Biodegradable magnetic nanocarrier for stimuli responsive drug release

Article abstract of DOI:10.1021/ma500384m

Biocompatible nanocarriers conjugated with magnetic nanoparticle, doxorubicin and poly(ethylene oxide) (PEG) motif have been designed (PVLPEG-PVLDOXI-PCL-PHOS) to create a magnetic vector under magnetic field. Acylhydrazine linker is used to release the drug exactly at the mild acidic conditions resembling the pH of the cancerous cells. All the monomers and polymers are characterized carefully by the routine analytical techniques. Thermogravimetric analysis (TGA), FT-IR spectroscopy and scanning electron microscope (SEM) techniques are employed to confirm the anchoring of iron particle (Fe₃O₄) to the PVLPEG-PVLDOXI-PCL-PHOS. Reservoir capabilities of the newly designed biodegradable nanocarrier are tested by both dynamic light scattering (DLS) and transmission electron microscopy (TEM). Drug release profile from nanocarrier is monitored by fluorimeter. The release profile shows the importance of having the acylhydrazine linker that helps to release the drug at the mild acidic conditions similar to cancerous cells. Confocal laser scanning microscopy (CLSM) and flow cytometry studies on 4T cells indicate that nanocarriers from PVLPEG-PVLDOXI-PCL-PHOS polymer are internalized efficiently. It is very interesting to note that the nanocarriers have exhibited both biologically and magnetically targeting abilities toward 4T cells in vitro.

Full text of DOI:10.1021/ma500384m

Article
pubs.acs.org/Macromolecules
Biodegradable Magnetic Nanocarrier for Stimuli Responsive Drug
Release
Mutyala Naidu Ganivada,† Vijayakameswara Rao N,† Himadri Dinda,† Pawan Kumar,†
Jayasri Das Sarma,*,‡ and Raja Shunmugam†,*
‡
†Polymer Research Centre, Department of Chemical Sciences and Department of Biological Sciences, Indian Institute of Science
Education and Research Kolkata, India
S
* Supporting Information
ABSTRACT: Biocompatible nanocarriers conjugated with
magnetic nanoparticle, doxorubicin and poly(ethylene oxide)
(PEG) motif have been designed (PVLPEG-PVLDOXI-PCL-
PHOS) to create a magnetic vector under magnetic field.
Acylhydrazine linker is used to release the drug exactly at the
mild acidic conditions resembling the pH of the cancerous
cells. All the monomers and polymers are characterized
carefully by the routine analytical techniques. Thermogravi-
metric analysis (TGA), FT-IR spectroscopy and scanning
electron microscope (SEM) techniques are employed to
confirm the anchoring of iron particle (Fe3O4) to the
PVLPEG-PVLDOXI-PCL-PHOS. Reservoir capabilities of
the newly designed biodegradable nanocarrier are tested by both dynamic light scattering (DLS) and transmission electron
microscopy (TEM). Drug release profile from nanocarrier is monitored by fluorimeter. The release profile shows the importance
of having the acylhydrazine linker that helps to release the drug at the mild acidic conditions similar to cancerous cells. Confocal
laser scanning microscopy (CLSM) and flow cytometry studies on 4T cells indicate that nanocarriers from PVLPEG-PVLDOXI-
PCL-PHOS polymer are internalized efficiently. It is very interesting to note that the nanocarriers have exhibited both
biologically and magnetically targeting abilities toward 4T cells in vitro.
inorganic nanoparticles.8 Aliphatic polyesters, with a well-
herapeutic drugs, once administrated, encounter a series
Tof transport barriers, namely, systemic barriers and cellular defined architecture, molecular weight, properties, and free of
barriers before they can start functioning at the tumor site.1a−c
residual catalyst fragments, are a most desirable choice for
developing hybrid materials toward biomedical applications.9a−c
Because of this, conventional small molecule therapeutic drugs
They can be potentially biodegradable through enzymatic and
chemical hydrolysis.10a,b However, due to its hydrophobic
nature, poly-ε-caprolactone (PCL) degrades much slower
compared to other polyesters. But this property can be altered
by appropriate variations in monomer structure, copolymer
composition, and reaction parameters. There are number of
examples reported on chain-end functionalized aliphatic
polyesters by ring-opening polymerization.11a−c However,
pendent functionalization of biodegradable PCL polymers are
not explored in detail for the stimuli responsive drug delivery
applications. Recently we have reported on pH-responsive
norbornene derived amphiphilic polymers to demonstrate the
stimuli responsive drug release.12a,b Herein, we report an
efficient method to prepare a lactone based smart biodegrad-
able nanocarrier (PVLPEG-PVLDOXI-PCL-PHOS-Fe3O4)
using ring-opening polymerization (ROP). In the present
work, we have designed unique copolymers having poly-
exhibit poor pharmacokinetic profiles, and unsatisfactory
therapeutic efficacy. The application of polymeric nanocarriers
to oncology explores the use of macromolecules to enhance
delivery of therapeutic agents.1a−c The improved efficacy and
decreased toxicity are the desired outcomes of using nano-
carriers.2a−d Polymeric nanocarriers, due to the enhanced
permeability and retention (EPR) effect, provide increased drug
solubility, extended drug half-life, and effective targeting to solid
tumors.3a Though there has been number of successful
−e
stimuli-responsive block copolymer based approaches in
nanomedicine,4a−d there are only few Food and Drug
Administration (FDA) approved examples available for the
actual treatment. This is because, in most of the systems, the
percent of drug containing nanocarriers (dose) that reaches the
tumor still remains low.5a,b So intricate approach and novel
strategies are warranted in delivering the cancer drugs.6 This
can be achieved by exploring the diverse self-assembled
structures and shapes with great drug loading capacity, which
can be in blood circulation for a longer time.7a−c
Received: February 20, 2014
Revised: March 27, 2014
Published: April 3, 2014
Long blood circulation due to desirable shape can be
achieved by making hybrid materials of block copolymers and
© 2014 American Chemical Society
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Scheme 1. Synthesis of PVL-PCL, PVL-PCL-PHOS, PVLPEG-PVLDOXI-PCL-PHOS, and PVLPEG-PVLDOXI-PCL-PHOS-
Fe3O4 Copolymers
(ethylene oxide) (PEG), doxorubicin (DOXI), and magnetic
particles all conjugated to the polymeric backbone. Among the
three functionalities, PEG and DOXI are grafted as pendant
motifs to the lactone copolymer, where as the phosphonoic acid
(PHOS) functionality, which is used for anchoring magnetic
particle, is functionalized as end group to the copolymer. We
believe that the newly designed biodegradable copolymer
(PVLPEG-PVLDOXI-PCL-PHOS-Fe3O4) having poly-
(ethylene oxide) (PEG), doxorubicin (DOXI), and a magnetic
nature is expected to behave as smart nanocarrier for both
magnetic resonance imaging (MRI) as well as efficient delivery
vehicle for cancer therapy.
The main objective of this study is to develop an effective
method to conjugate magnetic nanoparticle, doxorubicin and
poly(ethylene oxide) (PEG) motifs to the biodegradable
polyester backbone, which has not been explored. Toward
this goal, lactone 1 was synthesized from δ-valerolactone13,9c in
the presence of lithiumdiisopropylamide and propargyl bro-
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the lactone 1, the copolymerization condition with14 ε-
caprolactone was explored (Scheme 1). The copolymerization
of 1 and ε-caprolactone (CL) was carried out in an inert
nitrogen atmosphere at room temperature for 48 h by ring-
opening polymerization method which was initiated by ethanol
and catalyzed by Sn(OTf)2 in toluene. Three different types of
copolymers namely PVL-PCL1, PVL-PCL2, and PVL-PCL3
were synthesized by changing 1 and CL feed ratios, at constant
M/I = 100 and 2 mol % catalyst relative to the initiator. All the
copolymerizations were quantitative with more than 90%
conversion (Table 1). Among the three copolymers, PVL-
PCL3 copolymer ([lactone 1]:[ε-CL]: [Sn(OTf)2]:[EtOH] =
50:50:0.5:1) was further used for all the studies. The formation
of PVL-PCL3 copolymer was confirmed by GPC and 1H NMR
analysis (Figure 1 and S10 (Supporting Information)). The
GPC trace of PVL-PCL3 copolymer was observed as unimodal
with Mn = 11500 g/mol and PDI = 1.1 using polystyrene
standards (Figure 1a). The observed narrow PDI indicated the
Table 1. GPC Data of all Copolymers
b
c
Mn
Mn
a
d
run
feed ratio (1:CL )
(targeted)
(observed)
PDI
PVL-PCL1
PVL-PCL2
PVL-PCL3
10:90
75:25
11640
13200
12600
9800
12500
11500
1.25
1.38
1.11
50:50
a
b
ε-caprolactone. Theoretical number-average molecular weight
c
(Mn). Mn was determined by GPC in THF relative to linear
poly(methyl methacrylate) standards. Polydisperisty index (PDI) was
d
determined by GPC in THF relative to linear poly(methyl
methacrylate) standards.
mide at −78 °C. The crude product was purified first by
column chromatography, followed by distillation under reduced
pressure at 160 °C, produced lactone (1) as a colorless, viscous
liquid with 45% yield. The formation of 1 was confirmed by
1
observing a new signal at δ 2.0 ppm in H NMR spectroscopy
corresponding to the terminal acetylene protons. After isolating
Figure 1. (a) Representative gel permeation chromatogram of PVL-PCL3; (b) 1H NMR spectrum of PVLPEG-PVLDOXI-PCL-PHOS copolymer
in CD3OD.
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Figure 2. Comparative 31P NMR spectra of PHO, PHOS, and PVL-PCL-PHOS.
controlled polymerization of the monomers to produce PVL-
PCL3. The molar ratio of 1 in PVL-PCL3 was found by
measuring the integral value of acetylene proton at 2.0 ppm and
comparing it with the integral value of CH2−O protons at δ
butyl 2-(4-azidobenzoyl) hydrazine carboxylate was prepared
by coupling reaction between 4-azidobenzoic acid and boc-
protected hydrazine in the presence of catalysts N-(3-
(dimethylamino)propyl)-N′-ethylcarbodiimide hydrochloride/
1-hydroxybenzotriazole. In 1H NMR spectroscopy (Figure S3),
disappearance of signal at δ 13 ppm and appearance of a new
signal at δ 1.4 ppm (boc-methyl protons) suggested the
formation of tertiary butyl 2-(4-azidobenzoyl) hydrazine
carboxylate. It was further confirmed by FTIR and 13C NMR
spectroscopy (Figure S4). The boc-deprotection of tert-butyl 2-
(4-azidobenzoyl) hydrazine carboxylate was carried out by
treating it with trifluoroacetic acid in dichloromethane as
solvent. The absence of signal at δ 1.4 ppm for boc-methyl
proton and appearance of new signal due to -NH-NH2 proton
1
4.03 ppm in H NMR spectroscopy (Figure S10).
To incorporate PHOS functionality (Scheme 1) in PVL-
PCL3, 6-phosphonohexanoic acid was prepared by refluxing 6-
(diethoxyphosphoryl)hexanoic acid ethyl ester with concen-
trated hydrochloric acid. The formation of product was
1
confirmed by H NMR, 31P NMR, and FTIR spectroscopy.
Disappearance of signal at δ 32.7 ppm corresponds to
phosphate ester, and formation of a new signal at δ 26.5 ppm
corresponds to phosphonic acid in 31P NMR spectroscopy
confirmed the product formation (Figure 2). Next, PVL-PCL-
PHOS was prepared by coupling PVL-PCL3 with carboxylic
derivative of phosphonohexanoic acid in the presence of
dicyclohexylcarbodiimide (DCC)/4-dimethylaminopyridine
(DMAP) in dichloromethane solvent. The PVL-PCL-PHOS
copolymer was purified by reprecipitation method using cold
hexane as a solvent. The product formation was confirmed by
1H NMR (Figure S11), FTIR and 31P NMR spectroscopy. The
31P NMR spectrum of PVL-PCL-PHOS copolymer showed a
signal at δ 26.5 ppm which was due to phosphonic acid in
compound 5 (Figure 2). It indicated the attachment of
phosphonohexanoic acid with PVL-PCL3 copolymer. Also, in
FTIR spectroscopy the appearance of the characteristic IR
bands at 1261 and 1015 cm−1 were responsible for to PO
bond and the P−O bond, respectively (Figure S12c). Both 31P
NMR and FTIR spectroscopy confirmed the formation of PVL-
PCL-PHOS copolymer.
1
at δ 3.9 ppm in H NMR spectrum of (Figure S5), which
suggested the complete deprotection of tertiary butyl group
from compound 3. It was further confirmed by FTIR and 13C
NMR spectroscopy (Figure S6). Compound 4 with acylhy-
drazine linker was prepared by addition of doxorubicin
hydrochloride and 4-azidobenzodrazide in methanol in the
presence of catalytic amount of trifluoroacetic acid. The
1
product was confirmed by H NMR and FTIR spectroscopy.
The signals at δ 7.94−7.95 (m, 2H), 7.79−7.80 (m, 2H), 7.77−
7.78 (m, 1H), and 7.36−7.39 (m, 1H) ppm were responsible
for DOXI aromatic group protons which indicated the
formation of product (Figure S7). Also, in FTIR spectroscopy,
the stretching frequency at 2090 cm−1 (due to azide), that
indicated the formation of compound 4 (DOXI-N3) with
acylhydrazine linker. Finally, compound 6 (Scheme S2) was
prepared by coupling reaction of PEG-550 monomethyl ether
and 4-azidobenzodrazide in the presence of DCC)/DMAP in
Next, the possibilities of attaching DOXI motif15 to the PVL-
PCL-PHOS random copolymer was explored (Scheme 1).
Towards this motivation azide-containing doxorubicin with
acylhydrazine linker (DOXI-N3) was synthesized in four steps
as shown in the Supporting Information, Scheme S1. 4-
Azidobenzoic acid was prepared by using well-known
diazotization16 reaction of 4-aminobenzoic acid. The % of
yield was observed maximum (90%) when NaN3 was added
slowly or dropwise fashion for 1 h using additional dropping
1
DCM solvent. It was characterized by H NMR (Figure S8),
FTIR and 13C NMR spectroscopy. From the FTIR spectrum of
compound 6, the stretching frequency of azide was observed at
2095 cm−1, which confirmed the product formation. It was
further confirmed by 13C NMR spectroscopy (Figure S9). A
signal at δ 50.9 ppm corresponded to methylene protons, which
indicated the formation of the compound 6.
After successful synthesis of compounds 4 and 6,
experimental condition for the click chemistry between azide
(compounds 4 and 6) and acetylene (PVL-PCL-PHOS) was
1
funnel. The product formation was monitored by H NMR,
FTIR and 13C NMR spectroscopy (Figure S1 and S2). Tertiary
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1
PHOS copolymer was characterized by H NMR and FTIR
spectroscopy. In 1H NMR spectroscopy of PVLPEG-
PVLDOXI-PCL-PHOS copolymer revealed all characteristic
peaks of DOXI, PEG, phosphate, and the aliphatic polyester
backbone (Figure 1b). In addition, new signal at δ 7.37 ppm
observed due to the triazole proton which confirmed the
formation of PVLPEG-PVLDOXI-PCL-PHOS copolymer.
Also, in the FTIR spectroscopy (Figure S12a), the disappear-
ance of azide signal at 2095 cm−1 in PVLPEG-PVLDOXI-
PCL-PHOS copolymer confirmed the successful click reaction.
It was further confirmed by GPC analysis of PVLPEG-
PVLDOXI-PCL-PHOS copolymer using polystyrene as a
standards, which indicated a substantial increase in molecular
weight, from Mn = 11 500 g/mol (before conjugation) to Mn =
20 400 g/mol (after conjugation), with relatively narrow
polydispersity (PDI) = 1.9 (Figure S16).
The self-aggregation study of PVLPEG-PVLDOXI−PCL−
PHOS copolymer was carried out in water. One mg of
PVLPEG-PVLDOXI−PCL−PHOS copolymer was dissolved
in 1 mL of water. Then the solution was stirred until a clear
solution was obtained. The particle size was measured around
70 nm with 0.34 PDI using dynamic light scattering (DLS).
Further the morphology of the aggregates was determined by
SEM (Figure 3a and b) as well as TEM (Figure 3c, d and e).
From TEM and SEM analysis, the shape of nano aggregates
were observed as nanocapsules and the size of these
nanocapsules was around 70 nm, which was in good agreement
with DLS measurement (Figure S15a). During the magnifica-
tion of the image under the TEM, the capsules started
degrading due to beam daming under the electron beam
(Figure 3e). A recent report suggested that worm, or rod,
shaped aggregates showed a high loading capacity of hydro-
phobic objects per micelle as well as long in vivo circulation
capabilities.19 So the observed nanocapsules would be very
interesting to explore it further as these type of morphologies
would also exhibit in vivo circulation properties due to their
unique nanocapsule shape.
To test the stimuli responsive nature of newly designed
nanocarrier, in vitro drug release studies were carried out before
attaching the magnetic particles to PVLPEG-PVLDOXI-PCL-
PHOS copolymer. For the drug release profile of PVLPEG-
PVLDOXI-PCL-PHOS copolymer was monitored at pH 7.4,
as well as acidic condition. 1 mg sample of PVLPEG-
PVLDOXI-PCL-PHOS copolymer was dissolved in 1 mL of
distilled water. This solution was loaded in dialysis tube (3,500;
Dalton cutoff) and dialyzed against 100 mL of a buffer solution
whose pH was maintained to 6.0. From the above solution, 2.5
mL of solution was taken and its absorption spectrum was
obtained from UV spectroscopy. The absorbance was observed
at 480 nm, as an indication of the release of doxorubicin.
Fluorescence also recorded by exciting the solution at 510 nm.
Emissions of the free DOXI released from PVLPEG-
PVLDOXI-PCL-PHOS were observed at 560 and 590 nm.
The sample was then poured back to maintain the volume of
the solution constant. This procedure was repeated for every 1
h time interval up to 12 h. After 12 h it was observed that there
was no significance increase in the intensity of fluorescence.
Similar procedure was carried out for the drug release at pH
7.4. All the results are shown in Figure S14. The DOXI release
from PVLPEG-PVLDOXI-PCL-PHOS copolymer at pH 7.4
was observed less than 10%, it was interesting to note this
observation as it clearly demonstrated the PVLPEG-
PVLDOXI-PCL-PHOS system’s stability in the physiological
Figure 3. (a and b) SEM images of PVLPEG-PVLDOXI-PCL-PHOS
copolymer; (c−e) TEM images of PVLPEG-PVLDOXI-PCL-PHOS;
(f) Proposed cartoon representation for the observed nanocapsules.
explored. In general, most of the azide and acetylene coupling
reaction were performed either in water or mixed polar
solvents.9c For the first time, in our experiments, the click
reaction was performed in tetrahydrofuran and water (1:1)
solvent sytems. Click reaction was carried out by targeting 50
mol % DOXI and 50 mol % PEG, in the presence of sodium
ascorbate and copper(II) sulfate pentahydrate. The reaction
mixture was allowed to stirr at room temperature for 24 h. The
unreacted compounds 4 and 6 were removed by dialysis in
phosphate buffer solution. Water was evaporated using high
vacuum pump to get the pure and dry PVLPEG-PVLDOXI-
PCL-PHOS copolymer. The PVLPEG-PVLDOXI-PCL-
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Figure 4. (a) TGA data of PVLPEG-PVLDOXI-PCL-PHOS copolymer (A), PVLPEG-PVLDOXI-PCL-PHOS-Fe3O4 copolymer (B), PVLPEG-
PVLDOXI-PCL-PHOS copolymer + Fe3O4 physical mixture (C) and Fe3O4 nanoparticles alone (D); (b) SQUID data of PVLPEG-PVLDOXI-
PCL-PHOS-Fe3O4; insert-Demonstration of influence of permanent magnet on PVLPEG-PVLDOXI-PCL-PHOS-Fe3O4 solution; (c) EDX of
PVLPEG-PVLDOXI-PCL-PHOS-Fe3O4 in water; insert-SEM of PVLPEG-PVLDOXI−PCL−PHOS-Fe3O4; (d) SEM images of PVLPEG-
PVLDOXI-PCL-PHOS-Fe3O4 in water (scale bar =200 nm); (e−g) TEM images of PVLPEG-PVLDOXI-PCL−-PHOS-Fe3O4 in water (scale bar
=100 nm).
condition of human body. It was also very interesting to note
that the maximum 60% drug was release at pH 6.0 as compared
to pH 7.4, suggested the importance of having the acid-labile
acylhydrazine linker in the PVLPEG-PVLDOXI-PCL-PHOS
copolymer.
stability up to 190 °C, which was 40 °C higher than the
PVLPEG-PVLDOXI-PCL-PHOS copolymer. We believed
that the observed increase in stability was due to the
conjugation of particles to the −OH motifs of phosphonic
acid in PVLPEG-PVLDOXI−PCL−PHOS copolymer. The
−OH motifs of the copolymer got degraded at 150 °C, whereas
after attachment of Fe3O4 nanoparticles, thermal stability of
Fe3O4 conjugated copolymer increased up to 190 °C. Our
proposal was further confirmed by a control experiment. TGA
of physical mixture of Fe3O4 nanoparticles and PVLPEG-
PVLDOXI−PCL−PHOS was performed (Figure 4a (C)). It
was interesting to note that the initial degradation point of the
physical mixture was observed at 150 °C. Since it was a physical
mixture, the degradation pattern was similar to the individual
profiles. From wt % of the final residue, the amount of Fe3O4
conjugated to the polymer was calculated. The observed TGA
results were further confirmed by FTIR spectroscopy (Figure
S13). The appearance of the characteristic bands of Fe3O4 at
640 cm−1 in FTIR spectrum from PVLPEG-PVLDOXI-PCL-
PHOS-Fe3O4 copolymer confirmed the conjugation of Fe3O4
nanoparticles to the PVLPEG-PVLDOXI-PCL-PHOS copoly-
mer. The conjugation of Fe3O4 nanoparticles to the PVLPEG-
PVLDOXI-PCL-PHOS copolymer was further confirmed by
SEM, EDX, DLS and TEM. From SEM experiments (Figure
4d), it was observed that the nonocarrier size increases from 70
nm (before conjugation) to 110 nm (after conjugation). This
After demonstrating the stimuli responsive nature of
PVLPEG-PVLDOXI-PCL-PHOS copolymer, Fe3O4 nano-
particle conjugation was carried out following a known
procedure17,18 (Scheme 1). Magnetic nanoparticles were
purchased from Sigma-Aldrich which was having size less
than 40 nm. Fe3O4 nanoparticles were functionalized with the
PVLPEG-PVLDOXI-PCL-PHOS copolymer. Thermogravi-
metric analysis (TGA) on PVLPEG-PVLDOXI-PCL-PHOS-
Fe3O4 confirmed the conjugation of magnetic nanoparticle to
the PVLPEG-PVLDOXI-PCL-PHOS copolymer (Figure
4a(B)). To prove that, TGA analysis of Fe3O4 nanoparticles
alone was performed. It was observed that there was no thermal
degradation up to 500 °C (Figure 4a(D)). Similarly, TGA of
PVLPEG-PVLDOXI-PCL-PHOS copolymer alone was per-
formed. The copolymer was stable up to 150 °C, after that the
first degradation point was started (Figure 4a(A)). The weight
loss of the polymer was observed near about 65% when
it reached the temperature 280 °C. And then the second
degradation point started at 340 °C. The remaining 25% of the
polymer degraded between the temperatures 340 to 400 °C.
The TGA analysis of Fe3O4 conjugated copolymer showed
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Figure 5. (a) Cartoon representation of the magnetic field induced nanocarrier internalization; (b) CLSM images of control molecule. The nucleus
is stained with DAPI; (c) uptake of PVLPEG-PVLDOXI-PCL-PHOS copolymer in 4T cells with different time intervals (12, 24, 36, and 48 h); (d)
cytotoxicity profile of PVLPEG-PVLDOXI-PCL-PHOS copolymer in 4T cells.
was well supported by DLS (Figure S15b) and TEM (Figure
4e−g). The characteristic energy dispersive X-ray (EDX)
spectrum of PVLPEG-PVLDOXI-PCL-PHOS-Fe3O4 con-
firmed the presence of Fe (Figure 4c).
The magnetization study of PVLPEG-PVLDOXI-PCL-
PHOS-Fe3O4 nanocarriers was measured by magnetic property
measurement system (MPMS) magnetometry at 300 K (Figure
4b). From magnetization (M), it was confirmed that the
nanocarrier showed superparamagnetic nature, and hence it was
expected to produce gradient magnetic field. It was very
interesting to observe the movement of nanocarriers in aqueous
solution when they were introduced to the permanent magnet
(insert Figure 4b). We expected that newly designed PVLPEG-
PVLDOXI−PCL−PHOS-Fe3O4 nanocarrier would be effec-
tively utilized for enhanced tumor penetration (Figure 5a).
Nanocapsule from PVLPEG-PVLDOXI−PCL−PHOS with
pH linker provided the additional edge over the free drug as it
could be released slowly inside the cancerous cell. Next, to
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prove the magnetic field induced drug delivery, PVLPEG-
PVLDOXI-PCL-PHOS-Fe3O4 incubated 4T cells were ana-
lyzed using flow cytometer (Supporting Information, Figures
S17 and S18). It was studied in cell lines of a mouse mammary
gland cancer cell line (4T). 4T cells in suspension with
nanocapsule were taken in MACS magnetic column (Miltenyi
Biotec) and kept in the presence of magnetic field as shown in
(Figure 5a). 4T cells in suspension with nanocapsule without
magnetic field was treated as control. Since the nanocapsule
could give emission in the red spectrum due to DOX motif,
APC (Allophycocyanin; Ex- 640 nm and Em-660 nm) channel
was used to perform the flow cytometry analysis. Propidium
iodide experiment confirmed that only living cell populations
were being studied for the drug internalization under the
magnetic field (Supporting Information, Figure S17). It was
very interesting to note the clear intensity shift over cells while
comparing “with magnet” and “without magnet” in APC
channel. On the basis of the intensity shift, it was very
surprising to observe that the much greater internalization of
the nanocapsule in the presence of magnetic field (Supporting
Information, Figure S18). It is well-known that the internal-
ization concentration of a nanocapsule on a target cell actually
depends on the concentration of nanocapsules in extracellular
circumstance.20 So we hypothesized that due to the magnetic
field guiding transfer efficiency of drug vectors was improved.
The cellular uptake behavior of biodegradable nanocapsule was
analyzed using confocal laser scanning microscope (Zeiss, LSM
710). It was obvious from the CLSM images (Figure 5b and c)
that the amount of internalization of DOXI on 4T cells
increased with increasing time as well as magnetic field in
comparison with the control molecule. The cell growth effect
PVLPEG-PVLDOXI-PCL-PHOS copolymer was determined
using trypan blue exclusion method. The effect of cell viability
(Figure 5d) was tested in 4T cells by incubating the
nanocapsules from the copolymer with different concentration,
(250, 500, and 750 μg). Most interestingly, the DOXI
conjugated nanocapsules showed an enhanced antitumor
activity, which could be due to a prolonged retention of the
nanocarrier in the nucleus.
study. This material is available free of charge via the Internet at
http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*(R.S.) E-mail: sraja@iiserkol.ac.in.
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
G.M.N. thanks UGC for the research fellowship and N.V.K.
thank CSIR for the research fellowship. H.D. thanks the DST.
R.S. thanks the Department of Science and Technology, New
Delhi, India, for a Ramanujan fellowship. R.S. and J.D.S. thank
the IISER-Kolkata for providing the infrastructure and start up
funding. All authors thank Dr. Chiranjib Mitra, Department of
Physical Science, IISER-Kolkata, for the MPMS magnetometry
measurements and Koushik Chatterjee and Tanmoy Dalui for
the confocal studies.
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be used as a nanocapsule for intracellular sustained delivery of
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ASSOCIATED CONTENT
■
S
* Supporting Information
1
Experimental section, synthesis scheme of all monomers, H
NMR, 13C NMR and IR spectra of monomers, and a dialysis
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