Poly(N-isopropylacrylamide)-coated thermo-responsive nanoparticles for controlled delivery of

sulfonated Zn-phthalocyanine in Chinese hamster ovary cells in vitro and zebra fish in vivo

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IOP P

UBLISHING

N

ANOTECHNOLOGY

Nanotechnology 18 (2007) 415101 (5pp)

doi:10.1088/0957-4484/18/41/415101

Poly(

N-isopropylacrylamide)-coated

thermo-responsive nanoparticles for
controlled delivery of sulfonated
Zn-phthalocyanine in Chinese hamster
ovary cells in vitro and zebra fish in vivo

Jia He

1

, Ji-Yao Chen

1

,5

, Pu Wang

1

, Pei-Nan Wang

2

, Jia Guo

3

,

Wu-Li Yang

3

, Chang-Chun Wang

3

and Qian Peng

2

,4

1

Surface Physics Laboratory (National Key Laboratory) and Department of Physics,

Fudan University, Shanghai, People’s Republic of China

2

State Key Laboratory for Advanced Photonic Materials and Devices, Fudan University,

Shanghai, People’s Republic of China

3

Department of Macromolecular Science and Key Laboratory of Molecular Engineering of

Polymers, Fudan University, Shanghai, People’s Republic of China

4

Department of Pathology, The National Hospital-Norwegian Radium Hospital,

University of Oslo, Montebello, N-0310 Oslo, Norway

E-mail:

[e-mail]

Received 27 June 2007, in final form 23 August 2007
Published 12 September 2007
Online at

stacks.iop.org/Nano/18/415101

Abstract
Poly(N -isopropylacrylamide) (PNIPAM)-coated Fe

3

O

4

@SiO

2

@CdTe multifunctional

nanoparticles with photoluminescent (PL), thermosensitive and magnetic properties, were
investigated as carriers to deliver water-soluble, fluorescent sulfonated Zn-phthalocyanine (ZnPcS),
a photosensitizing drug for photodynamic therapy of cancer, in Chinese hamster ovary (CHO) cells
in vitro and zebra fish in vivo. PNIPAM is a well-known thermo-responsive polymer with a volume
phase transition temperature. This property allows it to be swollen in water at temperatures lower
than 32–34

◦

C to take up ZnPcS and shrunken to expel the drug at higher temperatures. Since the

PL band of CdTe quantum dots (QDs) as indicators for the nanoparticles is at 585 nm and the
emission band of ZnPcS is at 680 nm, it is possible to study the temperature-dependent release of
ZnPcS from the nanoparticles by fluorescence measurements. ZnPcS was embedded in the
PNIPAM of the nanoparticles at 25

◦

C in phosphate buffered saline (PBS) solution and released at

37

◦

C, measured with a spectrophotometer. When CHO cells had been incubated with the

ZnPcS-loaded nanoparticles at 27

◦

C, a similar intracellular localization pattern of CdTe QDs and

ZnPcS was seen by multichannel measurements in confocal laser scanning microscopy (CLSM),
but a diffuse pattern of only ZnPcS fluorescence was detected in the cytoplasm of the cells at 37

◦

C,

indicating a release of ZnPcS from the nanoparticles. Similar results were also found in the
intestinal tract of zebra fish in vivo after intake of the nanoparticles. Since the nanoparticles contain
magnetic (Fe

3

O

4

) material, the nanoparticles could also be manipulated to change their location in

the intestinal tract of the zebra fish with an external magnetic field gradient of 300 G mm

−1

. The

results presented suggest that such multifunctional nanoparticles may have combined potential for
temperature-dependent drug delivery, QD photodetection and magnetic manipulation in diagnosis
and therapy of diseases.

5

Author to whom any correspondence should be addressed.

0957-4484/07/415101+05

$30.00

1

© 2007 IOP Publishing Ltd

Printed in the UK

Nanotechnology 18 (2007) 415101

J He et al

1. Introduction

Quantum dots (QDs), nanosized semiconductors, with their
unique photoluminescence (PL) properties such as a size-
tunable emission band, a narrow and symmetric emission
profile with a high photoluminescence efficiency and a strong
photostability against photobleaching, have been used in
biomedical fields [

1–5

]. Due to the PL characteristics, QDs

are generally used as probes to label or track biological
targets. To widen biomedical applications, QD-conjugates [

6

]

and hybrids with composite nanostructures such as metal–
QD nanohybrids [

7

] or polymer–QD hybrids [

8

] have recently

been investigated. Furthermore, magnetic nanoparticles are
attractive [

9–12

] because they can be (a) manipulated by

an external magnetic field gradient to control a targeted
delivery and (b) detected by magnetic resonance imaging
as contrast agents.

In addition, some polymeric materials

with stimulus-sensitive characteristics have been explored as
carriers to release chemicals by controlled stimulus [

13

,

14

].

Actually, controlled drug delivery has become one of the most
important topics in the medical field [

15

,

16

]. Apparently, the

combination of these different characteristics would widen the
functions of nanoparticles as drug carriers, and the concept of
multifunctional nanocarriers has been proposed recently [

17

].

Photodynamic therapy (PDT) is a relatively new modality

for a number of diseases such as cancer. This therapy is based
on the combination of a lesion-localizing photosensitizer with
light exposure to destroy the lesion. One of the parameters
affecting the efficacy of this treatment is the selective uptake
of a photosensitizer by a diseased tissue. Samia et al firstly
studied the conjugated QD–silicon phthalocyanine (SiPcS)
for PDT [

18

] and Roy et al proposed that nanoparticle-

entrapped photosensitizing drugs were probably a novel drug-
carrier system for PDT [

19

]. Recent results have shown that

the encapsulation of photosensitizers in nanoparticles may
diminish the interaction of drugs with the biological milieu,
thus facilitating the systemic administration [

20

], enhance the

PDT activity on inactivation of cancer cells in vitro [

21

] and

eradicate tumors in mouse models in vivo [

22

]. A similar

finding was also reported recently with the use of the polymeric
nanoparticles consisting of a surface F3 peptide and imaging as
well as PDT agents. These nanoparticles were not only useful
for photodetection of tumors, but also effective for PDT of the
tumors in a glioma-bearing rat model [

23

]. In our previous

work, we designed and synthesized a novel multifunctional
nanoparticle of poly (

N

-isopropylacrylamide) (PNIPAM)-

coated Fe

3

O

4

@SiO

2

@CdTe possessing photoluminescent

(PL), thermosensitive and magnetic properties [

24

]. In this

study, such nanoparticles were investigated as carriers to
deliver fluorescent sulfonated Zn-phthalocyanine (ZnPcS), a
photosensitizing drug used for PDT, in Chinese hamster ovary
(CHO) cells in vitro and zebra fish in vivo.

2. Experimental details

2.1. PNIPAM-coated Fe

3

O

4

@SiO

2

@CdTe nanoparticles and

ZnPcS loading

The PNIPAM-coated Fe

3

O

4

@SiO

2

@CdTe nanoparticles (about

150 nm in diameter) were synthesized and characterized as

described in our previous report [

18

].

In brief, the pho-

toluminescent QDs (

λ

em

=

580 nm) and magnetic parti-

cles were synthesized, respectively, and then combined to
form Fe

3

O

4

@SiO

2

@CdTe particles with silicon shells. The

nanoparticles were finally coated with a thermosensitive poly-
mer (PNIPAM). In the present study, the water-soluble, fluo-
rescent ZnPcS (Frontier Scientific, Inc) with a concentration
of 0

.

1 mg ml

−1

was mixed with the PNIPAM-coated nanopar-

ticles (0

.

5 mg ml

−1

) in PBS solution and stirred for 24 h at

25

◦

C. The mixed solution was centrifuge-washed with dis-

tilled water at a speed of 10 000 rpm

(

12 000

g

)

for five times to

collect the pellet of ZnPcS-loaded nanoparticles by removing
the un-loaded ZnPcS supernatant. The characteristic emission
spectrum of ZnPcS was confirmed in the solution of the ZnPcS-
loaded nanoparticles, measured with a fluorometer. By com-
paring the emission intensity of ZnPcS in the ZnPcS-loaded
nanoparticle solution with the calibration curve of ZnPcS flu-
orescence intensities with known concentrations, the loading
amount of ZnPcS in the nanoparticles was calculated using the
following formula [

25

]. With this assay, the determined load-

ing content has an error of less than 5% of its value after re-
peating measurements:

Loading content

(

%

)

=

weight of ZnPcS in the nanoparticles

weight of the nanoparticles

×

100%

.

(1)

2.2. Biological systems

Chinese hamster ovary (CHO) cells obtained from the Cell
Bank of the Shanghai Science Academy [

26

] were incubated

in DMEM medium, containing calf serum (10%), penicillin
(100 units ml

−1

), streptomycin (100

μ

g ml

−1

) and neomycin

(100

μ

g ml

−1

), in a fully humidified incubator at 37

◦

C with

5% CO

2

. Zebra fish, an established model of a vertebrate,

was used in experiments in vivo due to the facts that the fish
can live in a relatively low temperature environment and the
body of the fish is so transparent that it is possible to directly
detect the nanoparticles under a microscope. The zebra fish
were raised at 28

◦

C according to the procedure as described in

the literature [

27

].

2.3. PL and fluorescence measurements

The PL of CdTe QDs as the indicators for the nanoparticles
and fluorescence emission spectra of ZnPcS both alone
and embedded into the PNIPAM-coated nanoparticles were
measured by a spectrophotometer (Hitachi, F-2500).

The

localization patterns of CdTe QDs and ZnPcS were studied
in the CHO cells and zebra fish with a confocal laser
scanning microscope (CLSM) (Olympus, FV-300, IX71) using
different channels to detect the 570–630 nm (band-pass filter)
luminescent QD signals and 680 nm ZnPcS fluorescence with
a 640 nm long-pass filter as well as the transmission signals for
simultaneously obtaining the differential interference contract
(DIC) image of cell morphology.

A water immersion

objective (60

×

) and a matched pinhole were used in the

imaging system with a

z

-scan mode to make images from

different layers to construct the three-dimensional distribution
of the nanoparticles and ZnPcS. A 405 nm semiconductor
laser (Coherent) was used as the excitation source in CLSM
measurements.

2

Nanotechnology 18 (2007) 415101

J He et al

2.4. ZnPcS released from the nanoparticles in PBS solution

The ZnPcS-loaded nanoparticles were suspended in PBS
solution (0

.

05 mg ml

−1

) at 25

◦

C as experimental samples

and some of them were heated at 37

◦

C for 30 min. These

suspensions were centrifuged at 10 000 rpm

(

12 000

g

)

to

separate the pellets of nanoparticles and the supernatants,
respectively. The PL of the CdTe QDs and fluorescence of
ZnPcS in both supernatants and the fresh PBS solutions in
which the precipitated nanoparticles had been resuspended
with the same concentration as those before centrifugation,
were measured.

2.5. ZnPcS released from the nanoparticles in the CHO cells

With cells already attached to the bottom of cell dishes with
normal morphology, the ZnPcS-loaded nanoparticles were
added to the cell dishes at the concentration of 0

.

1 mg ml

−1

and

incubated at 27

◦

C for 2 h. At this low incubation temperature

the PNIPAM layer of nanoparticles was so hydrophilic that
it was difficult for the nanoparticles to bind onto the plasma
membrane of cells. However, when a magnet was put beneath
the cell dishes producing a magnetic field gradient of about
400 G mm

−1

, cellular uptake of nanoparticles was achieved in

most cells. After incubation with the nanoparticles, cells were
washed with PBS for three times to remove the extracellular,
unbound nanoparticles. Then some cell dishes were moved
into an incubator with 37

◦

C for another 30 min. The release

of ZnPcS from the nanoparticles was studied by CLSM as
described above. All CLSM measurements were carried out
at room temperature (25

◦

C). The trypan blue exclusion assay

was also used to measure the toxicity of the nanoparticles in
the cells. When cells were incubated with the nanoparticles at
the above concentration, the cell survival rate was 93

±

2%,

comparable to that of control cells.

2.6. ZnPcS released from the nanoparticles in the intestinal
tract of zebra fish

The zebra fish aged around 1 week were used in the in vivo
experiments. The ZnPcS-loaded nanoparticles were added in
fish dishes at the concentration of 0

.

1 mg ml

−1

, and the dishes

were incubated in an incubator at 28

◦

C for 10 h. After the

incubation, the fish were washed and some fish samples were
additionally incubated at 37

◦

C for another 30 min. Finally,

the fish were dipped in pyroxylin on coverslips to limit their
movement during the CLSM measurement.

3. Results and discussion

3.1. ZnPcS embedded in the PNIPAM of nanoparticles

PNIPAM is a well-known thermosensitive polymer with a
volume phase transition temperature, which is swollen in water
at temperatures below lower critical solution temperatures
(LCST) (32–34

◦

C) and shrunken at temperatures higher than

the critical temperatures [

28

].

Such a swelling/deswelling

property of PNIPAM can be used to absorb a water-soluble
drug at a low temperature and release the drug at a higher
temperature. Herein, ZnPcS was added to the nanoparticles
in PBS solution at 25

◦

C which swells the PNIPAM to take up

the drug. The fluorescence emission spectra of nanoparticles
before and after the ZnPcS loading are shown in figure

1

. There

Figure 1. PL emission spectra of the nanoparticles (0

.05 mg ml

−1

)

before (solid) and after (dotted–dashed) the ZnPcS loading. The
dotted curve is the fluorescence of ZnPcS in PBS (pH 7.4) solution
(0

.03 mg ml

−1

). The nanoparticles were suspended in PBS and

excited at 350 nm.

is a typical emission of ZnPcS at 680 nm in the PBS solution
of the ZnPcS-loaded nanoparticles, indicating that ZnPcS had
been embedded in the PNIPAM of nanoparticles.

It was

estimated by comparing the fluorescence intensity of ZnPcS-
loaded nanoparticles at 680 nm band with the calibrating curve
of ZnPcS solutions with known concentrations, that the loading
content of ZnPcS was about 6% according to the formula (

1

),

indicating a micromole level of ZnPcS entrapped per mg
nanoparticles.

ZnPcS is an efficient photosensitizer and a

micromole concentration of the compound is high enough to
kill cells in vitro after light exposure [

29

].

3.2. Release of ZnPcS from the ZnPcS-loaded nanoparticles
in PBS solution

A small amount of ZnPcS was still seen in the supernatant
separated from the ZnPcS-loaded nanoparticle suspension by
centrifugation at 25

◦

C, probably due to the fact that some

ZnPcS molecules were not tightly bound to the PNIPAM
network of the nanoparticles. This was taken as the background
for further fluorescence measurements.

Figure

2

(a) shows

a remarkable increase in the ZnPcS fluorescence in the
supernatant of the ZnPcS-loaded nanoparticle suspension at
37

◦

C for 30 min, suggesting a release of ZnPcS from the

nanoparticles. Moreover, the precipitated nanoparticles were
resuspended in the solution at the same concentration as that
before heating. As shown in figure

2

(b), heating at 37

◦

C

did not affect the PL emission of CdTe QDs, but significantly
reduced the fluorescence intensity of ZnPcS, confirming a
release of the drug.

From the fluorescence data shown in

figure

2

(b) and similar measurements, about 80

±

6% ZnPcS

molecules were estimated to be released from the nanoparticles
by heating at 37

◦

C for 30 min, indicating that the PNIPAM-

coated nanoparticles can be carriers for the delivery of ZnPcS.

3.3. Release of ZnPcS from the ZnPcS-loaded nanoparticles
in CHO cells

The concentration (0

.

1 mg ml

−1

) of the ZnPcS-loaded

nanoparticles used in the present study was not toxic to the

3

Nanotechnology 18 (2007) 415101

J He et al

(a)

(b)

Figure 2. (a) Fluorescence emission spectra of ZnPcS in a
supernatant of the nanoparticle suspension before (solid) and after
(dotted) heating at 37

◦

C for 30 min. Excitation: 350 nm.

(b) Fluorescence emission spectra of the ZnPcS-loaded nanoparticles
before (solid) and after (dotted) heating to 37

◦

C for 30 min. The

inset is an enlarged view of the fluorescence emission spectra from
650 nm to 725 nm. The sample was suspended in PBS at the
concentration of 0

.05 mg ml

−1

and excited at 350 nm.

CHO cells. The release of ZnPcS from the nanoparticles in
the cells was studied by means of CLSM with different filtered
channels. As shown in figure

3

, the nanoparticles were taken

up by the cells after incubation at 27

◦

C with the help of a

magnetic field gradient. Figures

3

(a2) and (a3) show a similar

intracellular localization pattern of CdTe QDs and ZnPcS,
suggesting that ZnPcS is still bound to the nanoparticles.
However, when the temperature was increased to 37

◦

C for

only 30 min, a diffuse localization of ZnPcS differing from
that of CdTe QDs was clearly seen in the cytoplasm of
the cells (figure

3

(b3)), indicating that ZnPcS was released

from the nanoparticles. This finding demonstrates that such
nanoparticles can provide a temperature-dependent delivery of
ZnPcS in CHO cells.

3.4. Release of ZnPcS from the ZnPcS-loaded nanoparticles
in zebra fish

After intake of the ZnPcS-loaded nanoparticles by zebra fish at
28

◦

C, a usual living temperature for the fish, the nanoparticles

Figure 3. Subcellular localization patterns of the nanoparticles (CdTe
QDs as green) and ZnPcS (red) in the CHO cells. Upper panel: the
cells were kept at 27

◦

C. (a1), DIC; (a2), QDs; (a3), ZnPcS; and (a4),

merged image of (a2) and (a3). Lower panel: the nanoparticle-loaded
cells had been incubated at 37

◦

C for 30 min. (b1), DIC; (b2), QDs;

(b3), ZnPcS; and (b4), merged image of (b2) and (b3).

Figure 4. Localization of the nanoparticles (QDs as green) and
ZnPcS (red) in the intestinal tract of the zebra fish. The images were
the same as those described in figure

3

. Upper panel: the fish was

kept at 28

◦

C. Lower panel: the nanoparticle-loaded fish had been

incubated at 37

◦

C for 30 min.

were mainly localized as aggregates in the intestinal tract of the
fish (figure

4

, upper panel). After heating to 37

◦

C for 30 min,

the fluorescence of ZnPcS became more diffuse, while the PL
of the QDs was unchanged (figure

4

, lower panel), demonstrat-

ing a release of ZnPcS from the nanoparticles in the intestinal
tract of the fish. The result suggests that these nanoparticles
have the potential to be used for a drug delivery in vivo.

3.5. Movement of the nanoparticles in zebra fish by the
magnetic field gradient

Since the nanoparticles contain magnetic (Fe

3

O

4

) particles,

the location of the nanoparticles can be manipulated by an
external magnetic field gradient. When the nanoparticles were
in the intestinal tract of the fish, an external magnet with a
gradient of 300 G cm

−1

was used and a series of PL images

of the CdTe QDs as the indicators for the nanoparticles were
recorded by CLSM. Figure

5

shows that the nanoparticles were

effectively moved in the intestinal tract of the fish along the
gradient direction of the magnetic field, suggesting that such
multifunctional nanoparticles may also be used as promising
vehicles to transport loaded drugs toward the diseased cells and
tissues by an external magnetic field in a non-invasive way.

3.6. Phthalocyanines and PDT

PDT of cancer is a combination of light with a tumor-localizing
photosensitizing agent, resulting in photodamage to the tumor.

4

Nanotechnology 18 (2007) 415101

J He et al

Figure 5. Manipulation of the nanoparticles in the intestinal tract of
the zebra fish by an external magnet with the gradient of
300 G cm

−1

. Upper panel: PL images of the nanoparticles. Lower

panel: merged PL and DIC images. The image series from the left to
right were acquired at 0, 3, 12 and 20 s, respectively.

Phthalocyanines are a family of promising photosensitizers
for PDT used in both preclinical and clinical studies [

30–32

].

However, a selective delivery of a phthalocyanine, as for most
other photosensitizers, is one of the challenges for future PDT.
The finding in this investigation shows that the multifunctional
PNIPAM-coated Fe

3

O

4

@SiO

2

@CdTe nanoparticles can carry

and release the ZnPcS in CHO cells in vitro and zebra fishes in
vivo. However, these properties of the nanoparticles may vary
in different biological models. More work is thus needed to
further evaluate whether the nanoparticles are suitable carriers
to thermo-responsively deliver a photosensitizer for PDT.
Moreover, it should be pointed out that the thermosensitive
PNIPAM polymer in the nanoparticles should be modified to
increase the critical temperatures from 32–34

◦

C to the ones

slightly higher than 37

◦

C of the human body temperature, so

that such nanoparticles may be evaluated to see if they are
relevant to humans.

4. Conclusion

PNIPAM-coated Fe

3

O

4

@SiO

2

@CdTe multifunctional nanopar-

ticles with photoluminescent (PL), thermosensitive and mag-
netic properties, were evaluated as carriers to deliver water-
soluble fluorescent sulfonated Zn-phthalocyanine (ZnPcS), a
photosensitizing drug for PDT of cancer, in Chinese hamster
ovary (CHO) cells in vitro and zebra fish in vivo. ZnPcS can
be embedded in the PNIPAM-coated nanoparticles at 25–28

◦

C

and released at 37

◦

C in the PBS solution, CHO cells in vitro

and zebra fish in vivo, probably as a result of the thermody-
namic transition of the PNIPAM network. Moreover, the lo-
cation of such nanoparticles in the intestinal tract of the zebra
fish can be manipulated by an external magnetic field gradi-
ent. The results presented suggest that such multifunctional
nanoparticles may have an important combined potential for
temperature-dependent drug delivery, CdTe QD photodetec-
tion and magnetic manipulation in diagnosis and therapy of
diseases.

Acknowledgments

Financial support from Shanghai Municipal Science and
Technology Commission (06ZR14005 and 05QMX1404), the

National Natural Science Foundation of China (60638010,
50525310 and 50403011) and Visiting Scholar Foundation of
Key Lab in Fudan University is gratefully acknowledged.

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