Synthesis and cellular uptake of porphyrin decorated iron oxide nanoparticles - A potential candidate for bimodal anticancer therapy

Article abstract of DOI:10.1039/b507779f

This paper reports the synthesis, characterization, and cellular uptake of the conjugate of porphyrin and iron oxide nanoparticles, which may lead to a bimodal anticancer agent that can be used in the combinational treatment of photodynamic therapy (PDT)

Full text of DOI:10.1039/b507779f

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COMMUNICATION
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Synthesis and cellular uptake of porphyrin decorated iron oxide
nanoparticles—a potential candidate for bimodal anticancer therapy{
Hongwei Gu,a Keming Xu,b Zhimou Yang,a Chi K. Changa and Bing Xu*ab
Received (in Cambridge, UK) 2nd June 2005, Accepted 16th June 2005
First published as an Advance Article on the web 14th July 2005
DOI: 10.1039/b507779f
requirement of HT. We choose magnetite as the superparamag-
netic material because a recently developed synthetic procedure
offers a convenient means to control the sizes and shapes of the
magnetite nanoparticles from 6 to 50 nm in a relatively precise
manner,10,11 which should facilitate the search for the optimal sizes
of the nanoparticles for HT applications. We choose 3 as the
porphyrin derivative12 with which to develop the synthetic route
because 3 is readily available and bears two amino groups for
functionalization. Using these simple starting materials, we
obtained a stable conjugate of porphyrin and magnetite nano-
particles in four steps with an overall yield of 60%. This work not
only offers a method to obtain large amounts of the desired
conjugate, but also meets the most important challenge for HT,
that is, to develop versatile and robust surface chemistry to
functionalize superparamagnetic nanoparticles.3
This paper reports the synthesis, characterization, and cellular
uptake of the conjugate of porphyrin and iron oxide
nanoparticles, which may lead to a bimodal anticancer agent
that can be used in the combinational treatment of photo-
dynamic therapy (PDT) and hyperthermia therapy (HT).
Cancer, the uncontrolled growth and spread of malignant cells that
can affect almost any tissue of the body, remains a major burden
to human health. While the major research activities of
chemotherapy have focused on research and development of
new therapeutic drugs directed against specific targets in cancer
cells,1 substantial efforts have also been devoted to improve
minimal or non-invasive treatment methods against cancer.
Among the non-invasive protocols, hyperthermia (HT)2–4 and
photodynamic therapy (PDT)5–7 are receiving renewed interests
because of the recent advances in the chemistry of photosensitizers6
and the demonstration of HT by AC magnetic field-induced
excitation of superparamagnetic nanoparticles.3 Since HT can
increase the cellular uptake of oxygen molecules,2 which are crucial
for PDT,6 and the preferential uptake of porphyrin derivatives in
tumors6 may help localize the magnetic nanoparticles in tumors
for an HT protocol, it is logical to combine these two modalities to
maximize the efficiency of the treatment of cancer. To construct a
molecular conjugate of porphyrin derivatives (i.e., the photo-
sensitizers) and the nanoparticles of iron oxides (e.g., magnetite) as
the bimodal agents for PDT and HT, we chose porphyrin
derivatives and magnetite as the building blocks because (i) both
porphyrin derivatives and iron oxides are biocompatible; (ii)
biological processes allow both iron oxides and porphyrin
derivatives to be biodegradable after treatment; (iii) the well-
studied pharmacokinetics and low systemic toxicity have already
led to some clinical trials and usage of porphyrin derivatives in
PDT and iron oxides in HT;3,6 and (iv) porphyrin derivatives and
iron oxides are complementary in both properties and functions.
In order to form stable covalent bonds between the porphyrin
derivatives and iron oxide nanoparticles, we choose dopamine as
the molecular anchor because its dihydroxybenzene part binds
tightly to the surface of metal oxides (including iron oxide) via
M–O bonds.8,9 Moreover, the exceptional thermal stability of the
dopamine-based anchor on the iron oxide surfaces9 satisfies the
Scheme 1 illustrates the synthetic pathway for making the
conjugate Fe3O4-porphyrin nanoparticles (5). After an
N-hydroxysuccinimide (NHS) activated derivative of dopamine
(1) reacted with the diaminoporphyrin (3), a simple deprotection to
remove the benzyl groups afforded 4 in good yield (65%). Reacting
4 (5 mg, in 2 mL of MeOH/CHCl3 5 1:1) with magnetite
nanoparticles10 (30 mg, in 5 mL of hexane) in a ultrasonic bath for
60 minutes gave a red-brown mixture, which was centrifuged
and re-dissolved in methanol. After washing the methanol
solution three times using chloroform, high-speed centrifugation
(12000 rpm) afforded the final product 5.
We first used transmission electron microscopy (TEM) to
compare the morphologies of the as-prepared magnetite and 5.
TEM indicated a narrow size distribution (less than 5%) of the
magnetite nanoparticles made by using the procedures of
Sun et al.10 Selected area electron diffraction (SAED) patterns
also showed that the nanoparticles are highly crystalline; the
aDepartment of Chemistry, The Hong Kong University of Science &
Technology, Clear Water Bay, Hong Kong, China.
E-mail: chbingxu@ust.hk; Fax: +852 2358 1594; Tel: +852 2358 7351
bBioengineering program, The Hong Kong University of Science &
Technology, Clear Water Bay, Hong Kong, China
{ Electronic supplementary information (ESI) available: synthesis of 4;
EDP, XPS, ToF-SIMS and magnetic measurement of the conjugate (5).
See http://dx.doi.org/10.1039/b507779f
Scheme 1 Synthesis of Fe3O4-porphyrin (5).
4270 | Chem. Commun., 2005, 4270–4272
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diffraction rings match the crystal planes of magnetite ({220},
{311}, {400}, {422,}, {511} and {440}, Fig. 1A, inset), which was
further demonstrated by high resolution TEM (Fig. 1C). Although
the nanoparticles of 5 slightly aggregate due to the less hydrophilic
porphyrin molecules that are anchored on the surface of the
magnetite, the TEM image of 5 (Fig. 1B) indicates that the
morphology of these nanoparticles changes little. The high-
resolution TEM (HRTEM) image of 5 (Fig. 1D) also indicates
that the crystallinity of the nanoparticles is retained very well,
which assures that the reactions between the dihydroxybenzene
linkers and the surfaces of magnetite nanoparticles do not
drastically alter the magnetic properties of the nanoparticles.
Magnetic measurement (ESI{) on a SQUID magnetometer
indicates that the nanoparticles of 5 are superparamagnetic at
room temperature. The X-ray photoelectron spectrum (XPS) of 5
shows peaks at 712.0 and 726.1 eV (Fe2p), corresponding to the
binding energies of iron in an FeO environment (ESI{). The time-
of-flight secondary ion mass spectrum (ToF-SIMS) of 5 displays a
+
mass peak at m/z 5 177 (FeO2C7H5 ), proving that the
dihydroxybenzene group covalently anchors on the surface of
5 (ESI{).
In addition to the characteristic Q-band of porphyrin at
500–620 nm, the UV-vis spectrum of the methanol solution of 5
exhibits two absorption maxima, 280 nm and 400 nm. The former
originates from the phenyl group, and the latter belongs to the
Soret band of porphyrin. These peaks are similar to the absorption
spectra of the methanol solution of 4, indicating the presence of 4
on the magnetite nanoparticles (Fig. 2A). Being excited at the
wavelength of 400 nm, 5 exhibits two strong emission peaks
located at 580 and 632 nm, which are shifted significantly to the
blue region compared with the emission spectrum of 4 (lem 5 625
and 690 nm). The blue shift in the emission spectrum of 5 also
agrees with the increase of the baseline toward the blue in the
Fig. 2 (A) UV-vis spectra of 4 and 5 and (B) fluorescent spectra
(lex 5 400 nm) of 4, 5 and 5 after boiling for 30 minutes in H2O/MeOH
(inset: fluorescent image of 5 at lex 5 365 nm).
absorption spectrum of 5, which may result from a splitting of the
Soret band buried underneath.13 These blue shifts suggest that
these surface-anchored porphyrin moieties might form p–p dimers
whose porphyrin units exhibit stronger interaction for the
y-polarized transition dipoles.13 The bright-red fluorescent image
of 5 (inset, Fig. 2B) further demonstrates the existence of
porphyrin on the surface of the nanoparticles of 5. The
fluorescence of 5 proves that the presence of superparamagnetic
nanoparticles hardly affects the optical properties of the surface-
bound porphyrin moieties. In other words, the excited triplet state
of porphyrin, through which singlet oxygen is generated, is not
quenched by the superparamagnetic species in close proximity.
We also tested the thermal stability of 5. As shown in Fig. 2B,
after 5 was boiled in a water–methanol solution for 30 min, the
fluorescent spectrum of 5 remained unaffected, suggesting that 4
remains surface-bound on the nanoparticles. This result indicates
that 5 tolerates thermal treatment, which makes it an eligible
candidate for HT.
Before being used as a potential bimodal anticancer agent, this
molecular conjugate of porphyrin and magnetite nanoparticles
should be able to enter cells. To test if 5 meets this prerequisite, we
used cancer cell line HeLa to perform cellular uptake experiments
for 5. The HeLa cells were incubated with the nanoparticles (5) at
37 uC for 5 hours without showing observable dark toxicity. After
the cells were first washed with PBS buffer three times, to remove
residual nanoparticles in solution, the cells were observed under a
fluorescence microscope. Being excited by yellow light (545–
580 nm), 5 exhibits red emission (y610 nm). As shown in Fig. 3D,
Fig. 1 TEM images of the nanoparticles of (A) Fe3O4 and (B) Fe3O4-
porphyrin (5) (inset: SAED patterns); HRTEM images of the nanopar-
ticles of (C) Fe3O4 and (D) Fe3O4-porphyrin (5).
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the cell undergoes apoptosis. For the cells without the uptaken 5,
the same irradiation led to little change in their behaviors and
morphology. This result clearly and qualitatively demonstrates the
phototoxicity of 5.
In conclusion, we have developed a simple, general, and robust
strategy to construct the molecular conjugates of magnetic
nanoparticles and porphyrin derivatives, which already meets
several essential requirements of PDT and HT. This methodology
should lead to a variety of bimodal conjugates like 5 for further
investigations in PDT and HT as well as for other applications in
the emerging field of nanomedicine.
The support by RGC (Hong Kong), DuPont Asia and
European Young Faculty Grant, and HIA (HKUST) is
acknowledged.
Notes and references
Fig. 3 Phase (A, C) and fluorescence (B, D) microscope images of HeLa
cells before and after uptaking 5 intracellularly. Normal HeLa cells
without 5 were observed as the control (A, B). After incubating with 5 for
5 h, HeLa cells, which were cultured on a slide, were washed with PBS
buffer 3 times and then observed with an Olympus BX41 microscope at
61000 magnification (C, D). The fluorescence excitation wavelength
ranges from 545 nm to 580 nm, and the emission wavelength ranges from
610 nm to infrared.
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