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R ESEARCH A RTICLE
Vitamin E TPGS-emulsified poly(lactic-co-
glycolic acid) nanoparticles for cardiovascular
restenosis treatment
Si-Shen Feng1,2,3†,
Aims: Paclitaxel is one of the most effective antiproliferative agents and it has been applied
Wutao Zeng1,7,
in the development of drug-eluting stents. There are difficulties, however, in using paclitaxel
Yean Teng Lim5,6,
Lingyun Zhao1, in clinical applications owing to its poor solubility and side effects. We have synthesized
Khin Yin Win1, nanoparticles of biodegradable polymers for the effective and sustainable delivery of
Reida Oakley5, paclitaxel and other antiproliferative agents for restenosis treatment.
Swee Hin Teoh4, Methods & results: Paclitaxel-loaded poly(lactic-co-glycolic acid) (PLGA) nanoparticles were
Ronald Chi Hang Lee6 & prepared by a modified solvent extraction/evaporation method with D-α-tocopheryl
Shirong Pan7 polyethylene glycol 1000 succinate (TPGS) or polyvinyl alcohol (PVA) as an emulsifier.
†Author for correspondence Drug-loaded nanoparticles were characterized for size and size distribution, surface
1Department of Chemical & morphology, surface charge, drug-encapsulation efficiency and in vitro drug-release kinetics.
Biomolecular Engineering,
National University of Cellular uptake of fluorescent nanoparticles was investigated in vitro in coronary artery
Singapore, Block E5, 02–11, smooth muscle cells and in vivo in the carotid arteries of rabbits. The antiproliferative effects
4 Engineering Drive 4, of the nanoparticle formulations were assessed in vitro in close comparison with Taxol®.
117576 Singapore
Tel.: +65 6516 3835;
Both the PVA- and TPGS-emulsified nanoparticles have similar size and size distribution,
Fax: +65 6779 1936; surface morphology and dispersion stability and showed great advantages over paclitaxel in
E-mail: chefss@nus.edu.sg in vitro cellular uptake and cytotoxicity than Taxol. The TPGS-emulsified nanoparticle
2Division of Bioengineering,
formulation has higher drug-encapsulation efficiency, cellular uptake and cytotoxicity than
National University of
Singapore, Singapore the PVA-emulsified nanoparticle formulation. IC50 in 24-h culture with coronary artery
3Nanoscience &
smooth muscle cells is 748 ng/ml for paclitaxel, 708 ng/ml for PVA-emulsified nanoparticles
Nanoengineering Initiative
and 474 ng/ml for TPGS-emulsified nanoparticles, respectively. Conclusion: TPGS-emulsified
(NUSNNI), National
University of Singapore, PLGA nanoparticles have great potential for the effective and sustainable delivery of
Singapore antiproliferative agents and for the development of nanoparticle-coated stents, which may
4Department of Mechanical
become the third generation of cardiovascular stents.
Engineering, National
University of Singapore, Coronary atherosclerosis and heart attack are the polymers, for local delivery and a combination of
Singapore
5Department of Surgery, leading causes of mortality in the world. The most drug therapy and devices, such as drug-eluting
National University of common treatments so far include percutaneous stents; for example, the Cypher® stent, which
Singapore, Singapore transluminal coronary angioplasty (PTCA) with releases sirolimus, and the TAXUS® stent, which
6Cardiac Department,
or without intracoronary stents. However, releases paclitaxel.
National University Hospital,
Singapore 30–50% of patients experience restenosis within Paclitaxel (Taxol®) is one of the best anti-
7Division of Cardiology, 3–6 months of PTCA treatment [1]. There are two neoplastic drugs that has been found from nature
Cardiovascular Medical kinds of treatment for restenosis currently: in recent decades. It has excellent therapeutic
Department, The First
Affiliated Hospital,
mechanical treatment and drug therapy. The effects against a wide spectrum of cancers [4]. It
Zhongshan University former is stenting [2]. Although popular, stenting was approved by the US FDA for ovarian cancer
Medical School, Guangzhou, does not solve the problem becasue 10–15% of in 1992, for advanced breast cancer in 1994 and
China the patents will suffer from restenosis again within for early-stage breast cancer in 1999. The mech-
6 months. The latter treatment type includes the anism of its anticancer effects has been inten-
treatment by antiproliferative/antiplatelet/anti- sively investigated. It inhibits mitosis in tumor
coagulant agents, calcium channel antagonists, cells by binding to microtubules. Paclitaxel aids
inhibitors of angiotensin-converting enzyme, cor- polymerization of tubulin dimers to form micro-
Keywords: cardiovascular
stents, nanobiotechnology,
ticosteroids or a fish-oil diet [3]. Nevertheless, drug tubules and thus stabilizes the microtubules,
nanomedicine, paclitaxel, therapy is not that effective owing to the pure leading to cell death [5–7]. Paclitaxel is thus an
percutaneous transluminal pharmaceutical properties and the multidrug antiproliferative drug that could be beneficial for
coronary angioplasty,
sirolimus
resistance (MDR) effects of the antiproliferative many other diseases caused by a loss of control of
agents, such as paclitaxel and sirolimus. Research cell proliferation. Among them is cardiovascular
part of
is thus focused on more effective drug-delivery restenosis. Due to its difficulty in clinical admin-
devices, such as nanoparticles of biodegradable istration and MDR, various dosage forms of
10.2217/17435889.2.3.333 © 2007 Future Medicine Ltd ISSN 1743-5889 Nanomedicine (2007) 2(3), 333–344 333

2. RESEARCH ARTICLE – Feng, Zeng, Lim et al.
paclitaxel have been under intensive investiga- become a new (the third) generation of cardiovas-
tion. The dosage form used most often is Taxol, cular stents, which will solve the problems of the
which is formulated in Cremophor EL. This second-generation stents – the drug-eluting stents
adjuvant is responsible for serious side effects, [101]. Although successful, these drug-eluting
including hypersensitivity reactions, nephro- stents have some problems, such as low drug-load-
toxicity, neurotoxicity and cardiotoxicity. Some ing ability, slow and incomplete drug release, inef-
of the side effects are serious, even life-threaten- ficient uptake by vascular smooth muscle cells
ing [8–13]. A better dosage form, docetaxol (Taxo- (VSMCs) [17–20], late angiographic stent thrombo-
tere®), was developed later. Although it achieves sis (LAST) [21] and issues of long-term safety and
a higher survival rate (SR), the side effects are efficacy, which have raised the cost–effectiveness
still a problem and are probably caused by the problem [22,23].
adjuvant polysobate. PLGA nanoparticle formulation of anti-
Our research here has investigated the feasibil- proliferative drugs for the treatment of cardio-
ity of the formulation of antiproliferative agents vascular restenosis has had a history of more than
by biodegradable poly(lactic-co-glycolic acid) 10 years [24–31]. However, the reports of a PLGA
(PLGA) nanoparticles, which are prepared by the nanoparticle formulation of paclitaxel for resten-
solvent extraction/evaporation method by using osis treatment are few in the literature [32,33],
amphiphilic poly(vinyl alcohol) (PVA) or although this drug has been used widely in drug-
D-α-tocopheryl polyethylene glycol 1000 succi- eluting stents. The idea of TPGS-emulsified
nate (TPGS) as an emulsifier for the treatment PLGA nanoparticles for restenosis treatment is
and prevention of restenosis. The drug-loaded novel, coming from our research on TPGS used
nanoparticles were then characterized by various as an effective emulsifier or as a component of the
techniques, such as laser light scattering for novel PLA–TPGS copolymer in the nanoparticle
nanoparticle size and size distribution, field- formulation of anticancer drugs, which resulted
emission scanning electron spectroscopy in high drug EE, high cellular uptake of the
(FESEM) and atomic force microscopy (AFM) nanoparticles by cancer cells, long half-life in cir-
for surface morphology and zeta-potential for culation and high therapeutic effects demon-
surface charge. High-performance liquid chro- strated by high area-under-the-curve (AUC) of
matography (HPLC) was employed to measure the in vivo pharmacokinetic measurement [16].
the drug-encapsulation efficiency (EE) and the
in vitro drug-release kinetics. Cellular uptake of Materials & methods
fluorescent nanoparticles was investigated PLGA with L:G molar ratio of 50:50 and Mw of
in vitro in coronary artery smooth muscle cells 40,000–75,000, PVA with Mw of
(CASMCs) and in vivo in carotid arteries of rab- 30,000–70,000, fluorescence marker cou-
bits, which was visualized by confocal laser scan- marin-6, phosphate-buffered saline (PBS), mini-
ning spectroscopy (CLSM). The antiproliferative mum essential medium, penicillin–streptomycin
effects of the nanoparticle formulations were solution, trypsin–EDTA solution, Triton® X-
assessed in vitro by the MTS assay and analyzed 100, Hank’s balanced salt solution (HBSS) and
with consideration of the drug-release kinetics in 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymeth-
close comparison with Taxol. oxyphenyl)-2-(4-sulfonyl)-2H-tetrazoliumn
The drug-loaded PLGA nanoparticles can be (MTS) were purchased from Sigma (St Louis,
used either for local delivery by balloon catheter MO, USA). Paclitaxel was purchased from Yun-
or for the development of a novel type of cardio- nan Hande Biotechnology Inc (China). Taxol
vascular stent – the nanoparticle-coated stent was from Bristol-Myers Squibb Caribbean Com-
[101]. Why do we prefer the nanoparticle formula- pany (USA). Vitamin E D-α-tocopheryl polyeth-
tion? This is because pure paclitaxel is not bio- ylene glycol 1000 succinate (vitamin E TPGS or
adhesive to the cell membrane owing to its poor simply TPGS) was obtained from Eastman
pharmaceutical properties, including the MDR Chemical Company (USA). Dichloromethane
effects. Our pioneering research on the nano- (DCM, analytical grade) was from Merck
particle formulation of paclitaxel has demon- (Darmstadt, Germany) and acetonitrile (HPLC
strated that nanoparticles are more adhesive to, grade) was from Fisher Scientific (NJ, USA).
and thus easier to be taken up by, cancer cells, Fetal bovine serum (FBS) was received from
such as Caco-2 cells and HT-29 cells, than the Gibco (Life Technologies, AG, Switzerland).
microparticle formulation and the free drug itself Ultrapure water (Millipore, Bedford, MA, USA)
[14–16]. The nanoparticle-coated stents may was used throughout the experiment.
334 Nanomedicine (2007) 2(3) future science group

3. Nanoparticles of biodegradable polymers for restenosis treatment – RESEARCH ARTICLE
Preparation of nanoparticles Drug EE
PLGA nanoparticles loaded with paclitaxel or The amount of drug encapsulated in the nano-
fluorescent marker (0.5% coumarin-6) were particles was determined in triplicates by HPLC
prepared by a modified solvent extraction/evap- (Agilent LC 1100 series). 3 mg of nanoparticles
oration method (single emulsion) by using PVA were dissolved in 1 ml of DCM, and 3 ml of
or TPGS as the emulsifier [34,35]. In brief, 8 ml acetonitrile-water (50:50) was then added. A
of dichloromethane (DCM) solution of nitrogen stream was introduced to evaporate
110 mg PLGA and 11 mg paclitaxel was added the DCM until a clear solution was obtained.
drop by drop to a 120 ml aqueous phase, in The solution was put into vials to detect the
which PVA 600 mg or TPGS 36 mg was paclitaxel concentration by HPLC. For HPLC
added. The solution was then emulsified for analysis, a reverse phase Inertsil® ODS-3 col-
120 s using a microtip probe sonicator umn (150 x 4.6 mm i.d., pore size 5 µm, GL
(XL2000, Misonix Incorporated, NY, USA) at Science, Tokyo, Japan) was used and the mobile
50 W in pulse mode. The formed oil in water phase was delivered at a rate of 1 ml/min by a
(o/w) emulsion was stirred gently at room tem- pump (HP 1100 High Pressure Gradient
perature (22°C) by a magnetic stirrer overnight Pump). 50 µl of sample was injected by an auto
to evaporate the organic solvent. The resulting sampler (HP 1100 Autosampler) and the col-
sample was collected by centrifugation umn effluent was detected at 227 nm with a
(12,000 rpm, 15 min, 16°C; Eppendorf model variable wavelength detector (HP 1100 VWD).
5810R, Eppendorf, Hamburg, Germany) and The calibration curve was prepared for the
washed three times with ultrapure water. The quantification of drug in the nanoparticles and
product was freeze-dried (Alpha-2, Martin it was linear over the range of 50–10,000 ng/ml
Christ Freeze Dryers, Germany) to obtain a with a correlation factor of r2 = 0.9999. The
fine powder of nanoparticles, which was kept in measurement was performed in triplicate. The
a vacuum dessicator. We did not use any cryo- drug EE was obtained as the mass ratio between
protectant in the freeze-dry process because all the amount of paclitaxel incorporated in the
the materials, including paclitaxel and TPGS, nanoparticles and that used in the nanoparticle
are stable in lyophilization. preparation process [34,35].
Characterization of nanoparticles Surface charge
Size & size distribution Zeta-potential is an indicator of surface charge,
Nanoparticle size and size distribution were which determines particle stability in the dis-
determined by laser light scattering with a par- persion and redispersabliity of the nano-
ticle size analyzer (90 Plus, Brookhaven Insti- particles. Zeta-potential of nanoparticles was
tute, Huntsville, NY, USA) at a fixed angle of determined by a zeta-potential analyzer (Zeta
90° at 25°C. In brief, the dried nanoparticles Plus, Brookhaven Instruments, Huntsville,
were suspended in filtered deionized water and NY, USA) by dipping a palladium electrode in
sonicated to prevent particle aggregation and the sonicated particle suspension. The mean
to help form a uniform dispersion of nano- value of ten readings is reported.
particles. The size distribution was given by
the polydispersity index. In vitro drug-release kinetics
5 mg of the drug-loaded nanoparticles were put
Surface morphology in a centrifuge tube containing 10 ml PBS
Morphology of the drug-loaded nanoparticles (pH 7.4) with 0.1% tween 80. After dispersion
was observed by FESEM (JSM-6700F, by a vortex mixer (S0100–230V, Labnet Interna-
JEOL.LED, Japan), which requires an ion tional Inc., USA), the tube was placed in an
coating with platinum by a sputter coater orbital shaker water bath at 37°C. The well-
(JFC-1300, Jeol, Tokyo) for 40 s in a vacuum redispersed status of the nanoparticles for con-
at a current intensity of 40 mA after preparing tinuous release measurement can be confirmed
the sample on metallic studs with double-sided by the laser light-scattering measurement. At
conductive tape. AFM was conducted with designated time intervals, the tube was taken out
Nanoscope IIIa in the tapping mode. Before and centrifuged at 11,500 rpm for 15 min. The
observation, the nanoparticles were fixed on a supernatant was removed and extracted with
double-sided sticky tape that was stuck to the 5 ml DCM to determine the amount of drug
standard sample stud. released inside it. The pellets were resuspended
future science group www.futuremedicine.com 335

4. RESEARCH ARTICLE – Feng, Zeng, Lim et al.
in 10 ml of fresh PBS with 0.1% tween 80 for with HBSS for 30 min, the buffer was replaced
continuous release measurement. The analysis with a nanoparticle suspension (250 µg/ml in
procedure was the same as described in the HBSS) and the monolayers were further incu-
determination of the EE [34,35]. bated for 4 h. The monolayers were then washed
three times with fresh prewarmed transport
Cell culture buffer to eliminate excess nanoparticles. The
In the present study, CASMCs were provided by cells were fixed with 70% ethanol and the nuclei
Cambrex Bio Science Walkersville Inc, USA and were stained by propidium iodide (PI). The
passages between five and ten were used. samples were mounted in the fluorescent
CASMCs were cultured in Dulbecco’s modified mounting medium (Dako, CA, USA) until
Eagle’s medium (DMEM) supplemented with examination was performed by the CLSM (Zeiss
20% FBS (vol/vol %) and 1% penicillin–strep- LSM 410, Germany) equipped with an imaging
tomycin solution. The cells were seeded at software, Fluoview FV300.
4.3 × 104 cells/cm2 in 96-well black plates with
transparent bases (Costar, IL, USA) for quantita- In vitro antiproliferative effects of
tive measurement of the cellular uptake of the drug-loaded nanoparticles
fluorescent nanoparticles and cytotoxicity meas- The antiproliferative effects of the paclitaxel-
urement of the drug-loaded nanoparticles or on loaded PLGA nanoparticles were investigated
the Lab-Tek® chambered cover glasses (Nagle in vitro by culturing CASMCs with the nano-
Nunc International, Naperville, IL, USA) for particle formulation of paclitaxel in close com-
confocal microscopy. The cell monolayer was parison with Taxol at the same paclitaxel
cultured at 37°C in a humidified atmosphere concentration. The cell viability (survival rate)
containing 5% CO2 and the medium was was determined by the MTS assay, which is a
replaced every two days [34–36]. colorimetric method to determine the number of
viable cells that are proliferating. It is composed
Uptake of nanoparticles by CASMCs of a solution of tetrazolium, which is bioreduced
Quantitative study: microplate reader analysis by metabolically active cells into a soluble forma-
CASMCs were seeded in 96-well black plates zan product in the culture medium. Its absorb-
and were incubated for 48 h. Cultural medium ance can be measured at 490 nm by a microplate
was then replaced by transport buffer HBSS and reader (Genios, Tecan, Männedorf, Switzerland).
pre-incubated at 37°C for 1 h. After equilibra- The quantity of formazan is directly proportional
tion, cellular uptake of fluorescent nanoparticles to the number of living cells. After cells were
was initiated by exchanging the transport seeded in a 96-well plate (Costar, IL, USA) and
medium with 100 µl of the specific nanoparticle equilibrated with the DMEM medium (without
suspension and incubated the cells for approxi- FBS) for 1 h, the medium was removed and the
mately 1–6 h. The experiment was terminated nanoparticle suspension in DMEM with 10%
by washing the cell monolayer three times with FBS was added. After incubation for a scheduled
PBS to eliminate excess nanoparticles that were time, 20 µl of MTS inner salt was added to each
not entrapped by the cells. The cell membrane well of a 96-well assay plate containing the sam-
was permeated with Triton X-100 solution to ples in 100 µl of culture medium. The plate was
expose the internalized nanoparticles for quanti- incubated for 4 h at 37°C in a humidified atmos-
tative measurement. Cellular uptake of the fluo- phere containing 5% CO2 [34,35,37]. The cell
rescent nanoparticles was quantified by mortality (death rate) is defined as 100% viabil-
analyzing the cell lysate in a Genios microplate ity. It should be noted that it is the cell mortality,
reader. Uptake was expressed as the percentage of but not the cell viability, that should be propor-
the fluorescence associated with the cells versus tional to the area-under-the-curve of the drug
that present in the feed solution [36]. concentration versus time.
Qualitative study: CLSM Animal protocols
CASMCs were seeded on Lab-Tek® chambered The animal protocol was approved by the Insti-
cover glasses (Nagle Nunc International, Naper- tutional Animal Care and Use Committees
ville, IL, USA) and incubated at 37°C in a 95% (IACUC, Protocol #: 802/05), Office of Life Sci-
air and 5% CO2 environment. On the day of ence, National University of Singapore. Alto-
the experiment, the growth medium was gether, we used ten New Zealand white rabbits
replaced by HBSS (pH 7.4). After equilibration for the in vivo infusion experiment.
336 Nanomedicine (2007) 2(3) future science group

5. N an o p articles o f b iod egrad ab le p olym ers for resten osis treatm en t – R E S E A R C H A R T I C L E
Table 1. The size and size distribution, drug-encapsulation efficiency and
Zeta-potential of paclitaxel-loaded, PVA- or TPGS-emulsified PLGA nanoparticles.
Emulsifier Size (nm) Polydispersity EE (%) Zeta-potential (mv)
PVA (5.0% w/v) 257 ± 10.2 0.031 55.8 ± 4.98 -13.74 ± 1.94
TPGS (0.3 w/v) 288 ± 11.7 0.028 92.6 ± 10.0 -21.5 ± 3.57
EE: Encapsulation efficiency; PLGA: Poly(lactic-co-glycolic acid); PVA: Polyvinyl alcohol; TPGS: D-α-tocopheryl
polyethylene glycol 1000 succinate.
Rabbit anesthesia Statistical analysis
2.0–3.0 kg male New Zealand white rabbits were Results of the experiments are expressed as
anesthetized using Ketamine/Xylazine at a dos- mean ± SD. In the nanoparticle cellular uptake
age of 35 mg/kg/5 mg/kg subcutaneously fol- experiment, the student unpaired t-test was
lowed by tracheal incubation and were adopted for the comparison between the PVA-
maintained with 1.0–2.0 vol% isoflurance, 70% and TPGS-emulsified PLGA nanoparticles.
N2O and 30 vol% oxygen. The cytotoxicity study was tested by ANOVA.
Probability values of p < 0.05 and p < 0.01
Artery isolation were considered to be significant and highly
Carotid arteries (averaging 3–4 cm in length) significant, respectively.
were isolated with all side branches being ligated.
Results & discussions
Arteries injured by balloon catheter Physicochemical properties
The distal vessel was punctured by a 18 G trochar. of nanoparticles
The needle was withdrawn and the cannula was left Size, EE & surface charge of the
in the vessel. A balloon catheter (2.5 × 10–20 mm) drug-loaded nanoparticles
was introduced and advanced in a retrograde fash- The data reported in Table 1 represent the average
ion into the isolated artery segment via the cannula of five measurements. The emulsifier concentra-
distal in the vessel. Once positioned proximally, the tion needed to form the nanoparticles was
balloon was inflated with saline to achieve visual 5.0% w/v (emulsifier weight/water phase vol-
overstretch of the vessel and then withdrawn a dis- ume) for the PVA-emulsified nanoparticles and
tance of 2 cm. The process was repeated two 0.3% w/v for the TPGS-emulsified nano-
additional times. The catheter was then removed. particles. This means that TPGS was 16.7-times
more effective than PVA as an emulsifier for use
Infusion of nanoparticles in the emulsification process, that is, to make the
The proximal portion was clamped by a non- same amount of nanoparticles, the required
crushing vascular clamp. The nanoparticle suspen- amount of TPGS could be 16.7-times less than
sion was injected at the distal end of the segment, that of PVA. A more effective recipe was also
which was connected to a pressure pump via the reported [16]. This is a significant advantage of
cannula. The arterial lumen was filled with the TPGS over PVA. Also from Table 1, the mean size
nanoparticle suspension at 1 atm pressure for 60 s with 10% drug loading was 257 ± 10.2 nm for
and the vessel was harvested and the nanoparticles the PVA-emulsified nanoparticles and
flushed out by 0.9% saline water. 288 ± 11.7 nm for the TPGS-emulsified nano-
particles. The light-scattering measurement of
Histological examination of the particle size agrees well with that given by the
nanoparticle-infused arteries Smile View software from the FESEM images.
Nanoparticles loaded with the fluorescent marker The TPGS-emulsified nanoparticles achieved
coumarin-6 were used in this study. Each arterial much higher EE (92.6 ± 10.0%) than the PVA-
segment after nanoparticle infusion was flushed to emulsified nanoparticles (55.8 ± 4.98%). It is
remove the nanoparticles not taken up by clear that TPGS has an advantage over PVA,
CASMCs by 0.9% saline water and then frozen by resulting in much higher EE and, therefore, in
dry ice with an OCT (Mile, Inc, Elkhart, IN, much higher drug loading in the nanoparticles.
USA)-embedding compound. Cross sections of We mentioned earlier that 110 mg PLGA and
10 µm thickness were cut using a cryomicrotome 11 mg paclitaxel were added in the organic sol-
and mounted on the glass slides. The slides were vent. The theoretical drug-loading ratio should
observed under a confocal microscope. have been 10%. However, owing to incomplete
future science group www.futuremedicine.com 337

6. RESEARCH ARTICLE – Feng, Zeng, Lim et al.
Morphology of nanoparticles
Figure 1. Scanning electron microscopy images of
paclitaxel-loaded nanoparticles. Figure 1 shows the FESEM images of PVA-
(Figure 1A) and TPGS-emulsified (Figure 1B)
PLGA nanoparticles of 10% drug loading,
A B
which reveals their regular spherical shape and
smooth surface without any noticeable pinholes
or cracks within the instrument resolution. The
size distribution of all nanoparticles was unimo-
dal, with a range of 150–500 nm and a mean
diameter of 200–300 nm, as confirmed by the
laser light-scattering measurement. Figure 2 shows
1 µm 1 µm AFM images of paclitaxel-loaded, TPGS-emulsi-
fied PLGA nanoparticles and a magnified image
FESEM Images of (A) polyvinyl alcohol- or (B) D-α-tocopheryl polyethylene glycol of the nanoparticle surface, from which wrinkles
1000 succinate-emulsified, drug-loaded poly(lactic-co-glycolic acid) and a small hole can be observed. The advantage
nanoparticles of 10% drug loading. of AFM is that it can reveal the true structure
with much higher resolution than SEM since the
encapsulation, that is, less than 100% EE, the image is obtained by direct contact or tapping of
actual drug-loading ratio was modified by the the AFM tip on or over the particle surface.
EE values, which is thus 5.58% for the PVA-
emulsified nanoparticles and 9.26% for the In vitro drug-release kinetics
TPGS-emulsified nanoparticles. The in vitro release profiles of paclitaxel from the
Both types of drug-loaded nanoparticles PVA- or TPGS-emulsified PLGA nanoparticles
were stable in their dispersion, possessing neg- is shown in Figure 3, from which the effect of sur-
ative surface charges with high absolute values face coating on the in vitro drug-release behavior
of zeta-potential (Table 1), which are can be observed. The drug-release kinetics
-13.74 ± 1.94 mV for the PVA-emulsified exhibit a biphasic pattern characterized by a fast
nanoparticles and -21.5 ± 3.57 mV for the initial burst during the first 5 days, followed by a
TPGS-emulsified nanoparticles. The surface slow, sustained release. An initial burst during
charge determines stability of the nanoparticle the first 5 days of 67.9% for the PVA-emulsified
suspension and resuspensability of the nano- nanoparticles and 51.2% for the TPGS-emulsi-
particles. TPGS-emulsified nanoparticles thus fied nanoparticles was followed by a first order
have advantages over PVA-emulsified nano- release with a reduced rate afterwards. Approxi-
particles in their suspension stability and mately 81.2% of the PVA-emulsified nano-
resuspensability. This finding is in agreement particles and 66.2% of the TPGS-emulsified
with that of our earlier research of nanoparti- nanoparticles were released in 30 days. Please
cle formulation of paclitaxel for cancer note the release for the first 72 h is 58.6% for the
treatment [37]. PVA-emulsified nanoparticles and 43.4% for the
TPGS-emulsified nanoparticles. These data will
Figure 2. Atomic force microscopy images of paclitaxel-loaded be used later to interpret the cellular mortality of
nanoparticles. the drug formulated in the nanoparticles. It
seems that the drug release from the nano-
A B particles is much faster (∼1 month) than the
0.15 0.3 V
release of drugs from drug-eluting stents
0.1 V (∼6 months), which represent two different
0.10 treatments of restenosis: local drug delivery and
0.0 V device plus drug; each has advantages and disad-
vantages. For local drug delivery, the nanoparti-
0.05
cles are the reservoir of the drug after adsorption
by the CASMCs. The 1 month (or even faster)
0.1 0
0.2 0.3 µm 0 0.05 0.10 0.15 µm drug-release period may be appropriate for the
treatment. For drug-eluting stents, the stents
(A) Atomic force microscopy image of paclitaxel-loaded, D-α-tocopheryl themselves are the reservoir of the drug and a
polyethylene glycol 1000 succinate-emulsified poly(lactic-co-glycolic acid)
longer period would result in long-term treat-
nanoparticles and (B) magnified image of the nanoparticle surface.
ment. Unfortunately, one of the major problems
338 Nanomedicine (2007) 2(3) future science group

7. Nanoparticles of biodegradable polymers for restenosis treatment – RESEARCH ARTICLE
Figure 3. In vitro drug releases of PVA- or TPGS-emulsified, and the residues would affect the cellular
paclitaxel-loaded PLGA nanoparticles of 10% drug loading. uptake measurement of the fluorescent nano-
particles. The residues, however, would not
affect the CLSM images because CLSM has a
90 sectioning function.
Culmulative release (%)
80
70 Quantitative study
60 Figure 5 shows the effects of the incubation time
50 on the cellular uptake of the fluorescent PVA-
40 or TPGS-emulsified PLGA nanoparticles. The
Vitamin E TPGS
30 -emulsified NPs nanoparticle concentration used for incubation
20 PVA-emulsified NPs with the CASMCs was 500 µg/ml. The signifi-
10 cance of the TPGS-emulsified versus PVA-
0 emulsified nanoparticles is p < 0.01. Figure 4
0 5 10 15 20 25 30 35
Time (day) demonstrates that the cellular uptake of nano-
particles increased with the incubation time.
Each point represents mean ± SD (n = 3). At each designated time, the TPGS-emulsified
NP: Nanoparticle; PLGA: Poly(lactic-co-glycolic acid); PVA: Polyvinyl alcohol; nanoparticles could achieve much higher cellu-
TPGS: D-α-tocopheryl polyethylene glycol 1000 succinate. lar uptake than the PVA-emulsified nano-
particles. After incubation for 6 h, the
for drug-eluting stents is that the drug coated on CASMC uptake was 38% for the TPGS-emul-
the stent surface cannot be completely released. sified nanoparticles versus 21% for the
An ideal solution is thus to combine the two PVA-emulsified nanoparticles.
therapies, that is, to develop nanoparticle-coated Figure 6 shows the effects of the nanoparticle
stents [101]. concentration on the cellular uptake of the flu-
It should be pointed out that, although the orescent PVA- or TPGS-emulsified PLGA
surfactant molecules are supposed to be washed nanoparticles after 4 h incubation. We can see
away after formulation, incomplete washing will from this figure that the cellular uptake of
result in some residues remaining on the nano- nanoparticles increased with the nanoparticle
particle surface, which will affect the drug release. concentration and, at each designated nano-
Moreover, the release medium also plays a deci- particle concentration of 100, 250 500 µg/ml,
sive role in determining the drug-release kinetics. the TPGS-emulsified nanoparticles showed
The in vivo release could thus be much faster great advantages over the PVA-emulsified
than the in vitro release owing to the interactions nanoparticles in cellular uptake. For example,
between the plasma proteins and the drug. This at the 500 µg/ml nanoparticle concentration,
has been confirmed by the in vitro measurement the cellular uptake was 33% for the TPGS-
of drug release in plasma (data not shown). emulsified nanoparticles versus 13.5% for the
PVA-emulsified nanoparticles. This advantage
Cellular uptake of nanoparticles is significant (p < 0.01). The quantitative
Qualitative study study confirmed the results observed from the
Figure 4 shows confocal microscopic images of qualitative study, showing that the TPGS-
CASMCs after 4-h incubation with coumarin- emulsified nanoparticles have advantages
6-loaded, PVA- (Figure 4A) or TPGS-emulsified resulting in higher cellular internalization than
(Figure 4B) PLGA nanoparticles at 37°C. The the PVA-emulsified nanoparticles. Although
nuclei were stained by PI (red), and the cou- the detailed mechanism is unknown,
marin-6-loaded nanoparticles (green) in the vitamin E facilitates cellular uptake of drugs.
cytoplasm were visualized by overlaying images It may be concerning that the fluorescent
that were obtained by fluorescein isothio- coumarin-6 markers formulated in the nano-
cyanate (FITC) filter and PI filter. The images particles could leak, which may affect the result
show that most of the internalized nano- of the cellular uptake measurement. To address
particles are located in the cytoplasm. Some this problem, we have conducted an experi-
may have penetrated into the nuclei. ment to measure the in vitro release of cou-
It should be pointed out that the washing marin-6 from the nanoparticles and our results
procedure may not be able to wash the showed that the leakage, in up to 24 days, was
adhered nanoparticles out of the cell surface less than 5% and thus negligible [36].
future science group www.futuremedicine.com 339

8. RESEARCH ARTICLE – Feng, Zeng, Lim et al.
Figure 4. Confocal microscopic images of coronary artery CASMCs. Data represent the mean ± SD with
smooth muscle cells cultured with fluorescent nanoparticles. n = 6. There was no significant decease in the cell
viability for the two types of PLGA nanoparticles
compared with the control (p < 0.05), although
A B the placebo PVA-emulsified PLGA nanoparticles
showed a slightly larger decrease in cell viability
and such a decease becomes more significant at
high nanoparticle concentrations. This means
that the TPGS-emulsified nanoparticles are
more biocompatible than the PVA-emulsified
nanoparticles. This is another advantage of
TPGS versus PVA as an emulsifier.
50 µm 20 µm Figure 8 shows the effects of the drug concen-
tration on CASMC viability after 72 h incuba-
Confocal microscopic images of coronary artery smooth muscle cells after 4 h tion with the paclitaxel-loaded, PVA- or TPGS-
incubation with coumarin-6-loaded, (A) polyvinyl alcohol- or (B) D-α-tocopheryl emulsified PLGA nanoparticle suspension ver-
polyethylene glycol 1000 succinate-emulsified poly(lactic-co-glycolic acid) sus Taxol. The table in the figure shows the
nanoparticles at 37°C. The nuclei were stained by propidium iodide (PI) (red), measured CASMC mortality (viability + mor-
and the cellular uptake of fluorescent coumarin-6-loaded nanoparticles (green)
tality = 1) as well as that after the correction
in the cytoplasm were visualized by overlaying images obtained by a fluorescein
isothiocyanate filter and a PI filter. The cells look unhealthy because they were made by considering the 72 h drug release
being killed by the drug-loaded nanoparticles. found from the drug-release profiles (Figure 3).
The data represent mean ± SD of n = 6. The
In vitro antiproliferative effects of significance of the TPGS-emulsified nanoparti-
drug-loaded nanoparticles cles versus PVA-emulsified nanoparticles is
We first tested the cytotoxicity of the placebo p < 0.01 at 25 ng/ml drug concentration and
PVA- or TPGS-emulsified PLGA nanoparticles, p < 0.05 at 250 and 500 ng/ml drug concentra-
that is, the nanoparticles with no drug encapsu- tions. From the table it can be seen that the via-
lated. Figure 7 show the cytotoxicity of the placebo bility (the percentage of the CASMCs that
PVA- or TPGS-emulsified PLGA nanoparticles at survived) after 72 h culture at 25 ng/ml paclit-
nanoparticle concentrations of 2.5, 25 and axel concentration is 80.5% for Taxol, 79.1%
100 µg/ml after 72 h incubation with the for the PVA-emulsified nanoparticle formula-
tion and 78.2% for the TPGS-emulsified nano-
particle formulation. The mortality (the
Figure 5. Effects of the incubation time on the cellular
percentage of the CASMCs killed) after 72 h
uptake of the fluorescent PVA- or TPGS-emulsified
culture at 25 ng/ml paclitaxel concentration is
PLGA nanoparticles.
thus 19.5% for Taxol, 20.9% for the PVA-
emulsified nanoparticle formulation and 21.8%
for the TPGS-emulsified nanoparticle formula-
50 PVA
tion, which means that the PVA- and the
TPGS
CASMC uptake of
nanoparticles (%)
40 TPGS-emulsified nanoparticle formulations of
paclitaxel have 1.07- and 1.12-times higher
30
antiproliferative effects than the Taxol after 72 h
20 treatment. Such advantages of the nanoparticle
formulations versus the free drug should have
10 been even more significant if the sustainable
drug-release manner of the nanoparticle formula-
0
1 2 4 6 tion were further considered [16]. The drug release
Incubation time (h) from the nanoparticles for the first 72 h was
found to be 58.6% for the PVA-emulsified nano-
The nanoparticle concentration was 500 µg/ml. Each point represents particles and 43.4% for the TPGS-emulsified
mean ± SD (n = 4). The significance of the TPGS-emulsified versus nanoparticles, respectively (Figure 3). Moreover,
PVA-emulsified nanoparticles is p < 0.01.
the drug release is from 0% at t = 0 to 58.6 or
CASMC: Coronary artery smooth muscle cell; PLGA: Poly(lactic-co-glycolic acid);
PVA: Polyvinyl alcohol; TPGS: D-α-tocopheryl polyethylene glycol 1000 43.4% when Taxol was 100% immediately avail-
succinate. able to the cells. The corrected mortality after
72 h culture at 25 ng/ml paclitaxel concentration
340 Nanomedicine (2007) 2(3) future science group

9. Nanoparticles of biodegradable polymers for restenosis treatment – RESEARCH ARTICLE
Figure 6. Effects of the nanoparticle Another way to evaluate the antiproliferative
concentration on the cellular uptake of effectiveness of the drug in the various formula-
the fluorescent PVA- or TPGS-emulsified tions is to measure their IC50, which is defined
PLGA nanoparticles after as the drug concentration needed to kill 50% of
4 h incubation. the CASMCs at a given period, say in 24 h.
This can be obtained by finding the inter-
section of the viability versus the drug concen-
40 PVA tration curve with a horizontal line of viability
Cell uptake (%) TPGS at 50%. By extrapolation, we can find from
30 Figure 8 that the IC50 in 24 h would be
748 ng/ml for Taxol, 708 ng/ml for the PVA-
20
emulsified nanoparticle formulation and
10 474 ng/ml for the TPGS-emulsified nano-
particle formulation, which implies that the
0 PVA-emulsified nanoparticle formulation is
100 250 500
Nanoparticle concentration (µg/ml) 5.35% more effective than Taxol and the
TPGS-emulsified nanoparticle formulation is
The significance of the TPGS-emulsified 36.6% more effective than Taxol and 33.1%
nanoparticles versus PVA-emulsified is p < 0.01.
more effective than the PVA-emulsified nano-
PLGA: Poly(lactic-co-glycolic acid); PVA: Polyvinyl
alcohol; TPGS: D-α-tocopheryl polyethylene glycol
particle formulation in 24 h treatment. Consid-
1000 succinate. ering the sustainable-release manner of the
nanoparticle formulations, their advantage over
should thus be 0.209/0.586/0.5 = 0.713 for the the free drug should be even greater. If we used
PVA-emulsified nanoparticle formulation and the corrected data in Figure 8, the IC50 would
0.218/0.434/0.5 = 1.005 for the TPGS-emulsi- have been 748 ng/ml for Taxol, 209 ng/ml for
fied nanoparticle formulation, which means
that the PVA- and the TPGS-emulsified nano- Figure 7. Cytotoxicity of the placebo
particle formulations of paclitaxel should actu- PVA- or TPGS-emulsified PLGA
ally have 3.66- and 5.15-times higher nanoparticles (with no drug
antiproliferative effects than Taxol after the encapsulated inside the nanoparticles)
72 h treatment. at various nanoparticle concentrations
As can be seen from the table in Figure 8, the after 72 h incubation with CASMCs.
difference in the measured mortality of the
CASMCs after 72 h culture with the PVA- or
PVA
TPGS-emulsified PLGA nanoparticles at the
120 TPGS
same 25, 250, 500 ng/ml paclitaxel concentra-
Percentage of control
tions is not significant before corrected by drug 100
release, which is 20.9, 33.9 and 38.1% for the 80
PVA-emulsified nanoparticles versus 21.8, 60
38.8 and 48.7% for the TPGS-emulsified
40
nanoparticles. Nevertheless, the cellular uptake
of the nanoparticles after 6 h culture was 20
found before to be 21% for the PVA-emulsi- 0
2.5 25 100
fied nanoparticles compared with 38% for the Nanoparticle concentration (µg/ml)
TPGS-emulsified nanoparticles (Figure 5).
These two results seem to conflict. A fair expla- Data represent mean ± SD with n = 6. There were
nation, however, can be found from the drug- no significant changes in cell viability between
PLGA nanoparticles and the control (p < 0.05),
release kinetics. The 72 h drug release is 55%
although the PVA-emulsified PLGA nanoparticles
for the PVA-emulsified nanoparticles, which is showed a slight decrease in cell viability at high
much higher than the 24% for the TPGS- nanoparticle concentration.
emulsified nanoparticles. The effects of the CASMC: Coronary artery smooth muscle cell;
higher cellular uptake of the TPGS-emulsified PLGA: Poly(lactic-co-glycolic acid); PVA: Polyvinyl
nanoparticles might have been balanced by alcohol; TPGS: D-α-tocopheryl polyethylene glycol
that of the lower drug-release rate. 1000 succinate.
future science group www.futuremedicine.com 341

10. RESEARCH ARTICLE – Feng, Zeng, Lim et al.
Figure 8. Effects of the drug concentration on CASMC viability of the fluorescent nanoparticles from these fig-
after 72 h incubation with the paclitaxel-loaded, PVA- or ures: Figure 9A is the control, Figure 9B is the
TPGS-emulsified PLGA nanoparticle suspension versus Taxol®. PVA-emulsified PLGA nanoparticles, Figure 9C
is the TPGS-emulsified nanoparticles and
Figure 9D is the TPGS-emulsified nanoparticles
Taxol® at 100-times higher resolution. We can see little
100
PVA fluorescence in the control carotid artery wall
CASMC cell viability (%)
TPGS (Figure 9A). After the fluorescent nanoparticle
80 infusion, fluorescence could be clearly observed
in the carotid arteries walls (Figure 9B). The
60 TPGS-emulsified nanoparticles showed advan-
tages in cellular uptake compared with the
40 PVA-emulsified nanoparticles (Figure 9C & D).
As mentioned previously, the infusion time of
20 the fluorescent nanoparticle suspension in the
arteries was 60 s. Such a short period was
0 applied to address the concern of retention of
Mortality (%) 25 ng/ml 250 ng/ml 500 ng/ml the nanoparticles by the arteries in actual prac-
(1) Taxol 19.5 31.7 36.3 tice of local delivery by catheter. It is clear that
(2) PVA 20.9 33.9 38.1 nanoparticle-coated stents could have advan-
(3) TPGS 21.8 38.6 49.7 tages compared with local delivery, which could
(2)/(1) 1.07 1.07 1.05 result in higher nanoparticle retention. This
(3)/(1) 1.12 1.22 1.37 should be further investigated.
(4) 72 h 58.6% for PVA-emulsified nanoparticles
drug release 43.4 % for TPGS-emulsified nanoparticles
Discussion & future perspective
(5) PVA (Corr) 71.3 115.6 130.1
(6) TPGS(Corr) 229.0
Although our in vivo experiment showed effec-
100.5 177.9
(5)/(1) tive internalization of the paclitaxel-loaded,
3.66 3.65 3.58
(6)/(1) 5.15 5.61 6.31 TPGS-emulsified PLGA nanoparticles, further
experiments are needed to show the advantages
The attached table shows the measured CASMC mortality (viability + mortality
of the nanoparticle formulation versus the origi-
= 1) as well as that after the correction made by considering the 72 h drug nal drug in resulting in better therapeutic
release found from the drug-release profiles (Figure 3). The data represent effects. This means that an in vivo restenosis
mean ± SD of n = 6. The significance of the TPGS-emulsified nanoparticles model should be developed by balloon inflation
versus PVA-emulsified nanoparticles is p < 0.01 at 25 ng/ml drug injury, which should then be treated by the nan-
concentration and p < 0.05 at 250 and 500 ng/ml drug concentration. oparticle formulation of paclitaxel in close com-
CASMC: Coronary artery smooth muscle cell; PLGA: Poly(lactic-co-glycolic parison with Taxol. We shall continue this
acid); PVA: Polyvinyl alcohol; TPGS: D-α-tocopheryl polyethylene glycol
1000 succinate.
research as soon as possible.
Although the above research showed that the
nanoparticle formulation of antiproliferative
the PVA-emulsified nanoparticle formulation agents could have advantages versus the original
and 160 ng/ml for the TPGS-emulsified drug for cardiovascular restenosis treatment and
nanoparticle formulation. that the TPGS-emulsified PLGA nanoparticles
These in vitro experiments, of course, are just may have even better effects than the traditional
a preliminary evaluation of toxicity or therapeu- PVA-emulsified PLGA nanoparticles, it is still
tic activity of the nanoparticle formulation. Fur- unclear whether the MDR effects are involved in
ther in vivo study will determine if the the CASMC treatment by paclitaxel, that is,
formulation can be used for clinical trials before whether CASMCs are rich in multidrug pump
it can become a commercial product. proteins (P-glycoproteins). Paclitaxel-eluting
stents are effective in reducing restenosis and one
Arterial uptake of nanoparticles could argue that the current issue of late stent
Figure 9 shows confocal microscopic images of thrombosis could be related to a continued signif-
cross sections of the carotid arteries of rabbits icant reduction in smooth muscle cell prolifera-
that were injured by balloon catheter and then tion as well as endothelial coverage of the stent
infused by the fluorescent nanoparticle suspen- struts, certainly not lack of efficacy of the drug
sion. We can observe the carotid arterial uptake delivery. From this point of view, the nanoparticle
342 Nanomedicine (2007) 2(3) future science group

11. Nanoparticles of biodegradable polymers for restenosis treatment – RESEARCH ARTICLE
Figure 9. Confocal microscopic images of the uptake of the formulation may be more useful for local drug
drug-loaded nanoparticles by carotid arteries of rabbits. delivery for the treatment of cardiovascular
restenosis. Further investigations are needed.
A B Conclusion
We synthesized PVA- and TPGS-emulsified
PLGA nanoparticles to formulate antiprolifera-
tive agents with paclitaxel as a model drug for the
treatment and prevention of cardiovascular reste-
nosis. We found that the nanoparticle formula-
tions of paclitaxel can achieve much higher
cellular uptake and much better in vitro anti-
proliferative effects than Taxol. The emulsifier
150 µm 150 µm used in the nanoparticle preparation process
plays a key role in determining the drug EE,
C D drug-release kinetics, cellular uptake and thus
antiproliferative effectiveness of the formulated
drug. The TPGS-emulsified nanoparticles have
great advantages versus the PVA-emulsified
nanoparticles for local delivery of antiprolifera-
tive drugs, which can also be used in developing
nanoparticle-coated stents.
Acknowledgements
150 µm 20 µm
This research is supported by research grants
(A) Control. (B) Polyvinyl alcohol-emulsified poly(lactic-co-glycolic acid) R-397–000–014–112 (SS Feng: PI), National University
nanoparticles. (C) D-α-tocopheryl polyethylene glycol 1000 succinate (TPGS)- of Singapore (NUS). The authors are grateful of the review-
emulsified nanoparticles. (D) TPGS-emulsified nanoparticles (magnification ers for their thoughtful comments, without which this paper
100×). could not have reached its current status.
Executive summary
• Paclitaxel is one of the most effective antiproliferative agents and has been used in drug-eluting stents; however, owing to its
undesired physicochemical and pharmaceutical properties, it has difficulties in formulation and delivery. Nanoparticles of
biodegradable polymers can help to solve these problems.
• In this study, we prepared paclitaxel-loaded poly(lactic-co-glycolic acid) (PLGA) nanoparticles by a modified solvent
extraction/evaporation method with D-α-tocopheryl polyethylene glycol 1000 succinate (vitamin E TPGS or simply TPGS) as an
emulsifier, which was meant to have advantages versus those prepared by traditional emulsifiers, such as polyvinyl alcohol (PVA).
• Cellular uptake of fluorescent nanoparticles can be visualized and measured in vitro in coronary artery smooth muscle cells
(CASMCs) and in vivo in carotid arteries of rabbits. Both showed excellent effects of the TPGS-emulsified nanoparticles.
• The TPGS-emulsified nanoparticles had a higher drug-encapsulation efficiency, cellular uptake and cytotoxicity than PVA-
emulsified nanoparticle formulations. The IC50 in 24 h culture with CASMCs is only 474 ng/ml for the TPGS-emulsified
nanoparticles in comparison with 708 ng/ml for the PVA-emulsified nanoparticles and 748 ng/ml for Taxol®, respectively.
• TPGS-emulsified PLGA nanoparticles are of great potential for the effective and sustainable delivery of antiproliferative agents and
for the development of nanoparticle-coated stents, which may become the third generation of cardiovascular stents.
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