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RESEARCH PAPER
Dual targeting strategy of magnetic nanoparticle-loaded
and RGD peptide-activated stimuli-sensitive polymeric
micelles for delivery of paclitaxel
Meng Meng Lin
.
Yoon Joong Kang
.
Youngjoo Sohn
.
Do Kyung Kim
Received: 25 November 2014 / Accepted: 8 May 2015
Ó Springer Science+Business Media Dordrecht 2015
Abstract A double targeting strategy of anti-neoplas-
tic agent paclitaxel (PTX) was developed by incorpo-
rating magnetic nanoparticles and RGD peptide for
enhanced cell cytotoxicity effect at lower dosage. A dual
targeting mechanism including magnetic targeting and
RGD ligand-specific targeting enhanced the overall
cytotoxicity and reduced the effective dosage of PTX to
achieve enhanced and sustained release of PTX in vitro.
We addressed the issues of water-insolubility of oleic
acid (OA)-stabilized SPIONs and low incorporation
efficiency of hydrophobic PTX with SPION nanocarri-
ers by using an amphiphilic polymer poly[(N-isopropy-
lacrylamide-r-acrylamide) -b-
L-lac tic acid] (PNAL) as
micelle-forming materials. A targeting moiety,
GGGGRGD peptide, a RGD sequence-containing
peptide with a short linker, is attached to the surface of
PNAL-SPIONs via a homo-crosslinker. Confocal
microscopy image analysis revealed that the cellular
uptake was increased from (1.5 ± 0.5 % (PNAL) to
11.7 ± 0.8 % (RGD-PNAL-SPIONs) at 6 h incubation,
once both RGD peptide and magnetic force attraction
were incorporated into the carriers. Such multi-targeting
nanocarriers showed promising potential in cancer-
oriented diagnosis and therapy.
Keywords Superparamagnetic Iron oxide
nanoparticles Paclitaxel delivery RGD peptide
Nanomedicine
Introduction
In the field of cancer diagnostics and treatments, the
design of an ‘‘all-in-one’’ system that allows simulta-
neous tumor imaging and targeted anti-cancer drug
delivery is of particular interests. Accordingly, consid-
erable efforts have been addressed towards the devel-
opment of multifunctional nanocarriers that deliver the
anti-tumor agents and/or imaging moieties (i.e., metal,
metallic oxide, and semiconductor nanocrystals)
directly to the diseased tissues via sophisticated target-
ing strategies (Kwon et al. 2014; Lin et al. 2010).
Superparamagnetic iron oxide nanoparticles (SPIONs)
are promising candidates for multifunctional nanocar-
riers in cancer-oriented diagnosis and therapy, because
M. M. Lin
Department of Chemical Engineering, Tsinghua
University, Beijing 100084, People’s Republic of China
Y. J. Kang
Department of Biomedical Science, Jungwon University,
Goesan 367-805, South Korea
Y. Sohn
Department of Anatomy, College of Korean Medicine,
Kyung Hee University, Seoul 130-701, South Korea
D. K. Kim (&)
Industry Cooperation Foundation, Konyang University,
Daejeon 302-718, South Korea
e-mail: eurokorean@gmail.com; dokyung@konyang.ac.kr
123
J Nanopart Res (2015) 17:248
DOI 10.1007/s11051-015-3033-2
of their unique and fascinating superparamagnetism
(Lin et al. 2009;Lietal.2014). SPIONs have
demonstrated significant improvement as T
2
contrast
agents over conventional Gd complexes, (Qin et al.
2007) and they also showed promising potentials as
targeted drug carriers for in vivo drug delivery,
attributed to the capability of being localized and
concentrated at the target site by the applied magnetic
field once injected systematically (Yoon et al. 2005).
The primary concerns of cancer imaging/drug
delivery systems (DDSs) are that they should be non-
toxic and have long circulating times within the body.
Once they are injected into the blood stream it would be
ideal that they are not captured by macrophages before
they reach the diseased site to deliver the imaging/drug
moiety. Surface modification of SPIONs with inert-
coating substances with highly hydrophilic and anti-
fouling properties, such as, dextran (Santana et al.
2012) and poly(ethylene glycol) (PEG) (Lee et al.
2006), is a commonly adopted method for the protec-
tion of heterogeneous nanocarriers from the immune
system and prolonged blood circulation time. Biocom-
patible and long-circulating SPIONs have been applied
for magnetic resonance (MR) tracking and magnetic
force-assisted DDS. Peptide and peptidomimetics are
interesting targeting ligands for targeting cell receptors
and internalization, because hundreds of copies of
peptides can be conjugated onto one nanoparticle, thus
peptide-nanoparticles composites show greater binding
affinity toward target cells. TAT peptide has been
employed to conjugate onto mesoporous silica
nanoparticles (MSNs-TAT) with high payload for
nuclear-targeted drug delivery. MSNs-TAT with a
diameter of 50 nm or smaller can efficiently target the
nucleus and deliver the active anti-cancer drug dox-
orubicin (DOX) into the targeted nucleus, killing these
cancer cells with much enhanced efficiencies (Pan et al.
2012). Many potential malignancy-targeting ligands
have been identified as a significant development in
cancer biology, among which peptides containing Arg-
Gly-Asp (RGD) sequence are of particular interest in
tumor targeting, because RGD specifically binds to
integrin receptor a
m
b
3
which has been proved to be
overexpressed in certain cancer types (Zhang et al.
2012). The binding of magnetic nanoparticles to cancer
cells can be enhanced by RGD-integrin interaction
which leads to receptor-mediated endocytosis of RGD-
modified particles (Ito et al. 2007; Lee et al. 2009).
Chen et al. reported improved contrast of fluorescence
imaging of glioblastomas in mice models with RGD-
modified quantum dots (QD) (Cai et al. 2006).
Anderson et al. reported ligand-specific gene delivery
into human primary endothelial cells and embryonic
stem cells using RGD peptide-conjugated polymer/
DNA nanoparticles (Green et al. 2007, 2008). How-
ever, there are very few reports for receptor-mediated
targeting of anti-neoplastic agent delivery by SPION-
based nanocarriers. Moreover, most of the reported
cases are focused on water-soluble drugs, due to the
hydrophilic nature of coating substances of SPION
nanocarriers. It is very important to incorporate
hydrophobic drugs into magnetic nanocarriers.
Paclitaxel (PTX), a chemotherapeutic agent, showed
an exceptional activity against various solid tumors by
disrupting the normal tubule dynamics required for
cellular division thus provoking cell death (Zhang et al.
2013). Despite its proven efficacy and efficiency, PTX
is insoluble in water, which requires complicated
formulations. Currently, Taxol is the most commonly
used formulation for PTX, in which PTX is dissolved in
Cremophor EL and ethanol, as delivery agents. How-
ever, most of the adversary effects of Taxol in clinical
trials are associated with Cremophor EL (Campos et al.
2014). Alternatively, PTX was linked to albumin
nanoparticles, under the trademark Abraxane. In the
search of the more efficient delivery system for
hydrophobic anti-neoplastic agent like PTX, research-
ers have attempted to use SPIONs as potential drug
carriers, to achieve high specificity and selectivity of
the cancer tissues by manipulating the external mag-
netic field (Dilnawaz et al. 2011).
The experimental concept of this work is similar to
magnetofection. Magnetofection is a simple and highly
efficient transfection method that uses magnetic fields
to concentrate particles containing nucleic acid into the
target cells. Instead of nucleic acid, we introduced the
therapeutic molecules. The magnetic particles are then
concentrated on the target cells by the influence of an
external magnetic field generated by magnets. The
cellular uptake of the therapeutic molecules is accom-
plished by endocytosis and pinocytosis, two natural
biological processes. Consequently, membrane archi-
tecture and structure stays intact, in contrast to other
physical transfection methods that damage the cell
membrane. Coupling magnetic nanoparticles to the
therapeutic molecules results in a dramatic increase of
248 Page 2 of 18 J Nanopart Res (2015) 17:248
123
the uptake of the therapeutic molecules and conse-
quently high uptake efficiency.
Herein, we reported a double targeting strategy of
anti-neoplastic agent PTX by magnetic particles and
RGD peptide for enhanced cell cytotoxicity effect at
lower dosage. We addressed the issues of water-
insolubility of oleic acid (OA)-stabilized SPIONs
and low incorporation efficiency of hydrophobic
PTX with SPION nanocarr iers by using an amphi-
philic polymer poly[(N-isopropylacrylamide-r-acry-
lamide)-b-
L-lactic acid] (PNAL) as micelle-forming
materials. PNAL, as previously reported, above
certain concentration, is capable of forming
micelle-like nanostructures in water (Jo et al.
2004a; Qin et al. 2005). The hydrophobic PLLA
segments form aggregates inside the micelles in
which carboxylic acid stabilized-SPIONs and small
hydrophobic drugs can incorporate, while the
hydrophilic PNA interacts with aqueous environ-
ment. A targeting moiety, GGGGRGD peptide, a
RGD sequence-containing peptide with a short
linker, is attached to the surface of PNAL-SPIONs
via a homo-cr osslinker. We investigated the intra-
cellular uptake of several nanocarriers and the tumor
cell inhibitory effect of PTX- encapsulated RGD-
PNAL-SPIONs with the aid of an external magnetic
array, and demonstrated that the physical targeting
and biochemical targeting had a synergistic effect.
Materials and methods
Materials
L-lactide (99.5 %) was a free gift from Purac Bioma-
terials. N-isopropylacrylamide (NIPAAm, 98 %), acry-
lamide (AAm, 99 %), benzoyl peroxide (BPO, 40 wt%
blended in dibutyl phthalate), b-mercaptoethanol
(99 %), poly(vinyl alcohol) (PVA, Mw of 15 kDa),
tetrahydrofuran (THF, 99 %), stannous 2-ethylhex-
anoate (99 %), anhydrous toluene (99.5 %), dibutyl
ether (98 %), and methylene chloride (99 %) disuccin-
imidyl glutarate (DSG, 99.5 %) were purchased from
Sigma-Aldrich. PTX was purchased from Bristol
Meyer Squibb. Peptide NH
2
-GGGGR(Pbf)GD-OH
(RGD) was supplied by New England Peptide Ltd.
Iron (III) chloride hexahydrate (FeCl
3
6H
2
O,
98.0 %), iron (III) nitrate nonahydrate (Fe(NO
3
)
3
9H
2-
O, 97.0 %), cis-9-Octadecenoic acid (oleic acid, OA,
CH
3
(CH
2
)
7
CH=CH(CH
2
)
7
COOH, 90.0 %), sodium
oleate (NaOl, 82 %), hexane (99.0 %), ethanol
(95 %), 1-octadecene (CH
3
(CH
2
)
15
CH=CH
2
,ODE,
90.0 %) were obtained from Sigma-Aldrich. All the
chemicals were used without further purification except
ODE. Volatile substances, such as absorbed water and
organic impurities with a low Mw in ODE, were
evaporated by heating to 200 °C for 3 h prior to use.
Double distilled and deionized water (ddH
2
O) was used
throughout.
Synthesis of random copolymers PNA and diblock
terpolymer PNAL
The copolymer poly(N-isopropylacrylamide-r-acry-
lamide) (PNA) was synthesized according to the
reported procedure (Salehi et al. 2008). PNA copoly-
mers were synthesized at 70 °C for 12 h by free
radical polymerization under anhydrous condi-
tion. 4.5 g NIPAAm, 0.5 g AAm, and 0.075 g b-
mercaptoethanol were dissolved in 15 mL THF as a
solvent. 0.047 g BPO was added to the reaction
mixture as initiator, the reaction was carried out at
70 °C for 12 h under N
2
flow. Once the reaction was
completed, polymers were precipitated again with an
excess amount of diethyl ether; the precipitated
polymers were centrifuged at 4000 rpm for 2 min
and lyophilized for 8 h.
Thereafter, 1 g PNA, 5 g
L-lactide, and 0.22 g
stannous 2-ethylhexanoate were dissolved in anhy-
drous toluene and the reaction was carried out at
110 °C for 7 h under N
2
flowing. The poly[(N-
isopropylacrylamide-r-acrylamide)-b-
L-lactic acid]
(PNAL) was collected by the same procedure as
described in PNA synthesis.
Preparation of PNAL-based drug nanocarriers
OA-coated SPIONs (OA-SPIONs) were prepared
according to the previously published method (Jo
et al. 2004b). The lipid-coated SPIONs were dissolved
in 5 mL hexane, precipitated by 10 mL ethanol, and
centrifuged at 14,000 rpm for 10 min, after which the
supernatant was carefully decanted. The washing
process was repeated 5 times and the lipophilic
magnetic nanoparticles were re-dispersed in 5 mL
hexane, forming a stable magnetic fluid and kept at
4 °C for characterization and further experimentation.
J Nanopart Res (2015) 17:248 Page 3 of 18 248
123
PTX-loaded PNAL-SPIONs were prepared by the
emulsion method. 6 mg PNAL, 1 mg OA-SPIONs,
and 1 mg PTX were dissolved in 400 lL methylene
chloride and mixed with 5 mL aqueous PVA solution
(0.2 wt% in ddH
2
O, w/v %). The mixture was emul-
sified by a probe sonicator at 50 % amplitude and full
cycles for 30 s. The tip of the probe was kept in the
middle of the solution at maximum power. The
obtained PTX-loaded PNAL-SPIONs were cen-
trifuged at 6000 rpm for 30 s.The pellets were then
collected and lyophilized overn ight. The supernatant
containing the free drug was discharged. The PTX-
loaded PNAL nanoparticles were prepared with the
same procedure without adding OA-SPIONs. Drug-
free PNAL nanoparticles and PNAL-SPIONs were
prepared according to the above procedure without
adding PTX.
Functionalization of PNAL NPs and PNAL-
SPIONs
For the purpose of targeted delivery of PTX, a RGD
motif-containing peptide was conjugated to the PTX-
encapsulated PNAL nanoparticles and PNAL-
SPIONs, respectively. 11 lL DSG solution (5 mg/
mL, in DMSO, w/v %) and 30 lL RGD solution
(5 mg/mL , in ddH
2
O, w/v %) were added to 5 mL
PNAL nanoparticle or PNAL-SPION (both PTX-
loaded and drug-free samples) suspension and stirred
at room temperature for 4 h. The PTX-loaded and
PTX-free RGD-PNAL, RGD-PNAL-SPIONs, were
collected by centrifugation at 6000 rpm for 30 s to
remove the unbound RGD peptide. The RGD-attached
nanocarriers were re- dispersed in 0.1 N HCl for 4 h to
remove the 2, 2, 4, 6 , 7- pentamethyldihydrobenzo-
furan-6-sulfonyl (Pbf) protection groups at room
temperature, and centrifuged at 6000 rpm for 30 s
again. The activated RGD-modified particles were
collected and lyophilized.
In order to prepare fluorescent dye-labeled carriers
for tracking the cellular uptake, FITC-bonded particles
were obtained by the reaction between the amino
groups of PNAL and the isothiocyanate groups of
FITC (in excess). 10 lL FITC stock solution (5 mg/
mL, in DMSO, w/v %) was added to PTX-free PNAL,
PNAL-SPION, RGD-PNAL, and RGD-PNAL-
SPION suspension and stirred in dark at room
temperature for 4 h. The FITC-labeled particles were
washed and centrifuged at 6000 rpm three times to
remove the excess amount of unbound FITC, and
lyophilized in the dark.
Particle characterizations
The size and morphology of nanoparticles was exam-
ined on two transmission electron microscopes model
JEOL 2100F (200 kV) and 1230 (120 kV, Japan)
depending on different purposes: the physical sizes of
iron oxide cores were measured on JEOL 2100F
(200 kV); and both core and shell structures were
observed under JEOL 1230 (120 kV). TEM samples
were prepared by placing a few drops of nanoparticle
suspension onto a carbon-coated grid and air drying
under ambient conditions. The surface charge of
particles was characterized by electrophoresis mobil-
ity and zeta-potential using a Malvern HA-3000 Zeta-
sizer, all samples were diluted with ddH
2
O into
*10 lg/mL iron concentration, and the pH was
adjusted with 0.1 N NaOH or 0.1 N HCl to the range
of pH3 to pH12, respectively. The chemical compo-
sition of synthesized polymers was analyzed by an
ALPHA FTIR with a Platinum attenuated total
reflection (ATR) (Bruker Optics, USA). The samples
were dried in oven at 50 °C overnight and ground into
fine powder with a motor and pestle prior to measure-
ment. Spectra were measured with a resolution of
1cm
-1
and the wavenumber range was
500–4,000 cm
-1
.
Loading efficiency and cytotoxicity of drug-free
and PTX-loaded nanocarriers
The loadin g efficiency of PTX and F ITC was
determined by dissolving accurate amounts of the
samples in d ichloromethane. Standard PTX and
FITC soluti on was prepared by dilutin g PTX stock
solution (total 50 mg PTX (5 mg FITC) dissolved in
5 mL dichloromethane) (20, 40, 60, 8 0, and 100 lL)
with dichloromethane and adjusted total volume to
3 mL. The absorbance of a series standard iron
solution at k
max
= 229 nm was measured by a UV–
Vis spectrometer TG80, and plotted against blank as
a function of PTX or F ITC concentration of known
standard PTX or FITC solution.
HeLa cells were cultured in DMEM supplemented
with 10 % fet al bovine serum (FBS), 2 mM
L-glu-
tamine, and 100U/mL penicillin/streptomycin at
37 °C in a humidified atmosphere at 5 % CO
2
in
248 Page 4 of 18 J Nanopart Res (2015) 17:248
123
25 cm
2
cell culture flasks. Medium was changed every
2 or 3 days until 90 % confluence was achieved.
The cytotoxic effect of drug-free carriers (PNAL,
RGD-PNAL, PNAL-SPIONs, and RGD -PNAL-
SPIONs) and PTX-loaded PNAL nanoparticles,
RGD-PNAL, PNAL-SPIONs, and RGD- PNAL-
SPIONs at various concentrations for up to 5 day
incubation time was assessed by 3-[4, 5-dimethylth-
iazol-2-yl]-2, 5-diphenyl tetrazolium bromide (MTT)
assay, according to the supplier’s manual. Control
cells were cultured with complete medium only. Each
experiment was carried out in triplet and repeated
twice. The results were analyzed and plotted in Origin
9.0; one-way analysis of variance (ANOVA) was
performed to compare the means of each sample
groups, in which Tukey’s test was used as means
comparison test, and Levene’s test was used as equal
variance test.
Anti-proliferative activity assay by live/dead two-
coloured cell viability assay
Different dilutions of PTX were prepared in culture
medium using stock solutions of PTX (4.8 mg/mL,
w/v %) in 99 % ethanol. Sim ilarly, a stock suspension
of PTX-loaded PNAL nanoparticles, PNAL-SPIONs,
and RGD-PNAL-SPIONs was diluted appropriately in
culture medium to provide an equivalent amount of
PTX used in solution. Medium and control SPIONs
(without drug) were the respective controls for drug in
solution and drug-loaded carriers. The cytotoxicity of
PTX-loaded samples was verified by live/dead viabil-
ity staining kit (Molecular Probes) and visualized by
confocal microscopy. The live/dead viability kit
provides a two-colour fluorescence cell viability test
based on intracellular esterase activity and plasma
membrane integrity. Cells were seeded in a 24-well
plate at 12 9 10
5
cells/ well 20 h prior treatments
with PTX-loaded nanocarriers for cell attachment.
100 lL of PTX-loaded PNAL nanoparticles, RGD-
PNAL, PNAL-SPIONs, and RGD -PNAL-SPIONs
were added to each well at PTX conce ntration of
0.2 lg/mL , at various time intervals up to 5 days.
After appropriate treatment of cells, 100 lL2lM
calcein AM and 4 lM red fluorescent ethidium
homodimer-1 (EthD-1) solution (in PBS) were added
to each well to completely cover the surfaces, and
incubated for 30 min in dark at room temperature. The
stained cells were immediately examined under a laser
scanning confocal microscope (Olympus). Three cell
images were collected from each sample, and no post-
acquisition enhancing processing was performed.The
experiment was repeated twice at least. The number of
green cells in each micosropcy image was counted and
analyzed in Origin 9.0.
Cellular internalization study
HeLa cell membrane was stained by PKH 26 red
fluorescence cell linker kit (Sigma) according to the
standard protocol suppl ied by the manufacturer.
Briefly, HeLa cells were trypsinized, collected, and
counted on a heamocytometer. 2 9 10
7
cells were
washed once with medium without serum in a
polypropylene tube and collected by centrifuge at
400 rpm for 5 min. The cell pellet was re-suspended in
1 mL dilutent and quickly mixed with 1 mL freshly
prepared 4 9 10
-6
mol PKH26 dye in diluent C in a
polypropylene tube and incubated for 5 min at 25 °C.
2 mL FBS was added to the mixture to stop the
reaction, followed by addition of 4 mL complete
medium. Cells were centrifuged at 400 rpm for
10 min at 25 °C and the loose pellet was washed with
complete medium, for further three times. Finally,
10 mL complete medium was added to the washed cell
pellet and cells were counted again on a heamocy-
tometer. Cells were plated at 5000 cells/well in a
24-well flat-bottom plate in 2 mL complete cell
culture medium in each well for 24 h at 37 °Cin
5%CO
2
.
FITC-tagged drug-fr ee PNAL, RGD-PNAL,
PNAL-SPIONs, and RGD-PNAL-SPIONs were added
to HeLa cell cultures and incubated in dark at 37 °Cin
5%CO
2
atmosphere at different time intervals. Excess
and unbound particles were removed by washing in
PBS three times. Cells were fixed by 4 %
paraformaldehyde in PBS at 25 ° C for 20 min. Three
cell images were collected from each sample, under the
10 9 objective lenses, and no post-acquisition
enhancing processing was performed. The experiment
was repeated twice. The number of green dots and red
viable cells in each micosropic image was counted and
analyzed using Origin 7.0 software. The cellular
uptake of FITC-tagged drug carriers can be ex-
pressed as Cellular uptake %ðÞ¼green dots=red dots
100%.
J Nanopart Res (2015) 17:248 Page 5 of 18 248
123
Results
Morphological and structural characterization
of nanocarriers
Scheme 1a and b shows the synthetic pathway of
random copolymer PNA and diblock terpolymers.
Random copolymer PNA was prepared by free radical
polymerization of NIPAAm and AAm monomers at
60 °C under anhydrous condition, based on the previ-
ously reported method (Salehi et al. 2008). Poly(N-
isopropylacrylamide) (pNIPAAm)-based polymers,
have been frequently reported in heat-triggered drug
delivery nanocarriers in the form of hydrogel, micelles,
and micro/nanoparticles, because pNIPAAm undergoes
a phase transition from hydrophilic into hydrophobic in
water at lower critical solubility temperature (LCST).
Homopolymer pNIPAAm-based triggerable
Scheme 1 Reaction scheme of a synthesis of random copolymer PNA; and b diblock terpolymer PNAL; and c emulsion-freeze-drying
process of encapsulation of PTX into PNAL-SPIONs, followed by RGD peptide conjugation via DSG linkers
248 Page 6 of 18 J Nanopart Res (2015) 17:248
123
nanodevices constantly release the drug once injected
into the body (body temperature 37 °C), because the
LSCT of pNIPAAm was determined to be 32 °C. It is
important to manipulate the LSCT of the nanocarriers
to above 37 °C for two modes of drug release: slow and
sustained drug release at 37 °C and triggerable high-
dose drug release above 37 °C which can be induced by
any means of hyperthermia. Muhammed et al. reported
LSCT of 35.6 °C by controlling the weight ratio of
NIPAAm and AAm monomers (Jo et al. 2004a).
Entezami et al. engineered the LSCT of poly(N-
isopropylacrylamide-acrylamide-vinylpyrrolidone)
hydrogel from 35 to 42 °Cbyvaryingthemonomer
molar ratios (Salehi et al. 2008). Targeted nanocarriers
consisting of SPIONs and pNIPAAm-based copoly-
mers would be interesting but only a few reports have
described magnetically triggered drug release and long-
term sustained release to date. Kohane et al. reported
on-demand drug release from a composite membrane
based on SPIONs and pNIPAAm nanogel by an
external oscillating magnetic field (Hoare et al. 2009).
Ramanujan et al. prepared a core–shell nanocomposite
of pNIPAAm-SPIONs for hyperthermia-induced
release of anti-cancer DOX (Purushotham et al.
2009). However, it is essential to achieve sustained
anti-neoplastic release rather than high-dose release at
once in cancer treatment not only to achieve sufficient
cytotoxic efficacy but also to prevent side effects from
development of drug-resistance and relapsing.
In the bloodstream, opsonins interact with nanopar-
ticles by van der Waals, electrostatic , ionic, and
hydrophobic/hydrophilic forces. Consequently, they
are rapidly removed from the circulation mostly by the
mononuclear phagocyte system. Therefore, the sur-
face features of the nanocarriers have a key role in the
opsonisation process. It is well known that hydropho-
bic and charged particles undergo higher opsonisation
as compared to hydrophilic and neutrally charged
particles (Salmaso and Caliceti 2013).
In order to increase the circulation time, stealth
nanocarriers can be prepared by coating the surface
of nanoparticle with polymers, such as, PEG,
Poloxamines (Tetronics ), poloxamers (Pluronics),
and dextran.
Scheme 1c depicts the preparation route of PNAL-
SPIONs. Ca rboxylic acid-coated SPIONs, with aver-
age diameter of 10 nm, were prepared by a one-pot
thermolysis with strictly controlled molar ratio of Fe
salts and OA (Lin and Kim 2012; Yang et al. 2009; Qin
et al. 2009a). The hydrophobic carbon chain of OA-
SPIONs form an interstitial layer with PLA segment
via hydrophobic interaction, in which hydroph obic
low Mw drug PTX resides, and the PNA forms a
hydrated corona surrounding the hydrophobic core
(Qin et al. 2007, 2009b ). The freeze-drying process is
essential to remove the trace amount of chloroform
solvent, which may be present in the hydrophobic core
of the small micelle-like structure and cause further
harmful reaction. The freeze-dried TAX-PNAL-
SPIONs were readily dispersed in PBS.
Figure 1a and b shows the TEM images of practi-
cally monodispersed OA-SPIONs dispersed in chlo-
roform, with an average diameter of 10 nm; and that
after phase transfer and freeze-drying process,
SPIONs are well dispersed in water, possibly forming
small micelle-like nanostructures that contain only the
water-free hydrophobic aggregates formed by the
single iron oxide nanocrystals. Figure 1c and d shows
a photograph of PNAL nanoparticles (as a control,
prepared by the same protocol without SPIONs) and
PNAL-SPIONs. PNAL nanopa rticles, without incor-
porating magnetic particles, appeared as a white milky
colour. When incorporating OA-SPIONs, PNAL-
SPIONs solution appears as a brownish color. Fig-
ure 1
f shows all PNAL-SPIONs accumulated at the
side of the vial when placing an NdFeB magnet
outside of the vial, and the solution became colorless,
indicating that no PNAL-SPIONs were present in the
solution, and all the PNAL-SPIONs accumulated at
the glass surface next to the mag net. Figure 1g depicts
the chemical structures of the proposed multifunc-
tional nanocarriers of RGD-derived PNAL-SPIONs.
PNAL-SPIONs are assembled from OA-SPIONs with
an average size of 10 nm prepared , with as-prepared
PNAL via an emulsion/evaporation method. SPIONs
with an average diameter of 10 nm were prepared by a
one-pot thermolysis in the presence of carbox ylic acid.
OA-stabilized SPIONs prepared by this method can be
dispersed in non-polar or weakly polar solvents, such
as, hexane , toluene, chloroform, tetrahydrofuran, etc
(Lin and Kim 2012;Parketal.2004).
In this study, OA-SPIONs were transferred into
water with the assistance of the amphiphilic polymer
PNAL, in which OA-SPIONs and low Mw drug PTX
are encapsulated in the hydrophobic PLA segments
and hydrophilic PNA forms a hydrated corona
surrounding the PLA core (Qin et al. 2007, 2009b).
However, instead of forming small micelle-like
J Nanopart Res (2015) 17:248 Page 7 of 18 248
123
nanostructures that contain only the water-free
hydrophobic aggregates formed by the single iron
oxide nanocrystals, usually one micelle-like particle
encapsulates hundreds of SPIONs. Hydrophobic anti-
cancer agent PTX can be loaded into the hydrophobic
layer of OA/PLA; and RGD-derivatized peptide was
conjugated onto the pNIPAAm segment via homo-
crosslinker DSG. The high loading density of SPIONs
without PNAL micelle-like particles increases the
overall hydrodynamic size and often leads to fast and
strong response to an external magnetic field. The
amino functional groups on PNA segments are
available for further conjugation of other imaging
dyes or targeting ligands for sequent nanomedicine
applications. In this study, a peptide-containing RGD
sequence was attached onto the surface of PNAL-
SPIONs via a homo-crosslinker DSG, for enhanced
integrin receptor binding efficacy.
FTIR spectra of PNA and PNAL are compared in
Fig. 2 and Table 1. In the PNA spectrum, three bands
located at 2970, 2940, and 2880 cm
-1
are assigned to
the alkyl group stretching. There is a distinguished
peak appearing at 1540 cm
-1
attributed to the amide II
vibration. The peak at 1460 cm
-1
is assigned to the
Fig. 1 TEM images of a oleic acid-coated SPIONs dispersed in
hexane and b PNAL-SPIONs dispersed in water; and pho-
tograph of c OA-SPIONs dispersion in chloroform phase,
d PNAL-SPIONs dispersion in water phase (complete phase
transfer), e TAX-PNAL-SPIONs in water after 1 week storage,
and f TAX-PNAL-SPIONs in a proximity of a bench NdFeB
magnet. g Schematic illustration of the formation of paclitaxel-
loaded PNAL-SPIONs: the hydrophobic PLA segment of
copolymer PNAL interdigitated with the carbon tail of oleic
acid coating on thermolyzed iron oxide nanoparticles via
hydrophobic interaction; and the hydrophilic segment pNI-
PAAm (ambient temperature \ LSCT) increasing the particle
solubility in water
4000 3500 3000 2500 2000 1500 1000 500
1756
723
1463
2825
2925
1100
1130
1760
1622
1648
1460
1540
2940
Reflectance (%)
Wavelength (cm
-1
)
PNA
PNAL
OA-SPIONs
PNAL-SPIONs
2970
Fig. 2 FITR spectra of PNA, PNAL, OA-SPIONs, and PNAL-
SPIONs from wavelength 4000 to 500 cm
-1
248 Page 8 of 18 J Nanopart Res (2015) 17:248
123
vibration mode of methyl group, the two peaks at 1390
and 1370 cm
-1
correspond to the isopropyl groups
(Qin et al. 2005). Two sharp peak which appeared at
1648 and 1622 cm
-1
, were assigned to C=O stretching
mode. In the spectrum of PNAL, a new sharp peak that
can be assigned to carbonyl stretching appeared at
1760 cm
-1
, and peaks at 1,130 and 1100 cm
-1
can be
assigned to the C–O stret ching mode (Jo et al. 2004a;
Qin et al. 2005). In OA-SPIONs spectra, two bands
appearing at 1737 and 1711 cm
-1
are assigned to C=O
stretching bands of monomer and dimmer OA,
respectively. Two absorption bands appearing at
1588 and 1552 cm
-1
are assigned to asym metrical
carboxylate vibration, whereas the 1464 cm
-1
band
appearing is assigned to symmetrical carboxylate
vibration. A peak that appea red at 723 cm
-1
is due
to Fe–O bonding (Lin and Kim 2012). In PNAL-
SPIONs, the characteristic bands of C=O stretching at
1760 cm
-1
is still strong, indicating PNAL is present
on the surface of iron oxides.
The goal of this work is to develop a more efficient
DDS for anti-neoplastic agents using both magnetic
force and peptide surface activation as dual targeting
strategies for enhanced targeting efficiency, reduced
dosage of anti-neoplastic agent, and decreased side
effects. To investigate the peptide RGD targeting
efficacy, a synthetic RGD peptide was conjugated to
the PNA segment via a homo-crosslinker DSG, as
shown in Scheme 1.
The hydrodynamic sizes of micelle-like particles
with or without incorporation of SPIONs and/or PTX,
and with or without RGD surface activation (dispersed
in ddH
2
O), were in the range of 150–250 nm by DLS,
as shown in Table 2. The mean diameter of drug-free
PNAL micelle-like particles in water was determ ined
to be approximately 155.0 ± 6.2 nm, which is close to
the commercially available product dextran-coated
SPION, Endorem
Ò
(size distri bution from 120 to
180 nm). The hydrodynamic size of PTX-PNAL
particles was determined to be 162.0 ± 4.5 nm,
indicating that the incorporation of low Mw PTX
within the hydrophobic core did not change the
particle size. However, after attaching the RGD
peptide, the particle size increased to
199.0 ± 10.0 nm. This can be explained by the fact
that amine reactive homo-crosslinker DSG was used in
conjugation of the RGD peptide. Even though an
excess amount of DSG linkers and RGD peptides were
Table 1 Assignment of FTIR spectra shown in Fig. 2
Samples IR bands (cm
-1
) Assignment
PNA 2970,2940,2880 Alkyl group stretching
1648,1622 C=O stretching
1540 Amide II stretching
1460 –CH
3
stretching
1390,1370 –(CH
3
)
2
stretching
PNAL 1760 C=O stretching
1460 –CH
3
stretching
1390,1370 –(CH
3
)
2
stretching
1130,1100 C–O stretching
OA-SPIONs 2925, 2825 –CH
2
vibration
1743,1712 C=O stretching
1588,1552 Carboxylate vibration
723 Fe–O stretching
PNAL-SPIONs 1760,1712 C=O stretching
1460 –CH
3
stretching
1390,1370 –(CH
3
)
2
stretching
1130,1100 C–O stretching
1588,1552 Carboxylate vibration
723 Fe–O stretching
Table 2 Hydrodynamic size and zeta-potential (dispersed in ddH
2
O, pH 7.0) of various particles formulations PNAL, PTX-PNAL,
PTX-RGD-PNAL, PNAL-SPION, PTX-PNAL-SPIONs, and PTX-RGD-PNAL-SPIONs
Hydrodynamic particle size (nm) Zeta-potential (mV)
Mean SD Mean SD
PNAL 155.0 ±6.2 -0.7 ±0.6
PTX-PNAL 162.0 ±4.5 -0.7 ±0.5
PTX- RGD-PNAL 199.0 ±10.0 -3.4 ±0.4
PNAL-SPIONs 200.0 ±5.0 0.6 ±0.4
PTX-PNAL-SPIONs 210.0 ±6.0 1.0 ±0.6
PTX- RGD- PNAL-SPIONs 229.0 ±13.2 0.5 ±0.5
J Nanopart Res (2015) 17:248 Page 9 of 18 248
123
used to avoid the cross-linking of two adjacent PNAL
particles such a possibility cannot be completely
eliminated, which resulted in increasing the particle
size and broadeni ng of size distribution after RGD
peptide conjugation. The mean diameter of drug-free
PNAL-SPIONs was shown to be 200.0 ± 5.0 nm,
indicating that incorporation of SPIONs into the PLA
core increases the overall particle size up to 40 nm.
The hydrodynamic size of PTX-PNAL-SPIONs and
RGD-PTX-PNAL-SPIONs was determined to be
210.0 ± 6.0 and 229.0 ± 13.2 nm, showing a similar
trend in PNAL particles wi thout magnetic particles.
The incorporation of low Mw PTX does not increase
the hydrodynamic size of the micelle-like nanostruc-
tures, while conjugation of RGD peptides onto the
surface increases the hydrodynamic size. The zeta-
potential of both drug-free and PTX-loaded PNAL
particles was -0.7 mV in water, indicating that
incorporation of PTX within the hydrophobic core
does not influence the surface charge of the PNAL
nanoparticles. The RGD-PNAL particles showed a
decrease in zeta-potential to -3.4 ± 0.4 mV, con-
firming that RGD conjugation was successful. Inter-
estingly, the zeta-potential of PNAL-SPIONs showed
positive values in water, although the surface charges
were very close to neutral. The drug-free and PTX-
loaded PNAL-SPIONs showed zeta-potential of
0.6 ± 0.4 and 1.0 ± 0.6 mV, respectively, suggesting
that incorpor ation of PTX does not change the surface
charge of the particles. The zeta-potential value of
PTX-loaded RGD-PNAL-SPIONs was decreased
slightly to 0.5 ± 0.5 mV, confirming that addition of
the RGD peptides changes the surface charge of the
PNAL-SPIONs.
Neutral surface charges represent a major advantage
in ligand-specific delivery. Positively charged nanopar-
ticles can be internalized by cells non-specifically due
to the electrostatic interaction between positively
charged particles and negatively charged cell mem-
branes, while neutrally charged particles inhibit the
non-specific cellular uptake and enable only ligand-
specific receptor-mediated internalization.
Cytotoxicity and cellular uptake of the drug-free
nanocarriers
The cytotoxicity of drug-free nanocarriers PNAL
particles, PNAL-SPIONs and RGD-PNAL-SPIONs
(all prepared under the same condition with the PTX-
loaded ones, as negative control) were first assessed by
MTT cell viability assay using the HeLa cell line (a
human cervical adenocarcinomas cell line), up to
120 h incubation time. As shown in Fig. 3, at fixed
PNAL concentration of 120 lg/mL, the viability of
HeLa cells treated with drug-free PNAL, PNAL-
SPIONs, RGD-PNAL-SPIONs up to 120 h [ 95.0 %
does not show significant difference from negative
control (cell viability = 100 %) indicating that all
three drug carriers do not show significant adverse
effect toward the cells.
The cellular uptake of drug-free nanocarriers were
tracked by confocal microscopy, where HeLa cells
membranes were stained with red fluorescent cell
membrane linkers PKH 26 and nanocarriers were
labeled with green fluorescent FITC. As shown in
Fig. 4, the left column shows the general cell
morphologies, all of which represent typical HeLa
cells, indicating no significant adverse effect of the
drug-free nanocarriers to the cells.
This is consistent with t he previous MTT results.
In the negative control cells, no green fluorescence
was observed at all. The right column shows the
green fluorescence dots represe nting the FITC-
tagged carriers. Figure 5 shows the quantitative
24 48 72 96 120
0
20
40
60
80
100
120
Cell viability (%)
Incubation time (hrs)
PNAL
PNAL-SPIONs
RGD-PNAL-SPIONs
Fig. 3 Histograms of cell viability of HeLa cells incubated
with drug-free carriers PNAL (green), PNAL-SPIONs (red ),
and PTX-RGD-PNAL-SPIONs (blue) in the presence of an
external magnetic field at PNAL concentration of 20 mg/mL,
determined by MTT assay. Cells were seeded in 96-well plates
and incubated with different concentrations of all formulations
for 24, 48, 72, 96, and 120 h at 37 °C, 5 % CO
2
. (One-way
ANOVA was performed to compare the cell viability of
different treatments at different time points.) ( n C 5). a 6h,
b 12 h. (Color figure online)
248 Page 10 of 18 J Nanopart Res (2015) 17:248
123
analysis of cellular intake of the drug-free PNAL-
based delivery systems based on the confocal images,
in which the cellular uptake (%) was determined by
number of green dots (representing FITC-labeled
PNAL delivery v ehicles) divided by red dots (repre-
senting the cell number). In drug-free PNAL particle
group, very little cellular uptake (1.5 ± 0.5 %)
appeared at 6 h incubation, and slightly increased
cellular uptake (4.5 ± 0.7 %) at 12 h incubation.
Incorporation of the RGD s equence and magnetic
particles can increase the cellular uptake of nanocar-
riers to 6.8 ± 0.8 % (RGD-PNAL) and 6.6 ± 0.5 %
(PNAL-SPIONs) a t 6 and 12 h incubation, respec-
tively. Both incorporation of the RGD sequence and
magnetic and attachment of the RGD targeting
sequence and magnetic particles in the hydrophobic
core of the micelle-like nanostructure showed a
synergistic effect on cellular uptake (RGD-PNAL-
SPIONs) to 11.7 ± 0.8 % and 13.8 ± 0.5 % at 6 and
12 h incubation, respectively.
Negative
Control
Drug-free
PNAL
Drug-free
RGD-PNAL
Drug-free
RGD-PNAL-
SPIONs
Drug-free
PNAL-SPIONs
(a) (b)
Fig. 4 Representative
confocal images of PKH
26-stained HeLa cells
incubated with FITC-tagged
drug-free carriers PNAL,
PNAL-SPIONs, RGD-
PNAL, and RGD-PNAL-
SPIONs for time interval of
a 6 h, and b 12 h at 37 °C,
5%CO
2
.(Scale bar
represents 200 lm)
J Nanopart Res (2015) 17:248 Page 11 of 18 248
123
Anti-proliferative activity of PTX-loaded
nanocarriers
To investigate the efficacy of the above-mentioned
three drug carriers, the cytotoxicity of PTX (PTX)-
loaded PNAL, RGD-PNAL, PNAL-SPIONs, and
RGD-PNAL-SPIONs was also assessed by MTT
assay at certain time intervals. Differe nt concentra-
tions of all formulated particles were incubated with
HeLa cell in 96-well plates with PTX concentration
ranging from 0.02 to 2.0 lg/mL, which was chosen
according to the plasma level of the anti-neoplastic
drugs, that can reach in humans.[17] Combining both
the magnetic force attraction and peptide binding
affinity to target cells, drug carriers RGD-PNAL-
SPIONs showed the most cytotoxicity effect over
HeLa cells at all PTX concentration compared to the
other two carriers, however, the cell growth cannot be
suppressed completely at low PTX concentration
(0.02 lg/mL, Fig. 6a). Figure 6b shows the cell
survival pattern over 120 h incubation time at
increased PTX concentration of 0.2 lg/mL. Cell
viability of HeLa treated with PTX-PNAL only
decreased to 90.4 ± 9.4 % at 24 h and further reduced
to 57.6 ± 1.4 % at 48 h and remained unchanged
afterward. For those treated with all formulations at
120 h incubation were compared; PTX-PNAL group
showed significant reduction of cell survival of
56.7 ± 6.0 %, RGD-PNAL, PNAL-SPIONs, and
RGD-PNAL-SPIONs groups showed significant
suppression of cell growth (cell viabil-
ity = 13.0 ± 1.1, 20.8 ± 8.7, and 11.3 ± 1.5 %,
respectively) at 120 h, as shown in Fig. 6b. The total
inhibition of cell growth cannot be achieved at such
low PTX concentrations by using PNAL formulation
itself. Incorporation of magnetic particles improved
the cytotoxicity effect significantly, which as done
with the aid of a magnet force induced by incorpora-
tion of SPIONs in the formulation; the cell growth was
completely inhibited after 5-day incubation. Figure 6 c
showed the cell viability pattern of different treat-
ments at very high PTX concentration (2.0 lg/mL). At
such high PTX concentration, the survival rate of cells
with different treatments at 120 h was decreased to
\10 %, and ANOVA showed no significant differ-
ence between different formulations. At such high
PTX dosage, all formulations were able to decrease
the cell survival rate after 120 h effectively and
showed no significant differences according to
AVONA mean comparison Tukey’s test) in Fig. 6c.
To further confirm the efficiency of the three drug
carriers, live/dead cells were visualized by confocal
microscopy by the two-colour cell viability staining,
where green fluorescence represented the general
living cell morphology and red fluorescence showed
the dead cells. Intracellular esterase activity, a unique
feature of the live cells, was determined by the
conversion of non-fluorescent calcein AM into green
fluorescence calcein that is well retained inside the
live cells to produce a strong green fluorescence.
Nega
t
ive control
P
NAL
RGD-PNAL
PNAL-SPI
ONs
R
GD-PNAL-SPIONs
0
2
4
6
8
10
12
14
Cellular uptake (%)
(a)
*
*
*
Negative control
PNAL
RGD-PNAL
PNAL-SPIONs
RGD-PNAL-SPIONs
-2
0
2
4
6
8
10
12
14
16
Cellular uptake (%)
(b)
*
*
*
*
*
Fig. 5 Quantitative analysis of cellular uptake of FITC-tagged
drug-free carriers PNAL, RGD-PNAL, PNAL-SPIONs, and
RGD-PNAL-SPIONs in HeLa cells at time interval of a 6 h, and
b 12 h at 37 °C, 5 % CO
2
.(asterisk the mean of the group is
significantly different from the other groups at p = 0.05 level)
(n C 5)
248 Page 12 of 18 J Nanopart Res (2015) 17:248
123
(EthD-1) was actively excluded by the intact plasma
membrane of live cells, while the molecules can enter
the cellular membrane of dead cells and bind to
nucleic acid to produce a 40 times stronger red
fluorescence inside the dead cells. Viable cells (green)
were quantified in the images to analyze the efficacy
and efficiency of different treatments over 5-day
incubation time. Figure 7 shows that cell survival was
depressed by all treatments compared to the negative
control cells. Statistically, HeLa cells treated with
PTX-loaded PNAL-SPIONs showed lowered viability
compared to the ones with only PNAL; similarly, cells
treated with PTX- loaded RGD-PNAL-SPIONs
showed much lowered viability compared with the
ones treated with RGD-PNAL, suggesting that incor-
poration with SPIONs into the delivery vehicle
increases the cytotoxicity of the DDS, possibly by
the increased sedimentation rate of the whole delivery
system under the magnetic array. However, no signif-
icance was observed between the cell viability
between PTX-loaded PNAL and RGD-PNAL treat-
ment, indicating the RGD peptide conjugation has not
functioned as an effective targeting agent yet in the
first 12 h.
Figure 8 shows the quantitative analysis of cell
survival after being treated with PTX-PNAL, PTX-
PNAL-SPIONs, PTX-RGD-PNAL, and PTX-RGD-
PNAL-SPIONs at different time intervals. Figure 8a
0 20 40 60 80 100 120
30
40
50
60
70
80
90
100
110
Cell viability (%)
Incubation time (hrs)
Negative control
PTX-PNAL
PTX-RGD-PNAL
PTX-PNAL-SPIONs
PTX-RGD-PNAL-SPIONs
(a)
0 20 40 60 80 100 120
0
20
40
60
80
100
120
Cell viability (%)
Incubation time (hrs)
Negative Control
PTX-PNAL
PTX-RGD-PNAL
PTX-PNAL-SPIONs
PTX-RGD-PNAL-SPIONs
(b)
0 20 40 60 80 100 120
0
20
40
60
80
100
Cell viability (%)
Incubation time (hrs)
Negative control
PTX-PNAL
PTX-RGD-PNAL
PTX-PNAL-SPIONs
PTX-RGD-PNAL-SPIONs
(c)
Fig. 6 Viability of HeLa cells without any treatment (negative
control, black) and HeLa cells incubated with PTX-encapsulated
PNAL (red), RGD-PNAL (blue), PNAL-SPIONs (green),and
RGD-PNAL-SPIONs (pink) in the presence of an external
magnetic field at paclitaxel concentration of a 0.02 lg/mL,
b 0.2 lg/mL, and c 2.0 lg/mL, determined by MTT assay. Cells
were seeded in 96-well plates and incubated with different
concentrations of all formulations for 1, 24, 48, 72, 96, and 120 h
at 37 °C, 5 % CO
2
. Negative PTX-loaded PNAL RGD-PNAL
PNAL-SPIONs RGD-PNAL-SPIONs. (Color figure online)
J Nanopart Res (2015) 17:248 Page 13 of 18 248
123
Negative PTX-loaded PNAL
RGD-PNAL
PNAL-SPIONs RGD-PNAL-SPIONs
(b)
(c)
(d)
(e)
(f)
(a)
248 Page 14 of 18 J Nanopart Res (2015) 17:248
123
showed the average viable cell number in each field
after 12 h treat ment; cell survival was depressed by all
treatment compared to the negative control cells.
Figure 8b and c showed a further deduction of viable
cell number for all treatment compared to negative
control at 24 and 48 h time intervals. The difference
between DDS with or without SPIONs remains
statistically significant, suggesting SPIONs not only
function as an immediate targeting moiety, but also
work effectively over longer term. Moreover, the
difference between cells treated with PTX-PNAL and
PTX-RGD-PNAL was significant at p = 0.05 level,
and similarly the difference between PTX-PNAL-
SPIONs and PTX-RGD-PNAL-SPIONs has also
become significant statistically, indicating that RGD
peptide conjugation increases the cytotoxicity of the
DDS possibly due to the RGD-integrin interaction. As
shown in Fig. 8d and e, cell viability was further
dramatically decreased to less than 40 cells left in
average in each image at 72 and 96 h time interval.
After 120 h incubation, HeLa cells without any
treatment (negative control) become over-confluent,
and cell viability with all treatment become almost
zero as shown in Fig. 8f.
Discussions
The ideal anti-cancer agent delivery carrier should
fulfill the following requirements: (1) the carrier itself
should be non-toxic and non-immunogenic; (2) several
targeting strategies may be involved in a corporative
fashion to ensure high specificity and selectivity of the
cancerous tissues; (3) the polymer coating should
ensure a slow release of the drug in a controlled
fashion, or if possible, an external-force triggered flush
of the drug (such as, pH-responsive release or heat-
triggered release and etc.); (4) the carriers should be
monitored by the currently available imaging tech-
niques (such as MRI, ultrasound, PET, and etc.) to track
the release profile of the drug. SPIONs are very
promising anti-neoplastic agent delivery carriers, own-
ing to their biocompatibility and intrinsic superparam-
agnetic properties. It is hypothesized that hydrophobic
anti-cancer drugs can be loaded onto the surface of
SPIONs, which are administered intravenously at the
tumor site with the aid of an external magnetic field, and
meanwhile, the bio-distribution of the drug can be also
monitored by MRI in real time.
In this work the design, synthesis, and characteriza-
tion of a water-soluble PTX-encapsulated RGD-PNLA-
SPIONs was reported. Lipid-coated SPIONs were
firstly produced via thermolysis method, which resulted
in high monodispersity, spherical shape, and high
magnetic saturation. However, OA, which binds to the
iron oxide surface via bidentate coordination, is
extremely difficult to remove and render the particles
water-insoluble and so is incapable of being used in
biomedical applications. An amphiphilic polymer
PNAL, which comprises a hydrophobic poly(lactic
acid) segment and a hydrophilic PNA segment, is able
to form micelle-like structures containing both OA-
SPIONs and PTX in organic solvent like chloroform.
Poly(N-isopropylacrylamide) (pNIPAAm)-based poly-
mers, have been frequently reported in heat-triggered
drug delivery nanocarriers in the form of hydrogel,
(Destribats et al. 2011; Qin et al. 2009b) micelles, (Liu
et al. 2005) and micro/nanoparticles (Lien et al. 2011;
McKee et al. 2011). However, it is essential to achieve
sustained anti-neoplastic release rather than high-dose
release at once in cancer treatment not only to achieve
sufficient cytotoxic efficacy but also to prevent side
effects from development of drug-resistance and
relapsing. By manipulating the molar ratio of NIPAAm
and AAm in the random copolymer PNA, the low
critical solution temperature (LCST) was increased
from 32 °Ctoabove37 °C, to ensure the hydrophilicity
at physiological environment. After freeze-drying, the
residual solvents are completely removed. The as-
prepared PTX-encapsulated PNLA-SPIONs readily
dissolve in water or PBS. The available imide groups
in the PNA segment are abundantly available for
conjugation of bioactive molecules, in our case RGD
peptide. The zeta-potential change before and after
RGD conjugation indicated the successful attachment
of RGD peptide.
Effective biological activity of anti-cancer drug
carriers relies on (1) the biocompatibility of the carrier
itself; (2) the active cancer targeting and effective
b Fig. 7 Representative confocal images of HeLa cells negative
control and those incubated with PTX-encapsulated PNAL,
RGD-PNAL, PNAL-SPIONs, and RGD-PNAL-SPIONs in the
presence of the magnetic array at paclitaxel concentration of
1 lg/mL for time interval of a 12, b 24, c 48, d 72, e 96 h, and
f 120 h at 37 °C, 5 % CO
2
. Cells were stained with live/dead
viability staining kit prior to confocal microscopy examinations.
(Scale bar represents 200 lm)
J Nanopart Res (2015) 17:248 Page 15 of 18 248
123
cellular internalization; and (3) the slow drug release
via diffusion on once entering the cell. The drug-free
carriers, PNAL, RGD-PNAL, PNAL-SPIONs, and
RGD-PNAL-SPIONs showed no significant cytotox-
icity against cells, even at an escalating dosage
(120 lg/mL). The maximum tolerated dosage
Negative control
PTX-PNAL
PTX-RGD-PNAL
PTX-PNAL-SPIONs
PTX-RGD-PNAL-SPIONs
600
700
800
900
Viable cell number
Negative control
PTX-PNAL
PTX-RGD-PNAL
PTX-PNAL-SPIONs
PTX-RGD-PNAL-SPIONs
300
400
500
600
700
800
Viable cell number
(b)
(a)
(d)
(c)
(f)
(e)
Negative control
PTX-PNAL
PTX-RGD-PNAL
P
TX-PNAL-
S
PIONs
P
TX-RGD
-PNAL-S
PIONs
0
200
400
600
800
1000
Viable cell number
Negative control
PTX-PNAL
PT
X-RGD-PNAL
PTX-PNAL-
SPION
s
PT
X-RGD-PNAL-SPIONs
0
200
400
600
800
1000
Viable cell number
Negati
ve
control
PTX-PNAL
PTX-
RG
D
-P
N
A
L
PTX-PNAL-
S
PIONs
PTX-
RGD-P
NAL-SPIONs
0
200
400
600
800
1000
Viable cell number
N
e
gative control
PTX-PN
AL
PTX-RGD-PNAL
PTX-PNAL-SPIONs
PTX-RGD-PNAL-SPIONs
0
200
400
600
800
1000
Viable cell number
Fig. 8 Histograms of viable cell number of HeLa cells without
any treatment and those treated with PTX-PNAL, PTX-PNAL-
SPIONs, PTX-RGD-PNAL, and PTX-RGD-PNAL-SPIONs at
time intervals of a 12, b 24, c 48, d 72, e 96 h, and f 120 h at
37 °C, 5 % CO
2
(n C 5)
248 Page 16 of 18 J Nanopart Res (2015) 17:248
123
(MTD) of these carriers was not determined in this
work, because the tested dosages already exceed 10
times more the dosages required.
a
v
b
3
integrins are overexpressed on various cancer
cells and endothelial cells, which are a good candidate
for active cancer targeting. Hence, the thought of
combining these powerful approaches by developing
an anti-a
v
b
3
and SPIONs-PTX co-encapsulated tar-
geting the tumor cells and their endothelial microen-
vironment have been explored in this work. The
synergism of biochemical and physical targeting was
observed here, which resulted in forwarding a signif-
icant step within the field. The logical explanation lies
with the amount of free drug available to the localized
microenvironment where the cells reside in vitro.
Using PTX-encapsulated PNAL releases PTX from
the polymer particles via diffusion, hence, the con-
centration of free drug available to the cells is lower
when compared to the free PTX at any given time.
When coupling the PNAL particles with RGD target-
ing, no significant difference between RGD-conju-
gated PNAL and the PNAL was observed in the
cellular uptake and cell growth assays. Interestingly,
we showed that cell growth inhibitory activity and
intracellular uptake of our carriers was significantly
improved once SPIONs were incorporated into the
system together with magnetic arrays underneath the
cell culture disc, proving our hypothesis that the first
targeting strategy (direct physical contact) ensureing
the higher concentration of available PTX in the
microenvironment is the prelimi nary requisite for
enhanced cyto toxic effect. Notably, confocal micro-
scopy tracking of the fluorescent-labeled drug-free
nanocarriers has indicated significant increase in
cellular uptake of RGD-PNAL-SPIONs within 12 h,
compared to PNAL-SPIONs in the presence of an
external magnetic field. However, the cell growth
inhibitory effect of RGD-PNAL-SPIONs last for
5 days, among all the PNAL-based formulations,
which confirmed that the PTX is released slowly.
Such slow release profile of our nanocarrier system
provides certain advantages in cancer treatment, in
which the drug-loaded carriers are accumulated at the
tumor site rather quickly to eliminate the PTX release
in circulation, and slowly release to the local microen-
vironment for tumor inhibition
Another challenge of PTX therapy is overcoming
PTX-acquired resistance. Even though PTX is proved
to be effective in breast cancer treatment, still
significant amount of patients do not respond to PTX
well, or they acquired PTX resistance after treatment,
resulting in tumor recurrence. PTX resistance was
reported to be related to the up-regulation of a
v
b
3
and
its signaling pathway, hence, targeting PTX to the
tumors via this particular receptor, attracts attention to
tackle the efflux pumps’ related resistance. Our next
goal would be investigate the bio-distribution and anti-
tumor effect of these formulations in vivo.
Conclusion
A novel magnetic polymeric-based nanocarrier was
developed for cancer-specific delivery of a low Mw
hydrophobic anti-neoplastic agent PTX in this work. A
dual targeting mechanism including magnetic targeting
and RGD ligand-specific targeting enhanced the overall
cytotoxicity and reduced the effective dosage of PTX to
achieve enhanced and sustained release of PTX in vitro.
MTT cytotoxicity assays suggested that the all drug-
free carriers do not show adverse effect on HeLa cells.
At low PTX concentration 0.02 lg/mL, PTX-PNAL
showed reduction of cell viability to 38.0 ± 4.9 %,
while cell viability of PTX-RGD-PNAL-SPIONs group
was 84.8 ± 6.9 % at 120 h incubation. Confocal
microscopy image analysis revealed that the cellular
uptake was increased from (1.5 ± 0.5 % (PNAL) to
11.7 ± 0.8 % (RGD-PNAL-SPIONs) at 6 h incuba-
tion, once both RGD peptide and magnetic force
attraction were incorporated into the carriers. Such
multi-targeting nanocarriers showed promising poten-
tial in cancer-oriented diagnosis and therapy.
Acknowledgments This research i s financially supported by
Basic Science Research Program throug h the National
Research Foundation of Kor ea (NRF) funde d b y the Minist ry
of Education, Science and Technolo gy (Gra nt No. NRF-
2014R1A1A4A03005726).
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