference in water
solubility between ALN-MA-ami-DOX and ALN-MA-hyd-DOX.
Figure 4. Binding kinetics of 1 mg DOX, ALN-MA-ami-DOX, and
ALN-MA-hyd-DOX with the 100-mg bone mineral HA in 10 mL PBS.
Data are presented as the average standard deviation (n = 3).
the nature bone matrices were similar to those of ALN-MA-
hyd-DOX.
DOX Release from Natural Bone Matrices at Different pH Medium
The release of DOX from natural bone matrices that immo-
bilized ALN-MA-hyd-DOX and ALN-MA-ami-DOX was inves-
tigated at different pH medium. As shown in Figure 5b, the
release rate of DOX from immobilized ALN-MA-hyd-DOX was
closely related to the medium pH. Natural bone matrices that
immobilized ALN-MA-hyd-DOX released 73%, 43%, and 14%
of the immobilized DOX at pH 5.0, 6.0, and 7.4 in 28 h, respec-
tively. However, the rate that DOX released from immobilized
ALN-MA-ami-DOX was independent on the medium pH. As
shown in Figure 5c, after 28-h incubation, only 5% DOX was re-
leased from immobilized ALN-MA-ami-DOX in pH 7.4 medium,
and 11% DOX was released in pH 5.0 medium. The above re-
sults implied that ALN-MA-hyd-DOX bound quickly with bone
tissue and released DOX sustainedly at the bone tumor site.
HA-Binding Kinetics of DOX Conjugates
Cytotoxicity of DOX Conjugate
The binding kinetics of DOX conjugates with HA is shown in
Figure 4. Only a little amount of free DOX bounded with HA
(<10%) in 90 min. However, ALN-MA-ami-DOX and ALN-MA-
hyd-DOX bounded with HA very fast. About 62% of ALN-MA-
ami-DOX and ALN-MA-hyd-DOX in the solution was bounded
with HA in 20 min.
As shown in Figure 6a, the viability of A549 cells in the ALN-
MA-hyd-DOX-treated group was lower than that in ALN-MA-
ami-DOX-treated group in 24 h. There was no significant differ-
ence in cytotoxicity between free DOX and ALN-MA-hyd-DOX
on A549 cells in 24 h. However, as shown in Figure 6b, when
the A549 cells were treated with ALN-MA-hyd-DOX for 48 h,
the cell viability significant decreased, compared with the same
dose of free DOX. The effects of free DOX, ALN-MA-ami-DOX,
Binding Kinetics of DOX Conjugate with Natural Bone
As shown in Figure 5a, when free DOX was incubated with and ALN-MA-hyd-DOX on viability of MDA-MB-231/ADR cells
nature bone matrices at pH 7.4, only a little amount of DOX are shown in Figure 7. ALN-MA-hyd-DOX showed greater cy-
was bound with natural bone. In contrast, approximately 84% totoxicity than free DOX in 24 or 48 h on MDA-MB-231/ADR
of ALN-MA-hyd-DOX was bounded with bone matrices in cells. Compared with ALN-MA-ami-DOX, ALN-MA-hyd-DOX
30 min. The binding characteristics of ALN-MA-ami-DOX to exhibited higher cytotoxicity on MDA-MB-231/ADR cells.
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Figure 6. Cytotoxicity of DOX, ALN-MA-ami-DOX, and ALN-MA-
hyd-DOX on A549 cells for 24 h (a) and 48 h (b) incubation. Data are
presented as the average standard deviation (n = 5). *p < 0.05 versus
the same dose of ALN-MA-ami-DOX. #p < 0.05 versus the same dose
of DOX (n = 3).
Figure 5. Binding kinetics of 1 mg DOX, ALN-MA-ami-DOX, and
ALN-MA-hyd-DOX to 100 mg natural bone (a) in 10 mL PBS. The
accumulative release of DOX from natural bone immobilized with ALN-
MA-hyd-DOX (b) and ALN-MA-ami-DOX (c) at pH5.0, 6.0, or 7.4. Data
are presented as the average standard deviation (n = 3).
cence microscopy. As shown in Figure 9, the fluorescence was
observed mainly in nucleus but little in cytoplasm when the
A549 cells were incubated with free DOX for 4 h (Fig. 9a). How-
Apoptosis Induced by DOX Conjugates
The cell apoptosis, induced by free DOX, ALN-MA-ami-DOX, ever, as shown in Figure 9b, the red fluorescence was observed
and ALN-MA-hyd-DOX, was evaluated by measuring caspase- mainly in the cytoplasm and little in the nucleus when A549
3 activity of the cells. When the cells were treated with ALN- cells were incubated with ALN-MA-ami-DOX for 4 h. When
MA-hyd-DOX for 24 h, as shown in Figure 8a, the caspase-3 the A549 cells were incubated with ALN-MA-hyd-DOX for 4 h,
activity in A549 cells and MDA-MB-231/ADR cells markedly as shown in Figure 9c, a similar pattern of cellular distribu-
increased. When the cells were treated with free DOX for 24 h, tion with free DOX was observed. Little DOX was accumu-
the level of caspase-3 in A549 cells significantly increased, but lated in the cytoplasm and nucleus (Fig. 10a) when MDA-
the level of caspase-3 activity in MDA-MB-231/ADR cells re- MB-231/ADR cells were incubated with free DOX. The result
mained unchanged. When the cells were treated with ALN-MA- was consistent with MTT result from the MDA-MB-231/ADR
hyd-DOX for 48 h, as shown in Figure 7b, the level of caspase- cells. When MDA-MB-231/ADR cells were treated with ALN-
3 in A549cells and MDA-MB-231/ADR cells significantly MA-ami-DOX, the fluorescence was observed in cytoplasm and
increased, compared with 24 h treatment group. ALN-MA-ami- nucleus (Fig. 10b). Stronger fluorescence was observed in nu-
DOX did not significantly increase the level of caspase-3 in cleus when cells were incubated with ALN-MA-hyd-DOX for
A549 and MDA-MB-231/ADR cells after 24 or 48 h treatment. 4 h (10°C). The result indicated that ALN-MA-hyd-DOX could
These results were well consistent with the cytotoxicity of ALN- efficiently be uptaken by tumor cells and released DOX in tu-
MA-ami-DOX and ALN-MA-hyd-DOX on A549 and MDA-MB- mor cells.
231/ADR cells.
In Vivo Antitumor Activity of ALN-MA-hyd-DOX
Cellular uptake of DOX Conjugates
The experiment results indicated that ALN-MA-hyd-DOX
As DOX is fluorescent, cellular uptake of free DOX, ALN-MA- showed higher in vitro antitumor activity than ALN-MA-ami-
ami-DOX, and ALN-MA-hyd-DOX can be visualized by fluores- DOX did. So, ALN-MA-hyd-DOX was chosen to evaluate its
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Figure 8. The effect of ALN-MA-ami-DOX and ALN-MA-hyd-DOX
on caspase-3 activity in A549 cells and MDA-MB-231/ADR cells after
24 h (a) and 48 h (b) treatment. Data are presented as the average
standard deviation. *p < 0.05 versus control. #p < 0.05 versus DOX (n
= 3).
Figure 7. Cytotoxicity of DOX, ALN-MA-ami-DOX, and ALN-MA-
hyd-DOX on MDA-MB-231/ADR cells for 24 h (a) and 48 h (b) incuba-
tion. Data are presented as the average standard deviation (n = 5).
*p < 0.05 versus the same dose of ALN-MA-ami-DOX. #p < 0.05 versus
the same dose of DOX (n = 3).
tered to tumor-bearing mice, DOX accumulated in leg bone
tumor tissue, heart, and kidney. However, after ALN-MA-hyd-
DOX was intravenously administered to tumor-bearing mice,
ALN-MA-hyd-DOX was mainly distributed in leg bone tumor
in vivo antitumor activity by using free DOX as a control.
The in vivo antitumor activity of the DOX and ALN-MA-hyd-
DOX are shown in Figure 11. Figure 11a shows tumor growth
after tumor-bearing mice were treated with normal saline,
free DOX, and ALN-MA-hyd-DOX. The tumor volume in nor-
mal saline-treated mice increased very fast; ALN-MA-hyd-DOX
significantly slowed tumor growth, compared with free DOX.
Figure 11b shows body weight changes after tumor-bearing
mice were treated with normal saline, free DOX, and ALN-MA-
hyd-DOX. The decrease of body weight in free DOX-treated
mice was 28.6% of original weight, which indicated the severe
systemic side effects of DOX. In contrast, the body weight loss
in ALN-MA-hyd-DOX-treated group was 9.5% of the original
weight. This implied that the systemic side effects of ALN-MA-
hyd-DOX were lower than those of free DOX. Figure 11c showed
that ALN-MA-hyd-DOX significantly prolonged the survival
time of tumor-bearing mice, compared with the free DOX. From
Figure 11d, it can be clearly seen that ALN-MA-hyd-DOX sig-
nificantly delayed the tumor growth, compared with the control
mouse or free DOX-treated mouse.
In Vivo Targeting Ability of ALN-MA-hyd-DOX in Tumor-Bearing
Mice
Figure 9. Intracellular localization of DOX (a), ALN-MA-ami-DOX
(b), and ALN-MA-hyd-DOX (c) in A549 cells at an equivalent DOX
concentration of 10 :mol/L for 4 h incubation at 37°C. The pink region
shows the localization of DOX (red) in the nucleus (blue).
The biodistribution of DOX in tumor-bearing mice is shown
in Figure 12a. After free DOX was intravenously adminis-
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RESEARCH ARTICLE – Pharmaceutics, Drug Delivery and Pharmaceutical Technology
surgery is difficult because of the multiple metastatic sites and
inaccessibility to the metastatic nodules.29 Chemotherapy, the
only currently available method to treat bone metastatic tumor,
is limited by severe side effects to normal tissue.30
The bone microenvironment is composed of osteoblasts, os-
teoclasts, mineralized bone matrix, and many other kinds of
cells. Bone microenvironment is highly favorable for tumor in-
vasion and growth. Tumor cells that metastasize to bone usu-
ally adhere to the endosteal surface. Cross-talk between bone
microenvironment and tumor cells enhances a vicious cycle
of tumor growth and bone destruction.31,32 Some factors that
are secreted by tumor cells can stimulate osteoclast-mediated
bone destruction and the consequent release of numerous fac-
tors immobilized within the bony matrix, which can act on
tumor cells, promote more aggressive tumor metastasis and
bone destruction. Tumor metastasis leads to the acidosis within
bones.33 The pH value can decrease to 4.5 in bone resorptive
microenvironment.6 Recent studies have shown that the hydra-
zone bond between drug and targeting moiety is stable in blood
circulation, and it is easy to be broken in acidic microenviron-
ment. These characteristics can be used to control the release
of drug.34,35
Figure 10. Intracellular localization of DOX (a), ALN-MA-ami-DOX
(b), and ALN-MA-hyd-DOX (c) in MDA-MB-231/ADR cells at an equiv-
alent DOX concentration of 10 :mol/L for 4 h incubation at 37°C. The
pink region shows the localization of DOX (red) in the nucleus (blue).
It is well-documented that the BPs preferentially deposit in
metastatic bone lesions after systemic administration of BPs.
HA is a major component of bone. The binding mechanism of
BPs with HA is simple adsorption.18 In this paper, the HA ad-
sorption experiment was performed to evaluate the adsorption
capacity of ALN-MA-hyd-DOX with bone. The results indicated
that the adsorption capacity of the ALN-MA-ami-DOX and
ALN-MA-hyd-DOX with HA was significantly higher than that
of DOX. No significant difference was observed in adsorption
capacity between ALN-MA-ami-DOX and ALN-MA-hyd-DOX.
The ideal bone-targeted conjugates should not only have
high binding affinity with bone but can also release drug at
the desired site. The drug release characteristics from natu-
ral bone (immobilized ALN-MA-hyd-DOX and ALN-MA-ami-
DOX) in different pH medium were investigated. The results
showed that immobilized ALN-MA-hyd-DOX released DOX in
pH-dependent manner. The higher DOX release rate of ALN-
MA-hyd-DOX was observed at pH 5.0 and pH 6.0. This implied
that ALN-MA-hyd-DOX was able to release large amount of
DOX in sites of bone metastasis,6 thereby greatly enhancing
the efficacy of DOX. There was a little amount of DOX released
from immobilized ALN-MA-hyd-DOX in pH 7.4 medium. This
indicated that ALN-MA-hyd-DOX was stable in blood circu-
lation. On the contrary, a little amount of DOX was released
from immobilized ALN-MA-ami-DOX in different pH medium.
This implied that ALN-MA-ami-DOX could not exert obvious
antitumor activity in sites of bone metastasis.
tissue, and a relatively lower amount of DOX was distributed
in heart and kidney, compared with free DOX. The biodistri-
bution of DOX was further semiquantitatively analyzed. The
results are shown in Figure 12b. Compared with free DOX
treatment, the fluorescence intensity in leg bone tumor tis-
sue was significantly greater and the fluorescence intensity in
heart and kidney was obviously lower after ALN-MA-hyd-DOX
was intravenously administered to tumor-bearing mice. After
ALN-MA-hyd-DOX was intravenously administered to tumor-
bearing mice, the strongest fluorescence intensity was found in
leg bone tumor tissue; the fluorescence intensity decreased in
following order: liver, kidney, lung, heart, and spleen.
The Toxicity of ALN-MA-hyd-DOX in Heart and Kidney
The representative H&E staining sections of heart and kid-
ney of tumor-bearing nude mice with different treatments
are shown in Figure 13. Hearts from DOX-treated nude
mice showed characteristic cardiotoxic lesions, including mild
to moderate multifocal cardiomyocyte degeneration, vacuola-
tion, interstitial edema, and mild inflammatory cell infiltrates.
Hearts section from ALN-MA-hyd-DOX-treated mice appeared
similar to those of normal saline-treated tumor-bearing mice.
Compared with nude mice treated with normal saline, mice
treated with free DOX caused significant kidney tubules patho-
logical changes, including hyaline cast of kidney tubules. When
tumor-bearing nude mice were treated with ALN-MA-hyd-
DOX, the nephrotoxicity decreased as compared with the same
dose of free DOX. These results indicated that ALN-MA-hyd-
DOX treatment induced less cardiac and renal toxicity than
free DOX.
Alendronate-monoethyl
adipate-(hydrazone)-doxorubicin
conjugate showed much higher cytotoxicity on A549 cells and
MDA-MB-231/ADR cells than that of ALN-MA-ami-DOX.
ALN-MA-hyd-DOX showed the same cytotoxicity as free DOX
did on A549 cells in 24 h. However, ALN-MA-hyd-DOX exhib-
ited much higher cytotoxicity on A549 cells than free DOX
did in 48 h. This was because when ALN-MA-hyd-DOX was
uptaken by the tumor cell, the hydrazone bond was broken,
which resulted in the release of DOX and ALN-MA in the tu-
mor cell. It is reported that ALN inhibits farnesyl diphosphate
DISCUSSION
Bone is one of the most common sites of metastasis in several synthase, and subsequently inhibits protein prenylation of
Zoledronate,
epithelial tumors, such as breast tumor and prostate tumor.28 tumor cells, which leads to apoptosis of the cell.36
However, treatment of bone metastatic tumor by radiation or a new generation of BPs, can work synergistically with
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Figure 11. The in vivo antitumor activity of ALN-MA-hyd-DOX on tumor-bearing nude mice. (a) Tumor volume changes in tumor-bearing nude
mice. (b) Body weight changes in tumor-bearing nude mice. (c) Survival curve of tumor-bearing mice. (d) Tumor-bearing nude mice recorded by
camera at the end of the treatment. Female athymic nude mice were injected via intratibia with A549 cells. Treatment was initiated on the
10th day after tumor cell inoculation. Data are presented as the mean standard deviation. *p < 0.05 versus control group at the end of the
experiment (n = 3). #p < 0.05 versus DOX-treated group at the end of the experiment (n = 3).
DOX and decrease tumor growth in vivo in multiple tumor DOX is a substrate of P-glycoprotein, the structure of ALN-
types.37,38
MA-hyd-DOX and ALN-MA-ami-DOX is different from DOX.
The cellular uptake was observed by fluorescence mi- So, either ALN-MA-hyd-DOX or ALN-MA-ami-DOX is proba-
croscopy. After A549 cells were treated with free DOX, the bly not a suitable substrate of P-glycoprotein. Furthermore, it
fluorescence mainly localized in nucleus. When A549 cells were was reported that ALN-modified drug delivery system had a po-
treated with ALN-MA-ami-DOX, fluorescence was mainly local- tential of inhibiting P-glycoprotein by affecting ATPase activity
ized in the cytoplasm, which resulted in the lower antitumor ac- and MDR1 gene expression.41 Thus, ALN-MA-hyd-DOX and
tivity of ALN-MA-ami-DOX on A549 cells. However, when A549 ALN-MA-ami-DOX probably bypass P-glycoprotein-mediated
cells were treated with ALN-MA-hyd-DOX, the fluorescence lo- drug efflux, and efficiently accumulate in drug-resistant cells.
calized both in cytoplasm and nucleus. Previous studies demon- The exact mechanism that the cellular uptake of ALN-MA-hyd-
strated that cellular uptake of BP drugs required fluid-phase DOX increased compared with free DOX in drug-resistant cell
endocytosis, and BP usually localized in endolysosome.39 Thus, needs further investigation.
the hydrazone bond of ALN-MA-hyd-DOX was broken in en-
Although neither DOX nor ALN-MA-hyd-DOX could com-
dolysosome, and DOX was released and diffused to cytoplasma pletely stop the growth of tumor, the tumor growth in
and nucleus to exhibit its antitumor activity. When MDA-MB- drug-treated groups was obviously delayed. Compared with
231/ADR cells were treated with free DOX, little amount of DOX, ALN-MA-hyd-DOX exhibited stronger antitumor activ-
the fluorescence was found in the nucleus and cytoplasm. Com- ity, longer life span, and less toxicity on heart and kidney. This
pared with the free DOX, when MDA-MB-231/ADR cells were was because after ALN-MA-hyd-DOX was intravenously ad-
incubated with ALN-MA-hyd-DOX and ALN-MA-ami-DOX, a ministered to tumor-bearing mice, DOX was mainly distributed
large amount of the fluorescence was found in the nucleus and in leg bone tumor tissue; a little amount of DOX was distributed
cytoplasm, which resulted in the higher cytotoxicity of ALN- in heart and kidney. However, after free DOX was adminis-
MA-hyd-DOX and ALN-MA-ami-DOX than that of free DOX tered to tumor-bearing mice, besides distributed in leg bone
on MDA-MB-231/ADR cells. P-glycoprotein is an important pro- tumor tissue, some amount of DOX was distributed in heart
tein of the cell membrane that pumps many foreign substances and kidney, which resulted in significant damage in heart and
out of tumor cells and leads to drug resistance.40 Although kidney.
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Figure 13. The representative H&E staining sections of heart and
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CONCLUSION
Alendronate-monoethyl adipate-(hydrazone)-doxorubicin con-
jugate showed high adsorption capacity with HA and natural
bone. Natural bone immobilized ALN-MA-hyd-DOX released
DOX in a pH-dependent manner. ALN-MA-hyd-DOX induced
more apoptosis and showed high cytotoxicity on wild-type tu-
mor cells and DOX-resistant tumor cells. Compared with the
same dose of free DOX, ALN-MA-hyd-DOX significantly de-
layed tumor growth in tumor-bearing nude mice without obvi-
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ACKNOWLEDGMENT
This research was supported by the Shaanxi Science and
Technology Innovation Project (2012KTCL03-18).
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