Synthesis of a new pH-sensitive folate-doxorubicin conjugate and its antitumor activity in vitro

Article abstract of DOI:10.1002/jps.23381

Folate-aminocaproic acid-doxorubicin (FA-AMA-DOX) was synthesized and characterized by H NMR spectroscopy and mass spectrometry. Cytotoxicity and cellular uptake experiments were performed in KB and HepG2 cells, which express folic acid receptor, and the cell line A549, which does not express folic acid receptor. Cytotoxicity was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, and cellular uptake was monitored using fluorescence microscopy. The amount of DOX released from FA-AMA-DOX was much greater at pH 5.0 than that at pH 6.5 or 7.4. The cytotoxicity of FA-AMA-DOX toward KB and HepG2 cells was greater than that of DOX or AMA-DOX at the same concentrations, and cytotoxicity could be attenuated by FA in a dose-dependent manner. On the contrary, the cytotoxicity of FA-AMA-DOX and AMA-DOX toward A549 cells was lower than that of DOX at the same concentration, and cytotoxicity could not be reduced by FA. Compared with FA-AMA, FA-AMA-DOX increased the intracellular accumulation of DOX in KB cells. These results suggested that FA-AMA-DOX have suitable attributes for the active targeting of folate-receptor-positive tumor cells and for releasing the chemotherapeutic agent, DOX, in situ; it therefore has potential as a novel cancer therapeutic.

Full text of DOI:10.1002/jps.23381

Synthesis of a New pH-Sensitive Folate–Doxorubicin Conjugate
and its Antitumor Activity In Vitro
WEI-LIANG YE,1 ZENG-HUI TENG,1 DAO-ZHOU LIU,1 HAN CUI,1 MIAO LIU,1 YING CHENG,1 TIE-HONG YANG,2
QI-BING MEI,2 SI-YUAN ZHOU1
1Department of Pharmaceutics, School of Pharmacy, Fourth Military Medical University, Xi’an 710032, China
2National Key Lab of Gastrointestinal Pharmacology of Chinese Medicine, School of Pharmacy, Fourth Military Medical University,
Xi’an 710032, China
Received 1 August 2012; revised 11 October 2012; accepted 1 November 2012
Published online 20 November 2012 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/jps.23381
ABSTRACT: Folate–aminocaproic acid–doxorubicin (FA–AMA–DOX) was synthesized and
characterized by H NMR spectroscopy and mass spectrometry. Cytotoxicity and cellular uptake
experiments were performed in KB and HepG2 cells, which express folic acid receptor, and the
cell line A549, which does not express folic acid receptor. Cytotoxicity was determined using the
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, and cellular uptake was
monitored using fluorescence microscopy. The amount of DOX released from FA–AMA–DOX
was much greater at pH 5.0 than that at pH 6.5 or 7.4. The cytotoxicity of FA–AMA–DOX toward
KB and HepG2 cells was greater than that of DOX or AMA–DOX at the same concentrations,
and cytotoxicity could be attenuated by FA in a dose-dependent manner. On the contrary,
the cytotoxicity of FA–AMA–DOX and AMA–DOX toward A549 cells was lower than that of
DOX at the same concentration, and cytotoxicity could not be reduced by FA. Compared with
FA–AMA, FA–AMA–DOX increased the intracellular accumulation of DOX in KB cells. These
results suggested that FA–AMA–DOX have suitable attributes for the active targeting of folate-
receptor-positive tumor cells and for releasing the chemotherapeutic agent, DOX, in situ; it
therefore has potential as a novel cancer therapeutic. © 2012 Wiley Periodicals, Inc. and the
American Pharmacists Association J Pharm Sci 102:530–540, 2013
Keywords: folic acid; doxorubicin; folate receptor; drug delivery systems; controlled release;
conjugation; drug targeting; in vitro; cancer
Gonadotropin-releasing
hormone
receptors
INTRODUCTION
(GnRH-Rs) were found to be highly expressed
on various tumor types,4 GnRH derivatives can
serve as targeting moieties for the specific delivery
of chemotherapeutic agents. The first anticancer
drug–GnRH derivative bioconjugates were developed
by Schally AV’s group, among them AEZS-108 (for-
merly known as AN-152) consisting of the GnRH-I
derivative [D-6Lys]–GnRH-I as a targeting moiety
and DOX as an anticancer drug.5 This hybrid com-
pound linked [D-Lys6]–GnRH-I with DOX through
an ester bond via a glutaric acid linker [ester
DOX–(D-Lys6)–GnRH-I]. AEZS-108 was shown to
deliver DOX selectively to cancer cells, thereby
overcoming the drug resistance.6 Preclinical studies
demonstrate that the uptake of AEZS-108 is achieved
by receptor-mediated endocytosis. Results of Phase
I and II clinical trials in patients with gynecological
cancers demonstrated anticancer activity without
cardiotoxicity even in high-dose-treated patients.7
Doxorubicin (DOX) is a highly potent chemotherapeu-
tic agent, which is widely used to treat various types of
cancers, including breast and urothelial cancer. How-
ever, its severe side effects, such as cardiotoxicity, re-
main as a major problem.1,2 To improve the efficacy
of DOX, targeted therapies that maximize therapeu-
tic value while minimizing side effects, using different
drug delivery systems, have caught much attention. A
mechanism for tumor-specific drug delivery has been
determined based on current understanding of the
typical tumor microenvironment.3
Correspondence to: Si-Yuan Zhou (Telephone: +86-29-84776783;
Fax: +86-29-84776783; E-mail: zhousy@fmmu.edu.cn)
Wei-Liang Ye and Zeng-Hui Teng contributed equally to this
work.
Journal of Pharmaceutical Sciences, Vol. 102, 530–540 (2013)
© 2012 Wiley Periodicals, Inc. and the American Pharmacists Association
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531
However, the application of GnRH-I-based biocon-
jugates is limited because of their endocrine side
effects, which might cause an initial aggravation of
the disease.8
a short period. To our knowledge, this is the first
attempt to prepare a folate-based small molecular
tumor-specific drug delivery system with both active
targeting and a pH-sensitive covalently conjugated
anticancer drug.
The folate receptor (FR), a 38-kDa glycosyl–phos-
phatidylinositol membrane anchored glycoprotein
that is overexpressed on various human cancers, in-
cluding those of the ovary, kidney, uterus, testes,
brain, colon, lung, and in hematopoietic cells of myel-
ogenous origin9; however, FR is present at low levels
in most normal tissues.10 Folic acid (FA) binds to the
FR with high affinity (Kd = 0.1 nmol/L) and gets in-
ternalized via receptor-mediated endocytosis. Folate
is an attractive ligand because of its low immuno-
genicity, ease of modification, and low cost. For these
reasons, more and more folate conjugations have
been developed and used in tumor treatment, such
as biodegradable polymers drug carrier,11 liposomal
drug carriers,12 nanoparticals,13 and gene vectors.14
However, there is no report about folate-based small
molecular drug delivery system for DOX. Actually,
like AEZS-108, small molecular drug delivery system
is easier to prepare in large scale and control quality.
Compared with polymeric or nanopartical drug deliv-
ery system, small molecular drug delivery system is
better in drug-like properties.
MATERIALS AND METHODS
Materials
Folic acid, N-hydroxysuccinimide (NHS), di-
cyclohexylcarbodiimide
(DCC),
trifluoroacetic
acid, AMA, and 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide (MTT) were purchased
from Sigma–Aldrich (St. Louis, Missouri) and used
without further purification. DOX was purchased
from Hisun Pharmaceutical Company (Zhejiang,
China). The human squamous carcinoma cell line
(KB cells), the human liver hepatocellular carcinoma
cell line (HepG2 cells; both KB and HepG2 cells
overexpress FR), and the FR-deficient human lung
adenocarcinoma epithelial cell line (A549 cells)
were obtained from the Institute of Biochemistry
and Cell Biology, Chinese Academy of Science,
Shanghai, China. The medium Roswell Park Memo-
rial Institute-1640 (RPMI-1640), without FA, was
purchased from Invitrogen (Carlsbad, California).
It has been illustrated that the intracellular drug
release from the bioconjugates is required for their an-
titumor activity. Lysosomes, intracellular organelles
that have an internal acidic pH (pH < 5.0) and con-
tain a wide variety of hydrolytic enzymes, play a
crucial role in the intracellular drug release. Vari-
ous linkers have been developed and employed in the
preparation of drug delivery systems for targeted can-
cer chemotherapy. The most frequently used linkers
are (1) hydrazone linker, which can be cleaved under
acidic environment in lysosomes; (2) disulfide linkers
cleavable inside the tumor cells through disulfide ex-
change with thiol molecular, such as glutathione; (3)
peptide linkers designed for high stability in blood
and enzymatic hydrolysis in lysosomes.15
On the basis of these concepts, for the first time,
we designed and synthesized a new small molecular
FA-mediated targeted delivery system for DOX, us-
ing aminocaproic acid (AMA) as the linker. AMA was
conjugated to the targeting moiety FA via an amide
bond, and to the antitumor drug DOX via a hydrazone
bond, to produce the drug delivery molecule FA–A-
MA–DOX. Owing to the presence of the FA moiety,
it is anticipated that FA–AMA–DOX would preferen-
tially be internalized by tumor cells, largely via FR-
mediated endocytosis. Because the hydrazone linkage
between DOX and FA–AMA is acid labile, the release
rate of DOX would increase in the acidic environment
of the lysosomes following internalization by tumor
cells via folate-mediated endocytosis, thereby deliver-
ing a substantial amount of DOX to tumor cells in
Methods
Synthesis of FA–AMA–DOX
The synthesis of FA–AMA–DOX is shown schemati-
cally in Figure 1. First, AMA acetate was synthesized
by adding 25 mL fresh SOCl2, dropwise, to 150 mL
ethanol in a reaction vessel placed in an ice/salt bath.
The reaction was allowed to proceed for 10 h with
stirring. Next, 13.1 g AMA was added, and the re-
action mixture was heated and held at reflux for 7 h.
The SOCl2 and ethanol were then removed by rotary
evaporation, and the resulting powder was recrystal-
lized using a 5:1 (v/v) mixture of methanol–petroleum
ether.
To synthesize FA–AMA acetate, 882 mg FA was
dissolved in 10 mL dimethyl sulfoxide (DMSO), and
then 345 mg NHS, 618 mg DCC, and 120 :L triethy-
lamine were added. The reaction was allowed to pro-
ceed at room temperature for 6 h, with stirring, and
then 715 mg AMA acetate was added. The reaction
mixture was stirred for an additional 12 h. The 1,3-
dicyclohexylurea in the mixture was removed by fil-
tration and discarded. A 1:1 (v/v) mixture of ace-
tone–diethyl ether was added to the filtrate, and the
resulting precipitate was filtered and rinsed three
times with diethyl ether, then purified by flash chro-
matography on silica ge1.
In the next step, FA–AMA hydrazide was synthe-
sized. Hydrazine hydrate solution (4 mL) was added
to a solution of 400 mg of FA–AMA acetate dissolved
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Figure 1. Schematic representation of the synthesis of FA–AMA–DOX.
in 5 mL DMSO. The reaction mixture was stirred at
50◦C for 6 h. After cooling, 10 mL ethanol was added
to the reaction vessel, and the resulting precipitate
was filtered and rinsed three times with diethyl ether,
then purified by flash chromatography on silica ge1.
Finally, FA–AMA–DOX was synthesized: 230 mg
FA–AMA hydrazide was dissolved in 8 mL DMSO,
and then 75 mg of DOX and 60 :L of trifluoroacetic
acid were added. The reaction was allowed to pro-
ceed for 12 h at room temperature. Acetone–diethyl
ether (1:1, v/v) was added to the reaction mix-
ture, and the resulting precipitate was filtered and
rinsed three times with diethyl ether, then purified
by flash chromatography on silica gel as described
above.
Aminocaproic acid–DOX was also synthesized for
use as a control. AMA hydrazide (80 mg) was dis-
solved in 15 mL dichloromethane, and then 80 mg of
DOX and 60 :L of trifluoroacetic acid were added.
The reaction was stirred for 12 h at room temper-
ature. The dichloromethane was removed by rotary
evaporation, and the resulting powder was purified
by flash chromatography on silica ge1 as described
above.
Release of DOX from FA–AMA–DOX
Drug-release studies were performed at 37◦C using
solutions of sodium acetate buffer (pH 5.0 and 6.5)
and sodium phosphate buffer (pH 7.4) as the re-
lease buffers. FA–AMA–DOX (2 mg) was dissolved
in 20 mL of each of the three release solutions, and
the mixtures were incubated at 37◦C on a horizontal
shaker (100 rpm). Samples (50 :L) were taken at se-
lected incubation times and were replaced with the
same volume of fresh buffer. The quantity of released
DOX was determined by using high-performance liq-
uid chromatography–electrospray ionization–tandem
mass spectrometry (HPLC–ESI–MS/MS; Waters Cor-
poration, Milford, Massachusetts). The HPLC col-
umn used was a C18 analytical column (5 :m, 2.1 ×
150 mm2 XTerra; Waters Corporation). The column
was operated at a flow rate of 0.2 mL/min with mobile
phase of 70% aqueous acetonitrile containing 0.1%
acetic acid.
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coverglass. After 24 h, 20 :mol/L free DOX, AMA–
DOX, or FA–AMA–DOX (measured as free DOX
equivalent) was added, and the cells were incu-
bated for 4 h at 37◦C. The drugs were dissolved in
serum-free medium containing less than 0.1% DMSO.
After drug treatment, the medium was discarded
and cells were rinsed three times with PBS, then
stained with 1 :g/mL 4ꢀ,6-diamidino-2-phenylindole
for 10 min. Cells were finally rinsed three times with
PBS and fixed with 4% paraformaldehyde for 10 min.
Intrinsic fluorescence of DOX was observed using the
fluorescence microscope.
Cytotoxicity of FA–AMA–DOX
The cytotoxicity of FA–AMA–DOX to KB, HepG2,
and A549 cells was assessed using the MTT assay.
Cells were cultured in FA-free RPMI medium supple-
mented with 10% fetal bovine serum, 100 U/mL peni-
cillin/streptomycin at 37◦C under 5% CO2. The cells
were cultured and lifted before seeding into 96-well
plates (1 × 104 cells/well) and incubating for 24 h. The
medium then was replaced with fresh medium con-
taining serially diluted free DOX, FA–AMA–DOX, or
AMA–DOX, and plates were incubated for 24 h. The
wells were then washed three times with Phosphate
Buffered Saline (PBS) and incubated again for a fur-
ther 4 h with FA-free RPMI containing 5 mg/mL of
MTT. After discarding the culture medium, 150 :L of
DMSO was added to dissolve the precipitate, and the
absorbance at 570 nm of the resulting solutions was
measured using a CODA Automated EIA Analyzer
(Bio-Rad Laboratories, Hercules, California). Inhibi-
tion ratio was calculated as following formula16:
Statistical Methods
The statistical analysis of the data was analyzed by
Student t-test or ANOVA test with a p value of less
than 0.05.
RESULTS
(1 − absorbance of drug treated group/absorbance
Identification of FA–AMA–DOX
of control group) × 100%
The conjugate was characterized by mass spectrum
1
and H NMR spectroscopy, the results are shown in
Cellular Uptake of FA–AMA–DOX
Figures 2 and 3. Folate has "- and (-carboxylic acid
and both of them might be activated when using the
DCC/NHS as a catalyst. However, it is known that
the (-carboxylic acid was primarily conjugated be-
cause of its higher reactivity.17,18 Additionally, when
DOX was conjugated to FA–AMA by using the DCC/
NHS, unreacted "-carboxylic acid in FA might be ac-
tivated, producing DOX–FA–AMA as a by-product.
Cellular uptake of free DOX and FA–AMA–DOX
was investigated using a DS-5M-ui80i fluorescence
microscope (Nikon Corporation, Tokyo, Japan) to
observe the effect of the designed delivery sys-
tem on the intracellular distribution of DOX. Cells
(KB, HepG2, A549) were seeded into 24-well plates
(1 × 105 cells/mL), with each well containing a
Figure 2. Mass spectrum of FA–AMA–DOX.
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Figure 3. The 1H NMR spectrum of FA-AMA-DOX.
However, the reversed-phase HPLC result showed a
single peak, suggesting that the conjugate was homo-
geneous without producing undesirable side products.
Using ESI–MS/MS operating in negative ion mode,
[M-H]−, the molecular ion peak of FA–AMA–DOX was
observed at m/z = 1095, as predicted. The 500 MHz
1H NMR spectrum of FA–AMA–DOX confirmed the
presence of the AMA moiety (signals at 2.9, 2.5, 1.5,
and 1.3 ppm) and the FA moiety (signals at 8.8, 8.6,
7.6, 6.9, 6.6, 4.5, 4.3, 2.3, and 1.9 ppm). Conjugation of
DOX was confirmed by the presence of characteristic
DOX signals at 8.0, 7.8, 7.5, 4.2, 2.1, and 1.2 ppm.
Figure 4. The release profile of DOX from FA–AMA–DOX
in different media.
In Vitro Release of DOX from FA–AMA–DOX
Cytotoxicity of FA–AMA–DOX
The release of DOX from FA–AMA–DOX was investi-
gated under pseudophysiological conditions (PBS, pH
7.4) and also in acidic environments (acetate buffer,
pH 5.0 and pH 6.5) at 37◦C. As shown in Figure 4, the
release rate of DOX from FA–AMA–DOX was strongly
dependent on the pH of the medium. FA–AMA–DOX
showed a higher release rate of DOX at pH 5.0 than
that at pH 6.5 or pH 7.4. At pH 5.0, FA–AMA–DOX
released approximately 85% of the conjugated DOX
in 2 h, whereas at pH 6.6 and 7.4, FA–AMA–DOX re-
leased only 18% and 15% of the conjugated DOX in
2 h, respectively. These results showed that the re-
lease of DOX from FA–AMA–DOX was relied on the
acid-labile hydrazone linkage between DOX and the
folate moiety. Thus, DOX is released from FA–AMA–
DOX by hydrolysis of the hydrazone bond at the 13-
keto position of DOX.
The cytotoxic effects of free DOX, AMA–DOX, or
FA–AMA–DOX against the FR-positive cell lines (KB
and HepG2 cells) and the FR-negative line (A549
cells) were evaluated using the MTT assay. The re-
sults are shown in Figure 5. Cells were treated with
equimolar quantities of free DOX, AMA–DOX, or
FA–AMA–DOX. The cytotoxicity toward A549 cells
was much higher for free DOX than for AMA–DOX
or FA–AMA–DOX (Fig. 5c); the relatively faster dif-
fusion of free DOX into the cell nucleus could be the
reason for this. There was no significant difference
in viability between AMA–DOX- or FA–AMA–DOX-
treated A549 cells, indicating that the FA moiety in
FA–AMA–DOX had no influence on the cytotoxicity
in A549 cells. The cytotoxicity of free DOX, AMA–
DOX, or FA–AMA–DOX toward KB cells showed that
FA–AMA–DOX and DOX were more cytotoxic than
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Figure 5. The in vitro cytotoxicity of DOX, AMA–DOX,
and FA–AMA–DOX toward KB cells (a), HepG2 cells (b),
and A549 cells (c) after 24 h.
AMA–DOX (Fig. 5a). The cytotoxic effect of free DOX,
AMA–DOX, or FA–AMA–DOX toward HepG2 cells is
shown in Fig. 5b, the result of which was similar to
that in KB cells. These results suggested that the FA-
targeting ligand present in FA–AMA–DOX played an
important role in enhancing the cytotoxic effect by
binding FA–AMA–DOX to the overexpressed FR lo-
cated on the surface of KB cells, thereby increasing
the intracellular uptake of FA–AMA–DOX via FR-
mediated endocytosis. Hydrolysis of the acid-labile
AMA–DOX hydrazone bond results in the release of
free DOX and a greater accumulation of DOX in tu-
mor cells compared with treatment with free DOX
alone.
Figure 6. The effects of free FA on the cytotoxicity of
FA–AMA–DOX toward KB cells (a), HepG2 cells (b), and
A549 cells (c). Statistically significant decreases in inhibi-
tion ratios were detected (P < 0.05) when KB and HepG2
cells were incubated with FA–AMA–DOX and different con-
centrations of FA, compared with FA-free medium.
The presence of free FA in the medium is antic-
ipated to compete with FA–AMA–DOX in binding
to cell-surface FRs. To test the effect of exogenously
added free FA on the cytotoxicity of FA–AMA–DOX,
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KB, HepG2, and A549 cells were incubated with
FA–AMA–DOX (at a DOX-equivalent concentration
of 30 :g/mL) in medium containing various concen-
trations of FA. The results are shown in Figure 6.
The viability of A549 cells in the presence of FA–
AMA–DOX was unchanged when free FA was added
(Fig. 6c), whereas that of KB and HepG2 cells were in-
creased in a dose-dependent manner. This indicated
that the cytotoxicity of FA–AMA–DOX against KB
and HepG2 cells was inhibited by excess free FA
(Figs. 6a and Figs. 6b) and confirmed that free FA
inhibits cellular uptake of FA–AMA–DOX by compet-
itively binding to FRs on the cancer cell surface.
than in the cytoplasm for FA–AMA–DOX-treated KB
and HepG2 cells, whereas AMA–DOX-treated KB
cells had low DOX fluorescence in the nucleus, being
mostly localized in cytosolic regions. For A549 cells
treated with FA–AMA–DOX or AMA–DOX, DOX flu-
orescence was mostly localized in the cytoplasm. By
using fluorescence microscopy, the effects of exoge-
nous free FA on the cellular uptake of FA–AMA–DOX
were investigated. The results are shown in Figure 8.
The exogenous free FA attenuated the intracellular
DOX fluorescence when the FA–AMA–DOX was incu-
bated with KB and HepG2 cells. These results clearly
indicate that cellular uptake of FA–AMA–DOX by
KB cells is facilitated by active, receptor-mediated
endocytosis.
Cellular Uptake of FA–AMA–DOX
The uptake of FA–AMA–DOX, free DOX, and AMA–
DOX by the FR-positive cell lines (KB and HepG2
cells) and FR-negative cell line (A549 cells) was in-
vestigated using fluorescence microscopy. The results
are shown in Figure 7. Following treatment with free
DOX for 4 h, DOX was predominantly accumulated
in the nucleus of cells from each cell line. The accu-
mulation of FA–AMA–DOX in KB and HepG2 cells,
however, was significantly increased compared with
that of AMA–DOX and free DOX, whereas the up-
take of FA–AMA–DOX and AMA–DOX was signifi-
cantly reduced in A549 cells. In addition, there was
significantly greater DOX fluorescence in the nucleus
DISCUSSION
One of the most important features of drug delivery
systems is the stability of the chemical bond between
drug and targeting moiety during the blood circula-
tion and its susceptibility to acidic environment or
enzymatic hydrolysis upon internalization into the
cancer cells. This can potentially be achieved by
liberating the drug from its conjugate through
pH-controlled hydrolysis.19–21 In our present
study, we have synthesized hydrazone-bond-linked
DOX–folate bioconjugate (FA–AMA–DOX) and
Figure 7. Intracellular localization of DOX and FA–AMA–DOX. DOX (a1), FA–AMA–DOX
(b1), and AMA–DOX (c1) in KB cells (A); DOX (d1), FA–AMA–DOX (e1), and AMA–DOX (f1)
in HepG2 cells (B); DOX (g1), FA–AMA–DOX (h1), and AMA–DOX (i1) in A549 cells (C) at
an equivalent DOX concentration of 50 :mol/L for 4 h at 37◦C. The pink region shows the
localization of DOX (red) in the nucleus (blue). (Continued)
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Figure 7. Continued.
characterized it structurally and biologically. Our
study is largely based on the following advantages of
the hydrazone-linked bioconjugates: (1) easy synthe-
sis because of chemoselectivity without leading to the
loss of the antitumor effect of DOX, (2) high chemical
stability of hydrazone bond containing bioconjugates
in blood, and (3) acidic cleavable of hydrazone bond
in lysosomes.
lease rate of DOX at physiological blood pH (pH 7.4)
greatly reduces the likelihood of premature drug re-
lease during circulation in the bloodstream. Owing to
the presence of the FA moiety, it is anticipated that
FA–AMA–DOX would be preferentially internalized
by tumor cells, largely via FR-mediated endocytosis.
Once inside tumor cells, the acidic endosomal or lyso-
somal compartments would cause DOX to be cleaved
from the conjugate, leading to a burst release of DOX,
which would subsequently diffuse into the cytosol via
the proton sponge effect and also into the nucleus.22,23
Strong pH-dependent release of DOX in FA–
AMA–DOX is a highly desirable characteristic for
a potential cancer therapeutic because the slow re-
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Figure 8. The effects of exogenous free FA on the cellular uptake of FA–AMA–DOX. Panel (A)
shows the fluorescence of KB cells treated by FA–AMA–DOX with folate (k1) and without folate
(j1) in the medium. Panel (B) shows the fluorescence of HepG2 cells treated by FA–AMA–DOX
with folate (m1) and without folate (l1) in the medium. Cells were treated with equivalent DOX
concentration of 50 :mol/L for 4 h at 37◦C. The final concentration of folate is 10 :g/mL. The
pink region shows the localization of DOX (red) in the nucleus (blue).
The higher DOX-release rate observed at pH 5.0,
which is comparable to the physiological pH of en-
docytic compartments of tumor cells, would lead to a
higher level of DOX inside tumor cells, thereby greatly
enhancing the efficacy of such a cancer therapy. Our
results show that the release rate of DOX from FA–
AMA–DOX is strongly dependent on the medium
pH, and FA–AMA–DOX shows a much higher re-
lease rate of DOX at pH 5.0 than that at pH 6.5
and 7.4.
As DOX itself is intrinsically fluorescent, cellu-
lar uptake could be directly measured without ad-
ditional markers; the fluorescence intensity repre-
sents the amount of DOX internalized by cells. The
intense fluorescence arising from DOX accumula-
tion in the nucleus from treatment with free DOX
occurs because intracellular free DOX (in the cy-
tosol) is rapidly transported to the nucleus where it
strongly binds to chromosomal DNA.24 It has been
established that bioconjugates localize in endosomes
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or lysosomes after its uptake is achieved through
receptor-mediated endocytosis.6,25,26 Therefore, when
FA–AMA–DOX was incubated with KB cells, it under-
went FR-mediated endocytosis, the hydrazone bond
between DOX and AMA could be hydrolyzed prob-
ably in lysosomes, releasing a large amount of DOX.
When AMA–DOX was incubated with KB cells, it was
uptaken into the cells and then localized in the cyto-
plasm, where the pH is approximately 7.4. At this
pH, the hydrazone bond between DOX and AMA is
relatively stable, and only small amounts of DOX
were released into the cytosol with subsequent local-
ization in the nucleus. Compared with AMA–DOX,
FA–AMA–DOX increased the intracellular accumu-
lation of DOX in KB cells. As a result, the cytotoxicity
of FA–AMA–DOX toward KB cells was greater than
that of AMA–DOX. On the contrary, when FA–AMA–
DOX and AMA–DOX were incubated with A549 cells,
the cytotoxicity of FA–AMA–DOX and AMA–DOX to-
ward A549 cells was thus lower than that of DOX
alone. In addition, FA–AMA–DOX is more cytotoxic
toward KB cells than DOX or AMA–DOX at the same
concentration, but its cytotoxicity can be inhibited, in
a dose-dependent manner, by the addition of FA. The
cytotoxicity of FA–AMA–DOX and AMA–DOX toward
A549 cells could not be inhibited by the addition of FA.
This indicates that the uptake of FA–AMA–DOX by
KB cells is mediated by FR. In a word, owing to the
presence of FA, FA–AMA–DOX can deliver DOX di-
rectly to FR-positive cancerous cells, thereby increas-
ing the concentration of the drugs in tumor tissue and
sparing normal, noncancerous cells from unnecessary
exposure.
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The C-13 keto group of DOX can be used for the conju-
gation with targeting moieties without leading to the
loss of the antitumor effect. FA–AMA–DOX appear to
be more efficacious and has suitable attributes for the
active targeting of FR-positive tumor cells and for re-
leasing the chemotherapeutic agent, DOX, in situ; it
therefore has potential as a novel cancer therapeutic.
On the basis of the results obtained in this
study, the in vivo antitumor activity of FA-AMA-
DOX is currently further evaluated on KB cells and
MDA-MB-231 cells bearing mice.
ACKNOWLEDGMENTS
This research was supported by the Shaanxi Science
and Technology Innovation Project (2012KTCL03-18).
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