Characterization, stability, and pharmacokinetics of sibutramine/β

Biomaterials 32 (2011) 8635e8644
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Biomaterials
journal homepage: www.elsevier.com/locate/biomaterials
Intracellular delivery of paclitaxel using oil-free, shell cross-linked
HSA e Multi-armed PEG nanocapsules
Jeong Yu Lee a, *, Ki Hyun Bae a, Jee Seon Kim a, Yoon Sung Nam a, b, Tae Gwan Park a, c, y
a
Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejeon 305-701, Republic of Korea
Department of Materials Science and Engineering, Korea Advanced Institute of Science and Technology, Daejeon 305-701, Republic of Korea
c
Graduate School of Nanoscience and Technology, Korea Advanced Institute of Science and Technology, Daejeon 305-701, Republic of Korea
b
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 24 May 2011
Accepted 20 July 2011
Available online 23 August 2011
Various approaches to increase the solubility of water-insoluble anti-cancer drugs in aqueous formulations have been undertaken with the aim of treating solid tumors through intravenous drug administration. Nanoscale drug carriers are particularly attractive for cancer therapy because of their passive
targeting effect to enhance the therapeutic efficacy of drugs. Here we introduce an oil-free, shell crosslinked nanocapsule as an efficient intracellular delivery system for paclitaxel. The nanocapsules are
prepared by emulsifying amine-reactive six-arm-branched polyethylene glycol (PEG) in dichloromethane
into aqueous solution of human serum albumin (HSA), followed by cross-linking at the organic/aqueous
interface. Paclitaxel is successfully incorporated into the HSA/PEG nanocapsules having a spherical shape
with an average diameter of about 280 nm. In several types of cells, the surface modification of nanocapsules with a cell-penetrating peptide, Hph1, greatly facilitates cellular uptake and apoptosis-inducing
effects of paclitaxel. Furthermore, the targeted anti-tumor activities of the paclitaxel-loaded nanocapsules in a mouse tumor model suggest that the shell cross-linked nanocapsules are very promising
oil-free nanoscale delivery vehicles for water-insoluble anti-cancer agents.
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
Paclitaxel
Drug delivery system
Human serum albumin
Nanocapsule
Cell-penetrating peptide
1. Introduction
Paclitaxel, a well-known water-insoluble anti-cancer drug, has
been formulated using polyethylated castor oil (Cremophor EL) and
ethanol as delivery vehicles to increase its solubility [1,2]. However,
this oil-based formulation often shows acute side effects, such as
hypersensitivity, neurotoxicity, and neuropathy [2e4]. To reduce
these side effects, a new formulation was developed by utilizing the
high affinity of paclitaxel to human serum albumin (HSA) to
produce a nanoscale, physical complex [5]. In 2005, AbraxaneÒ,
containing the paclitaxel/HSA complex, was approved by FDA [6].
To further improve the efficacy of anti-cancer drugs via tumor
targeting, multiple functionalized nano-carrier systems have also
been proposed, including liposomes incorporating drug-polymer
conjugates, lipid nano-emulsions, polymeric micelles, and biodegradable nanoparticles [7e11]. It has been proved that these nanocarriers can increase the drug solubility in aqueous solution,
colloidal stability, and injectability [9e12].
* Corresponding author. Tel.: þ82 42 350 2661; fax: þ82 42 350 2610.
E-mail address: [email protected] (J. Y. Lee).
y
Deceased, April 10, 2011.
0142-9612/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.biomaterials.2011.07.063
Recently we reported a new single oil-in-water (O/W) emulsion
technique to fabricate shell cross-linked nanocapsules encapsulating drugs and inorganic nanocrystals within nano-reservoir
structures [13e15]. An organic phase containing chemically activated multi-arm polyethylene glycol (PEG) or Pluronic copolymers
was emulsified into aqueous solution of cross-linkable, aminecontaining polymers, leading to the formation of O/W emulsion
droplets. As the dispersed organic phase was evaporated, the
polymers were covalently cross-linked with each other at the O/W
interfaces and generated a cross-linked shell nanostructure
[14e16]. Our previous work also demonstrated that this type of
shell cross-linked nanocapsules can be used to encapsulate paclitaxel into the core region using hydrophobic, non-volatile oil (e.g.,
Lipiodol) [14].
In this study, HSA and amine-reactive multi-arm PEG were
employed to fabricate shell cross-linked nanocapsules incorporating paclitaxel with using no oil. Through the strong binding of
paclitaxel to HSA, the HSA/PEG nanocapsules were expected to
effectively encapsulate paclitaxel into the oil-free hydrophobic
interior stabilized by a cross-linked shell layer. Moreover, the drugloaded nanocapsules were conjugated with Hph1, a cell-penetrating peptide derived from a human transcriptional factor, to
promote the intracellular uptake of the nanocapsules and thus
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enhance the drug-mediated cytotoxic effect [17e19]. The therapeutic potentials of the paclitaxel-loaded HSA/PEG nanocapsules
were then evaluated using a mouse tumor model in terms of in vivo
accumulation in tumor sites and tumor volume regression.
(Cy5.5-NHS) was obtained from GE Healthcare (Piscataway, NJ), and Cell-Counting
Kit-8 (CCK-8) from Dojindo laboratories (Kumamoto, Japan). All other chemicals
and reagents were of reagent grade and used without further purification.
2. Experimental section
Paclitaxel-loaded HSA/PEG nanocapsules were synthesized using a modified
emulsification/solvent evaporation method [20]. In brief, 2.5 mg of paclitaxel and
6.8 mg of 6-arm PEG-NHS were first dissolved in 2 mL of dichloromethane. The
mixture was added, in a dropwise manner, to 10 mL HSA solution (1 mg mL1, pH 9)
saturated with dichloromethane, and then sonicated at ambient temperature for
5 min using a Branson SonifierÒ 450 at a frequency of 20 kHz with a duty cycle of 20
and an output control of 3.5. The prepared O/W emulsion was transferred to a rotary
evaporator, and the organic solvent was rapidly evaporated under reduced pressure
at 45 C. The produced HSA/PEG nanocapsules were subjected to dialysis against
deionized water (Mw cutoff of 300 kDa) to remove free drugs, and then stored at 4 C
until use.
2.1. Materials
Paclitaxel (>97%) was obtained from Wako (Osaka, Japan) and used without
further purification. HSA (purity 96%), phosphate-buffered saline (PBS), fluorescamine, 4,6-diamidino-2-phenylindole (DAPI), and propidium iodide (PI) were
purchased from SigmaeAldrich (St. Louis, MO). Maleimide-poly(ethylene glycol)succinimidyl carbonate (MAL-PEG-NHS, Mw ¼ 5 kDa) was obtained from NOF
Corporation (Tokyo, Japan). N-hydroxysuccinimide-functionalized six-armbranched poly(ethylene glycol) (6-arm PEG-NHS, Mw ¼ 15 kDa) was obtained
from Sunbio Inc. (Walnut Creek, CA). A cell-penetrating peptide, Hph1
(CGGYARVRRRGPRR), originated from a human transcriptional factor, HPH1, was
obtained from Peptron Inc. (Daejeon, Korea). Dulbecco’s modified Eagle’s medium
(DMEM), BODIPYÒ 564/570-paclitaxel conjugates, and fetal bovine serum were
purchased from Invitrogen (Carlsbad, CA). Cy5.5-N-hydroxysuccinimide ester
2.2. Preparation of paclitaxel-loaded HSA/PEG nanocapsules
2.3. Conjugation of Hph1 to HSA/PEG nanocapsules
Hph1 was conjugated to the paclitaxel-loaded HSA/PEG nanocapsules using
a hetero-bifunctional cross-linking agent, MAL-PEG-NHS. Hph1 (7 mg, or 200 mmol)
Fig. 1. Schematic illustration of the formation of the paclitaxel-loaded shell cross-linked HSA/PEG nanocapsules decorated with Hph1.
J.Y. Lee et al. / Biomaterials 32 (2011) 8635e8644
with a cysteine-modified N-terminal end was dissolved in anhydrous dimethylformamide (DMF) and then mixed with MAL-PEG-NHS (47 mg, or 5 mmol). The
mixture solution was incubated at ambient temperature for 4 h to allow the
formation of a thioether bond. The synthesized conjugate, Hph1-PEG-NHS, was
added to 5 mL PBS containing the HSA/PEG nanocapsules (2 mg mL1), followed by
incubation at ambient temperature for 1 day. Unreacted materials were removed by
dialysis against deionized water for 1 day (Mw cutoff of 300 kDa). The amount of
conjugated Hph1 was determined using the fluorescamine assay according to the
method reported previously [21].
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Technologies, Palo Alto, CA) equipped with a Waters Spherisorb ODS2 column
(4.6 mm 250 mm). Acetonitrile was used as a mobile phase with a flow rate of
1.0 mL min1. Eluted peaks were monitored at 227 nm. A calibration curve was
obtained using a series of paclitaxel solutions at different concentrations.
2.6. Evaluation of cytotoxic effect of HSA/PEG nanocapsules
The hydrodynamic diameter of the prepared the HSA/PEG nanocapsules was
measured by dynamic light scattering (DLS) (Zeta-Plus, Brookhaven, NY) in triplicate
at a concentration of 1 mg mL1 at 20 C. The size and shape of the nanocapsules were
determined using scanning electron microscopy (SEM) and atomic force microscopy
(AFM). The nanocapsules were freeze-fried, placed on a clean plate, and examined on
a Hitachi S-4800 scanning electron microscope. For AFM analysis, 50 mL of the sample
solution was deposited and dried in air on a fresh mica surface, and then images were
taken on a PSIA XE-100 AFM system. The average particle size was determined by
measuring the diameters of 70 nanocapsules in the SEM and AFM images.
The inhibition of cell growth was examined to evaluate the cytotoxicity of the
paclitaxel-loaded HSA/PEG nanocapsules. Four different human cells were used:
human breast adenocarcinoma (MCF-7), human ovarian carcinoma (OVCAR-3),
human nasopharyngeal epidermal carcinoma (KB), and human coronary artery
smooth muscle cells (hCASMCs). Each type of cells was plated over a 96-well plate at
a density of 1 105 cells per well and grown in DMEM supplemented with 10% fetal
bovine serum for 24 h at 37 C. The nanocapsule solution was diluted using PBS to
a wide range of the paclitaxel concentration (10 ng mL1 w 100 mg mL1). For
comparison, a Taxol formulation was also prepared as reported previously [1]. The
culture medium was replaced with DMEM containing different paclitaxel formulations, and further incubated for 48 h at 37 C. The number of viable cells was
determined by the CCK-8 cell viability assay. Briefly, 10 mL of CCK-8 solution was
added to 100 mL of serum-free DMEM in each well of the plate. After 1 h incubation
at 37 C, the absorbance at 450 nm was measured using a Bio-Rad microplate reader.
2.5. Determination of paclitaxel loaded within HSA/PEG nanocapsules
2.7. Confocal microscopy and cell cycle analysis
In order to determine the loading amount of paclitaxel within the HSA/PEG
nanocapsules, the nanocapsules were freeze-dried and re-dispersed in acetonitrile
with shaking for 12 h to extract paclitaxel. After filtration through a 0.45 mm
cellulose filter, the amount of paclitaxel in the filtrate was analyzed using reversephase high-performance liquid chromatography (HPLC 1100 series, Agilent
To visualize the intracellular uptake of paclitaxel, fluorescent BODIPYÒ 564/570paclitaxel conjugates were incorporated into the HSA/PEG nanocapsules. MCF-7
cells were seeded in a chamber slide (4-well CultureSlides, BD Falcon, MA) at
a density of 1 105 cells per well and grown in DMEM supplemented with 10% fetal
bovine serum for 24 h at 37 C. The cells were then incubated with the HSA/PEG
2.4. Characterization of HSA/PEG nanocapsules
Fig. 2. (A) Hydrodynamic diameter of the paclitaxel-loaded HSA/PEG nanocapsules. (B) Time course changes of the hydrodynamic diameter of the paclitaxel-loaded HSA/PEG
nanocapsules and paclitaxel/HSA mixture. The inset photographs show the dispersion of the paclitaxel-loaded HSA/PEG nanocapsules after incubation in PBS for 3 h (left) and 30
days (right). The concentration of the nanocapsule dispersion was 1 mg mL1. SEM (C) and AFM (D) images of the paclitaxel-loaded HSA/PEG nanocapsules.
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nanocapsules containing the fluorescently-labeled paclitaxel (1 mg mL1) at 37 C
for 48 h. The cells were washed with PBS and fixed with 1 wt-% formaldehyde. After
washing with PBS, the cell nuclei were stained with DAPI (1.5 mg mL1 in PBS) for
10 min. The cells were examined on a LSM510 confocal laser scanning microscope
(Carl Zeiss, Germany). For cell cycle analysis, the fixed cells were dehydrated by
slowly adding ethanol to 70%, stored at 4 C overnight, and then incubated with PI
staining solution (0.25 mg mL1 PI and 0.1 mg mL1 RNase A in PBS) for 30 min at
37 C. The PI fluorescence of each nucleus was determined using a FACSCalibur flow
cytometer (BD Biosciences) and CellQuest software (PharMingen).
2.8. In vivo tumor accumulation, biodistribution, and anti-tumor effect
Male BALB/C nude mice (7e8 weeks of age, about 20 g) were housed in
a pathogen-free environment at 4e5 mice/cage. They were supplied with autoclaved and non-fluorescent mouse chow and water. All animal experiments were
conducted in accordance with the guidelines provided by Institutional Animal Care
and Use committee of KAIST. The mouse tumor model was developed by injecting
100 mL of MCF-7 cell suspension (3 106 cells) into the subcutaneous region of each
mouse. Tumor growth was monitored daily until its volume reached to 100 mm3. For
in vivo optical imaging, the paclitaxel-loaded HSA/PEG nanocapsules were labeled
with a near-infrared fluorescent dye, Cy5.5-NHS. In brief, 10 mM of Cy5.5-NHS was
reacted for 5 h with the nanocapsule solution (1 mg mL1) in PBS (pH 7.4). Cy5.5labeled nanocapsules were purified by dialysis (Mw cutoff ¼ 300 kDa). Each
mouse was intravenously (i.v.) injected with 200 mL of the fluorescently labeled
nanocapsules at a given dose of paclitaxel (5 mg kg1 body weight). PBS was used for
the control group of mice. At predetermined time intervals, fluorescence images of
each mouse were captured with the IVISÒ Lumina imaging system (Caliper Life
Sciences, Hopkinton, MA) in a sequential acquisition mode. The fluorescence
intensity was calculated from the obtained fluorescence signals using the software
(Living ImageÒ Software) provided by the manufacturer. A digital caliper was used to
determine the perpendicular diameter of the tumor. The respective tumor volume
was calculated from the following formula: tumor volume ¼ 0.5 major
axis2 minor axis. After 2 weeks, the mice were sacrificed and dissected to obtain
the ex vivo fluorescence images of their organs and tumor. Excised tissues were also
histologically examined to determine the tumor accumulation of the nanocapsules.
Tissue samples were fixed using 4 wt-% formaldehyde in PBS (pH 7.4), embedded in
a paraffin block, and sectioned into 10-mm-thick slices. The sections were co-stained
with terminal deoxynucleotidyl transferase-mediated 20 -deoxyuridine 50 -triphosphate-biotin nick end labeling (TUNEL) and with hematoxylin and eosin (H&E).
Images were then taken on a Nikon TE300 inverted microscope equipped with
a digital microscope camera (Polaroid DMC2, USA).
2.9. Statistical analysis
Statistical analysis was performed using a standard Student’s t-test with
a minimum confidence level of 0.05 for significant statistical difference. All experiments were performed over triplicate.
3. Results and discussion
HSA has multiple hydrophobic binding sites that can strongly
bind to a variety of molecules, including unesterified fatty acids,
bile acids, and water-insoluble drugs (e.g., paclitaxel, ketoprofen,
doxorubicin, and rapamycin) [5,22e24]. The number of drugs that
bind to each HSA depends on the molecular structure of the drug
[25]. Paclitaxel is known to have seven binding sites in a single HSA
[26]. Accordingly, it was conceivable that the nanocapsules
composed of HSA and PEG could incorporate paclitaxel into the
hydrophobic interior of nanocapsules through the HSA-paclitaxel
interactions. Fig. 1 illustrates the preparation procedures of the
paclitaxel-loaded HSA/PEG nanocapsules. Paclitaxel and NHSfunctionalized six-arm-branched PEG were co-dissolved in
dichloromethane, transferred to aqueous HSA solution, and
immediately ultrasonicated to prepare nano-sized O/W emulsions.
During this process, the terminal NHS groups of the branched PEG
chains were covalently conjugated to the primary amines of HSA at
the interface, leading to the formation of a cross-linked shell layer
comprising PEG and HSA [20]. Stable paclitaxel-loaded HSA/PEG
nanocapsules were successfully produced as dichloromethane was
evaporated under reduced pressure at 45 C, which is below lower
than the critical temperature above which HSA is irreversibly
denatured [27]. The prepared paclitaxel-loaded HSA/PEG nanocapsules had an average hydrodynamic diameter of 280.8 9.8 nm
Fig. 3. Cytotoxicity profiles of the paclitaxel-loaded HSA/PEG nanocapsules (black bar),
the Hph1-decorated HSA/PEG nanocapsules (Gy bar), and the Taxol formulation
(dotted bar) against MCF-7 cells (A) and hCASMCs (B) after incubation for 48 h. (C)
Cytotoxicities of different paclitaxel formulations against MCF-7 cells, OVCAR-3 cells,
KB cells, and hCASMCs at an equivalent drug concentration of 100 mg mL1. Statistically
significant difference between two groups, *p < 0.01 (n ¼ 3).
J.Y. Lee et al. / Biomaterials 32 (2011) 8635e8644
with a narrow size distribution, as determined by DLS (Fig. 2A).
Blank nanocapsules account for the smaller peak around 100 nm
because paclitaxel-free HSA/PEG nanocapsules prepared as a separate batch had the similar average diameter, 118.5 19.6 nm. HPLC
analysis showed that approximately 9.58 wt-% of paclitaxel was
incorporated into the nanocapsules. As a control sample, paclitaxel/
HSA complexes were prepared without NHS-functionalized sixarm-branched PEG at a drug concentration of 12.95 wt-%; however,
the complex did not exhibit good colloidal stability in aqueous
solution. After 3-h incubation at ambient temperature, the hydrodynamic diameter of the prepared paclitaxel/HSA complex
increased from ca. 400 nm to ca. 1.2 mm (Fig. 2B). It seems likely that
the large aggregates were generated by unbound, hydrophobic
paclitaxel molecules that can serve as cross-linking agents between
the paclitaxel/HSA complexes. By contrast, the paclitaxel-loaded
HSA/PEG nanocapsules showed no significant change in the size
distribution for 30 days at ambient temperature (Fig. 2B). This
result demonstrates the excellent structural stability of the HSA/
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PEG nanocapsules for paclitaxel encapsulation. In order to facilitate
the intracellular translocation of the HSA/PEG nanocapsules, a cellpenetrating peptide, Hph1, was conjugated to the nanocapsules
using a flexible PEG linker (Mw ¼ 5 kDa) [28]. The Hph1 conjugation increased the size of nanocapsules to 385.7 23.3 nm in
diameter, as determined by DLS. This dramatically increased size,
which is unusual in PEGylated proteins, seems likely to be caused
by a combination of two effects: 1) the conformation transition of
the grafted PEG chain from mushroomlike to brushlike structure
due to a high grafting density of PEG (0.58 PEG nm2, see Supplementary Material) and 2) the increased swelling of the PEG layer
induced by the charged Hph1 peptide via the Donnan equilibrium.
Despite the increased size, the colloidal stability of nanocapsules
was not significantly affected by the Hph1 conjugation. The surface
morphology and size distribution of the prepared nanocapsules
were also examined using SEM and AFM. When the sample was
prepared by freeze-drying for SEM, the individual nanocapsules
maintained a spherical shape without any significant aggregation,
Fig. 4. Confocal microscopic images of MCF-7 cells following treatment with the HSA/PEG nanocapsules (A), Hph1-decorated HSA/PEG nanocapsules (B), and Taxol formulation (C)
at an equivalent drug concentration of 10 mg mL1. To investigate the intracellular distribution, red fluorescent BODIPYÒ 564/570-paclitaxel conjugate was incorporated into the
nanocapsules. Cell nuclei were also visualized with DAPI (blue fluorescence) (For interpretation of the references to colour in this figure legend, the reader is referred to the web
version of this article.)
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Fig. 5. Flow cytometric analysis of the cell cycle of hCASMCs following incubation with PBS (A), the paclitaxel-loaded HSA/PEG nanocapsules (B), and the Hph1-decorated HSA/PEG
nanocapsules (C) at an equivalent drug concentration of 10 mg mL1.
J.Y. Lee et al. / Biomaterials 32 (2011) 8635e8644
indicating that the shell cross-linked layer effectively prevented the
formation of large paclitaxel aggregates (Fig. 2C). The measured
average diameter of the nanocapsules was about 412 nm from the
SEM image (the sample size, n ¼ 100), which matched very well
with the DLS result. Fig. 2D shows the AFM image of the nanocapsules prepared by air-drying on a mica surface. The
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nanocapsules were severely deformed, and their average diameter
was about 207 nm (n ¼ 70), which was much smaller than the
values obtained from the DLS and SEM analyses. This structural
change seems to be caused by the shrinkage of the hydrophilic
polymer networks of the nanocapsules during the air-drying
process [29].
Fig. 6. (A) In vivo near-infrared fluorescence images of a MCF-7 tumor-bearing mouse after i.v. injection of the Cy5.5-labeled HSA/PEG nanocapsules. The fluorescently labeled
nanocapsules were injected intravenously at a given dose of paclitaxel (5 mg per kg body weight), and whole body images of each mouse were acquired by an IVISÒ Xenogen
imaging system at various time intervals. The area pointed by a black arrow indicates the implanted tumor site. (B) Quantification of the in vivo tumor accumulation of the
nanocapsules (n ¼ 3 mice per group). (C) Ex vivo images of major organs (liver, lung, spleen, kidney, and heart) and a tumor harvested from mice on days 7 (middle row) and 14
(lower row) post-injection of the Cy5.5-labeled HSA/PEG nanocapsules. The upper row shows the ex vivo images of tumor and organs excised from mice treated with PBS on days 7.
(D) In vivo distribution of the HSA/PEG nanocapsules expressed as fluorescence intensity per gram of each excised organ. All data represent mean s.e. (n ¼ 3). (E) Time course
changes of a relative tumor volume following a single i.v. injection of the paclitaxel-loaded HSA/PEG nanocapsules or PBS.
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J.Y. Lee et al. / Biomaterials 32 (2011) 8635e8644
Therapeutic potential of the paclitaxel-loaded nanocapsules for
cancer treatment was evaluated by measuring the inhibition
activity of cancer cell growth. Fig. 3A shows that the HSA/PEG
nanocapsules reduced the viability of MCF-7 cells, and the effect
increased with the increased drug concentration. It should be noted
that the Hph1-decorated HSA/PEG nanocapsules exhibited
a dramatic anti-cancer activity (40.9% cell viability) at 10 mg mL1,
while the unmodified nanocapsules still showed high cell viability
(83%) at the same concentration. This enhanced cytotoxic effect
could be induced by the Hph1-facilitated translocation of the
nanocapsules into the cell, as Hph1 has been previously shown to
enable the efficient penetration of nano-carriers through the cell
membrane [18,30]. We also tested a clinically-available TaxolÒ
formulation (Bristol-Myers Squibb), which contains 30 mg paclitaxel dissolved in a 1:1 (v/v) mixture of Cremophor EL and ethanol
[1]. The Taxol formulation exhibited a significant level of cytotoxicity at >10 mg mL1, comparable to the Hph1-decorated HSA/PEG
nanocapsules. However, the cytotoxic effect of the Taxol formulation was considerably derived from non-specific toxicity of Cremophor EL: the Cremophor EL/ethanol mixture containing no
drugs had 42.1% cell viability of MCF-7 cells under the above
condition [2]. By contrast, the paclitaxel-free HSA/PEG nanocapsules showed 98.1% cell viability, demonstrating that our nanocarriers are relatively biocompatible, and the cytotoxic effect was
induced mainly by the paclitaxel incorporated within the
nanocapsules.
Paclitaxel has been also explored as a highly potent drug to
prevent intimal hyperplasia after balloon angioplasty because of its
powerful inhibitory effect on the proliferation and migration of
hCASMCs [31,32]. However, the efficient intracellular delivery of
paclitaxel into hCASMCs is very challenging because hCASMCs are
known as difficult-to-transfect primary cells [33]. Hence, it was
investigated whether the Hph1 conjugation can facilitate the
translocation of the HSA/PEG nanocapsules into the cytoplasm of
hCASMCs and exhibit an anti-proliferative activity. Fig. 3B shows
that the Hph1-decorated HSA/PEG nanocapsules can inhibit the
growth of hCASMCs at 100 mg mL1, while the unmodified nanocapsules showed only marginal anti-proliferative activity. This result
indicates that the anti-proliferative effect was enhanced through the
increased intracellular uptake of the nanocapsules mediated by the
cell-penetrating activity of Hph1. Fig. 3C summarizes the effects of
different formulations on cell viability, demonstrating that the
surface conjugation of Hph1 onto the nanocapsules greatly
enhanced the therapeutic performance of the loaded paclitaxel
against three different cancer cell lines and hCASMCs.
Confocal laser scanning microscopy was used to visualize the
cellular uptake of the paclitaxel-loaded HSA/PEG nanocapsules
(Fig. 4). In order to monitor the subcellular drug distribution, red
fluorescent BODIPYÒ 564/570-paclitaxel conjugates were incorporated into the HSA/PEG nanocapsules. While the cancer cells
treated with the Hph1-decorated HSA/PEG nanocapsules displayed
intense red fluorescence within the cytoplasm, only weak fluorescence was observed in the cells incubated with the unmodified
nanocapsules or the Taxol formulation. Since HSA is a highly
negatively charged protein at a neutral pH (the isoelectric
point ¼ 5.3), the HSA/PEG nanocapsules could hardly enter the cells
through the negatively charged cell membrane because of strong
electrostatic repulsion [34e36]. By contrast, Hph1-decorated
nanocapsules showed strong red fluorescence signals from the
BODIPYÒ-paclitaxel conjugates, which were homogeneously
distributed throughout the cytoplasm without marked localization
in the endosomal compartments. These results confirmed that the
Fig. 7. Histological cross-sections of tumor tissues excised from the mice on days 7 and 14 post-injection of the Cy5.5-labeled HSA/PEG nanocapsules. These sections were also
stained with TUNEL and H&E.
J.Y. Lee et al. / Biomaterials 32 (2011) 8635e8644
facilitated transport of the nanocapsules was mediated by the
Hph1-mediated transcytosis pathway, contributing to the
enhanced therapeutic efficacy of paclitaxel [17].
To determine whether the cytotoxic/anti-proliferative activity of
the paclitaxel-loaded HSA/PEG nanocapsules is correlated to their
apoptosis-inducing capability, a cell cycle analysis was performed
using flow cytometry with PI staining. It is well known that the anticancer effect of paclitaxel is directly related to its inhibitory effect on
the formation of mitotic spindle, which leads to the initiation of the
cell cycle arrest in the G2/M phase and apoptotic cell death [37]. The
extent of apoptosis can be approximately quantified from the
percentage of cell population in the G2/M phase. Fig. 5 shows that
the treatment of MCF-7 with the paclitaxel-loaded HSA/PEG nanocapsules induced the substantial accumulation of the G2/M cell
population (27.4%), while only 9.8% of the cells were presented in the
G2/M phase in the untreated sample. The Hph1-decorated nanocapsules had a higher percentage of the cells arrested at the G2/M
checkpoint (35.2%) as compared to the nanocapsules without Hph1
(27.4%). The highest apoptotic activity of the Hph1-decorated
nanocapsules was in agreement with the cytotoxicity profiles, confirming that the dramatic cytotoxic effect of the nanocapsules was
attributed to the paclitaxel-induced apoptosis.
In vivo accumulation of the nanocapsules in a tumor tissue was
examined using the average fluorescence intensity, defined the
total photon count per unit area of tumor. Fig. 6 A and B show that
the fluorescence intensity was maintained on its maximum level in
the tumor site for up to 2 days post-injection, and then gradually
decreased until day 14. This result indicates that the injected
nanocapsules were targeted to and stayed in the tumor tissue for an
extended period of time presumably because of the absence of an
active lymphatic system that is necessary for clearing macromolecules [38]. To more precisely evaluate the spatial distribution of
the HSA/PEG nanocapsules in the body, we obtained the ex vivo
fluorescence images of the organs excised from the mice (Fig. 6C). A
strong fluorescence signal was dominantly found in the tumor
tissue, while only marginal fluorescence was detected in the liver
and lung. No fluorescence was observed in the spleen, kidney, and
heart, indicating that the injected nanocapsules can preferentially
accumulate in the tumor region with no severe non-specific uptake
by the normal tissues. The accumulation of the HSA/PEG nanocapsules in the tumor was about 8 times higher than the uptake by
the liver on day 14 post-injection (Fig. 6D). This tumor targeting
effect suggests that the HSA/PEG nanocapsules are hardly recognized and cleared by the macrophages distributed in the liver [9].
Tumor volume variation was also monitored to examine the antitumor activity of the paclitaxel-loaded HSA/PEG nanocapsules
(Fig. 6E). The untreated control mice had about 8-fold increase in
tumor volume in 14 days; however, the mice treated with a single
i.v. administration of the paclitaxel-loaded HSA/PEG nanocapsules
only showed about 1.7-fold increase. This remarkable effect of the
HSA/PEG nanocapsules on the suppression of tumor growth is well
supported by the histological cross-sections of the excised tumors.
Fig. 7 shows that the Cy5.5-labeled HSA/PEG nanocapsules were
homogeneously distributed throughout the tumor tissues on days 7
and 14 post-injection, providing the direct evidence of the accumulation of the nanocapsules in the tumor tissue. TUNEL analysis
also revealed that the treatment with the HSA/PEG nanocapsules
induced the apoptotic death of cancer cells to a greater extent, as
compared to the untreated control that showed no significant sign
of apoptosis [39].
4. Conclusions
This study demonstrated that shell cross-linked nanocapsules
composed of HSA and multi-armed PEG can be used as an efficient
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nanoscale carrier for the targeted delivery of paclitaxel. The
nanocapsules successfully encapsulated paclitaxel into the covalently cross-linked framework without significant aggregation.
Surface modification with a cell-penetrating peptide, Hph1, facilitated the intracellular delivery of paclitaxel via a Hph1-mediated
transcytosis and efficiently induce the apoptotic death of cancer
cells. Furthermore, animal tumor model studies revealed that the
paclitaxel-loaded HSA/PEG nanocapsules can preferentially accumulate in the tumor site, and thus can effectively suppress tumor
growth upon i.v. administration. Because of the unique nanoreservoir structure for stable drug encapsulation and high tumor
targeting specificity, the HSA/PEG nanocapsules can be potentially
extended as delivery vehicles for other water-insoluble anti-cancer
drugs to achieve effective cancer therapy by reducing the nonspecific side effects to normal tissues.
Acknowledgments
The late Professor Tae Gwan Park supervised the overall work as
a principal investigator. All co-authors deeply appreciate his
invaluable contribution and educational efforts. This research was
financially supported from the Ministry of Health, Welfare and
Family Affairs and the Ministry of Education, Science and Technology (Republic of Korea) through Basic Science Research Program
(2010-0027955), the World Class University project, and the
National Research Laboratory program. We thank Dr. Earl Choi and
Prof. Won-il Jeong in the Graduate School of Medical Science and
Engineering for their kind assistance in animal experiments.
Appendix. Supplementary material
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.biomaterials.2011.07.063.
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