Cationic porphyrin@SPION nanospheres as multifunctional anticancer therapeutics: Magnetic targeting, photodynamic potential and bio-safety research

Article abstract of DOI:10.1039/c6ra19697g

The synthesis and characterization of carboxyl-functionalized superparamagnetic iron oxide nanoparticles (SPION)-attached cationic porphyrins with different positive charges (porphyrin@SPION) are described as pH-sensitive, multifunctional photosensitization. These nanospheres exhibit excellent targeted cell accumulation under external magnetic field and outstanding cell photocytotoxicity in weak acidic solution, which is the pH for most tumors. The dicationic porphyrin@SPION has the smartest pH-responding release and lowest half-maximal inhibitory concentration (IC₅₀) values after visible light irradiation against HeLa cells. Moreover, the delivery safety of these porphyrin@SPION nanospheres was considered by investigating their behaviors in human plasma during the delivery process. The results elucidate that these nanospheres have no significant deleterious influence on the frame conformation of protein in their delivery process and thus are relatively safe in the drug delivery process. Our study demonstrates that the combination of targeted magnetic-loading drug design methodology with the PDT potential of cationic porphyrins may be a very useful strategy to develop novel tumor-oriented multifunctional anticancer therapeutics.

Full text of DOI:10.1039/c6ra19697g

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Cationic porphyrin@SPION nanospheres as
multifunctional anticancer therapeutics: magnetic
targeting, photodynamic potential and bio-safety
research†
Cite this: RSC Adv., 2016, 6, 103137
Ping Zhao,*ab Min-Chao Liu, Thushara W. Madanayake, Chawla Reena, Min Zheng,
a
b
b
c
a
a
a
Zhen-Feng Cheng, Yu-Min Huang and Xia-Hong Wang
The synthesis and characterization of carboxyl-functionalized superparamagnetic iron oxide nanoparticles
SPION)-attached cationic porphyrins with different positive charges (porphyrin@SPION) are described as
(
pH-sensitive, multifunctional photosensitization. These nanospheres exhibit excellent targeted cell
accumulation under external magnetic field and outstanding cell photocytotoxicity in weak acidic
solution, which is the pH for most tumors. The dicationic porphyrin@SPION has the smartest
pH-responding release and lowest half-maximal inhibitory concentration (IC50) values after visible light
irradiation against HeLa cells. Moreover, the delivery safety of these porphyrin@SPION nanospheres was
considered by investigating their behaviors in human plasma during the delivery process. The results
elucidate that these nanospheres have no significant deleterious influence on the frame conformation of
protein in their delivery process and thus are relatively safe in the drug delivery process. Our study
demonstrates that the combination of targeted magnetic-loading drug design methodology with the
PDT potential of cationic porphyrins may be a very useful strategy to develop novel tumor-oriented
multifunctional anticancer therapeutics.
Received 4th August 2016
Accepted 9th October 2016
DOI: 10.1039/c6ra19697g
www.rsc.org/advances
hydrophobic and strongly aggregate in aqueous media, which
Introduction
signicantly reduces their therapeutic efficacy because only
11
In recent years, the well-described photosensitizing properties monomeric species are appreciably active. Moreover, it is
of porphyrins have led to their potential use as sensitizers and frequently reported that this therapeutic method, PDT, is not
adjuvants in a variety of biomedical applications, including highly selective to tumorous tissue. One of the side effects of
1
diagnostic imaging (e.g. uorescence imaging, magnetic reso- PDT is prolonged skin photosensitivity due to sensitizer accu-
2
12
nance imaging (MRI) ), therapies (e.g. photodynamic therapy mulation in normal cells. These drawbacks signicantly limit
3
4
(
PDT), sonodynamic therapy (SDT) and boronneutron capture the biomedical applications of porphyrin and its related
13
5
therapy (BNCT). Cationic porphyrins, represented by meso- compounds in PDT.
tetrakis(4-N-methylpyridiniumyl)porphyrin (TMPyP4), have
The basic design to overcome these drawbacks is to incor-
attracted special attention for their additional tight interaction porate certain factors (such as drug loading, pH controlling,
and efficient photocleavage with DNA. Numerous cationic bioaffinity modication, etc.) into a conventional sensitizer to
porphyrins have been designed and some of them exhibit control the competition between the accumulation in normal
6,7
excellent anticancer abilities. Our group has also devoted cells and the targeted tumor tissues. Recently, researchers have
great efforts to this research area and successfully contributed been paying more attention to smart PDT treatments of cancer
8–10
more than 10 series of cationic porphyrins.
However, most by integrating drug loading technology and pH-base trigger. Li
porphyrins used clinically or in preclinical development are et al. prepared a pH-activatable smart nanoparticle by adsorb-
ing a cationic photosensitizer of meso-tetra(N-methyl-4-pyridyl)
porphine (TMPyP) onto the surface of SiO or TiO2 nano-
2
a
School of Chemistry and Chemical Engineering, Guangdong Pharmaceutical
12,14
particles.
Wang, Zhang and co-workers prepared a pH-
University, No. 280, Waihuandong Road, Education Mega Centre, Guangzhou
responsive nanoparticle-based platform where both hydro-
phobic porphyrin and pH indicator bromocresol purple (BCP)
5
10006, PR China. E-mail: zhaopinggdpu@outlook.com
b
College of Pharmacy, University of Arizona, Tucson, Arizona 85721, USA
c
School of Basic, Guangdong Pharmaceutical University, No. 280, Waihuandong Road, were encapsulated in organically modied silica nano-
15
Education Mega Centre, Guangzhou 510006, PR China
particles. This work conrms that the use of organically
modied silica nanoparticles retains both photosensitization
†
Electronic supplementary information (ESI) available. See DOI:
0.1039/c6ra19697g
1
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efficacy and selective accumulation in tumor cells due to the This contribution also concerned the bio-safety of delivery
nanopermeability. process for this system. The interactions between porphyr-
However, although individuals have already shed light on in@SPION and human serum albumins (HSA), which are
incorporating nano-supporting and pH-base skills in PDT abundantly found in blood plasma and function as carriers for
therapeutics, as far as we know, the magnetic targeting factor numerous exogenous and endogenous compounds in the body,
has not been integrated with them. Among kinds of targeted were investigated using the spectroscopic techniques and
drug nano-deliverers, the superparamagnetic iron oxide nano- molecular modeling methods at the molecular level.
particles (SPION) are particularly interesting since they are
devoid of magnetic remanence due to their small size. In the
last decades, SPION-based ferrouids have been developed as
contrast agents for MRI and as heating intermediates for
Experiments
Materials
18
magnetic hyperthermia. The enhanced interest of the SPION– SPION was prepared by the reported method and porphyrins
drug associates is mostly related to the potentially combined were synthesized in our lab. Succinic anhydride and triethyl-
16,17
roles of targeted therapy and diagnosis.
amine, which were used in the preparation of SPION–COOH,
Meanwhile, despite of the various efforts on the loading work were obtained from J&K Chemical (China). RPMI 1640 medium,
of cationic porphyrins, currently only tetracationic TMPyP was fetal bovine serum (FBS), trypsin-EDTA solution and Penicillin-
intensively employed. The inuence of positive charges on the streptomycin solution were purchased from Gibco Life Tech-
PDT efficiency has not been considered. Herein in the present nologies. Water-soluble tetrazolium (WST), Hoechst 33258, and
work, a series of porphyrin with different positive charges (see N,N-dimethyl-4-nitrosoaniline (RNO) were obtained from
structures in Fig. 1) were successfully loaded on the surface of Sigma-Aldrich. Reactive Oxygen Species Assay Kit was obtained
carboxyl-functionalized SPION (SPION–COOH, see schematic from Beyotime Biotech (China). The human blood was supplied
diagram in Fig. 2), which has deprotonated and thus negatively by People's hospital of Zhongshan, Guangdong, China. All the
18
charged carboxylic groups at physiological pH. The pH cell lines were supplied by ATCC company of America. Human
controlled release, cellular uptake activity, magnetic cell accu- serum albumins (HSA) were obtained from Sangon Biotech
mulation and photocytotoxicity of these nanospheres were (China). Measurements at xed pH were made using 0.02 M
ꢀ
comparatively studied under the pH conditions of tumor and phosphate buffer. The solutions were stored at 0–4 C and
normal cells. The number of positive charges of the porphyrin shaken gently as needed to redissolve the contents. All the other
molecules were proved to have signicant inuence on the chemicals were of analytical grade, and Millipore water (18.2
photocytotoxicity efficiency and dicationic porphyrin loaded on MU) was used throughout the experiments.
SPION–COOH exhibit even better PDT potential than TMPyP.
Instrumentation
The scanning electron microscopy (SEM) analyses were per-
formed with a JSM-6330F eld emission scanning electron
microscope. The transmission electron microscope (TEM)
analyses were performed with a JEM-2010HR transmission
electron microscope. Solid UV-Vis and FTIR spectra were recor-
ded on a Shimadzu UV-3150 spectrophotometer and an Equinox
55 Fourier transformation infra-red spectrometer. Magnetic
measurements were performed using a XL-7 magnetic property
measurement system. Fluorescence microscopy of apoptosis
assays was performed with an OX31 uorescence microscope
(Olympus, Japan). Electronic absorption spectroscopy was
recorded on a Perkin-Elmer-Lambda-850 spectrophotometer
and uorescence spectroscopy was recorded on a Perkin-Elmer
L55 spectrouorophotometer. CD spectra were measured on
a J-810 CD-Shade spectrophotometer (Jasco, Japan). Statistical
analysis was performed using origin 9.0. Comparisons between
two groups were performed by an unpaired t test. Multiple
comparisons between more than two groups were performed by
one-way analysis of variance. Signicance was accepted at a P
value lower than 0.05. The pH values were measured using
a BeckmanpH meter (United States).
Fig. 1 The synthesis scheme of cationic porphyrins loaded on SPION.
Preparation of SPION–COOH
To a solution of 50 mg of succinic anhydride in distilled DMF,
50 mg aminated SPION nanoparticles dispersed in 10 mL DMF
Fig. 2 The schematic diagram of porphyrin@SPION.
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were added dropwise with ultrasonication. A catalytic amount 3, with 4-pyridinecarboxaldehyde was replaced by 1.4 mL (0.02
of distilled triethylamine was added to the resulting suspension mol) 4-pyridinecarboxaldehyde and 2.4 g (0.02 mol) p-hydrox-
and the reaction was stirred for 24 h. The particles were nally ybenzaldehyde, and the eluent was replaced by CH Cl /EtOH ¼
2
2
recovered by magnetic concentration, washed with distilled 18 : 1. The purple powder obtained from the second band was
water and dried using acetone. SPION–COOH was thus ob- then puried by thin layer chromatography (TCL) use CH
tained as dark brown powder.
2 2
Cl /
EtOH ¼ 20 : 1 as eluent to get 5,15-di(1-pyridinium-4-yl)-10,20-
The carboxyl groups on the surface of SPION–COOH was di(4-methoxyphenyl)porphyrin, which was then methylated by
determined as following: to a solution of 50 mg of SPION–COOH, an excess amount of methyl iodide afforded target compounds.
ꢁ
1
1
2
3
0 mL 0.1 mol L NaHCO solution was added. Aer shaking Yield: 93.7%. The spectroscopic results obtained from H NMR
overnight, the magnetic nanospheres were removed, and 3 drops (500 MHz, DMSO): chemical shi d: 9.34 (d, J ¼ 7.75 Hz, 4H, 2,6-
of 0.1% methyl orange solution was added to the supernatant. pyridinium), 9.04 (d, J ¼ 7.70 Hz, 4H, 3,5-pyridinium), 9.01 (d, J
ꢁ1
The supernatant was then titrated with 0.09458 mol L dilute ¼ 9.90 Hz, 8H, pyrrole), 8.27 (d, J ¼ 8.41 Hz, 4H, 2,6-phenyl),
+
hydrochloric acid solution. The carboxyl groups were thus calcu- 6.96 (d, J ¼ 5.55 Hz, 4H, 3,5-phenyl), 4.72 (s, 6H, N –Me), 2.56 (s,
lated to be 18.14% according to the titration results.
4H, –CH
Anal. calcd for C50
Found: C, 55.24; H, 4.25; N, 8.17. ES-MS EtOH, m/z: 790 (M ),
2
), 1.29 (d, J ¼ 7.71 Hz, 6H, –CH
3
), ꢁ2.89 (s, 2H, NH).
42 6 2 4 2
H N I O $H O: C, 56.51; H, 4.17; N, 7.91.
+
Synthesis of cationic porphyrins
2+
395 (M ). UV-Vis (10 mM in Tris buffer), lmax (nm) (log 3):
19
Classical Adler–Longo method was used to synthesize the 422(5.01), 520(3.91), 561(3.72), 582(3.41), 644(3.28).
porphyrins, whose synthetic pathway was given in Fig. 1.
5-(4-Methoxyphenyl)-10,15,20-tri(1-methylpyridinium-4-yl)
,10,15,20-Tetra(N-methyl-4-pyridyl)porphyrin (TMPyP4, 1). porphyrin (4). Similar method as 1 was employed to synthesize
5
A mixture of 150 mL propionic acid, 7 mL acetic anhydride was 4, with 4-pyridinecarboxaldehyde was replaced by 0.7 mL (0.01
ꢀ
heated at 130 C with stirring. 2.7 mL (0.04 mol) 4-pyr- mol) 4-pyridinecarboxaldehyde and 3.6 g (0.03 mol) p-hydrox-
idinecarboxaldehyde and 3.8 mL (0.04 mol) pyrrole were then ybenzaldehyde, and the eluent was replaced by CH
2
Cl
2
/EtOH ¼
added separately from dropping funnels to the reuxing 15 : 1. The purple powder obtained from the third band was
mixture. The resulting mixture was reuxed for 1.5 h. The then methylated by an excess amount of methyl iodide afforded
solvent was subsequently evaporated under reduced pressure. target compounds. Yield: 92.7%. The spectroscopic results ob-
1
The residue was then puried by column chromatography with tained from: H NMR (500 MHz, DMSO): chemical shi d: 9.33
CH
2
Cl
2
/EtOH ¼ 10 : 1 as eluent. The purple powder obtained (d, J ¼ 7.75 Hz, 2H, 2,6-pyridinium), 9.04 (d, J ¼ 7.70 Hz, 2H, 3,5-
from the main band was 5,10,15,20-tetra(4-pyridyl)porphyrin, pyridinium), 9.01 (d, J ¼ 9.90 Hz, 4H, b-pyrrole), 8.80 (d, J ¼
which was then methylated by an excess amount of methyl 9.90 Hz, 4H, b-pyrrole) 8.76 (d, J ¼ 9.90 Hz, 4H, b-pyrrole), 8.30
1
iodide afforded target compounds. Yield: 95.6%. H NMR (500 (d, J ¼ 8.41 Hz, 4H, 2,6-phenyl), 6.96 (d, J ¼ 5.55 Hz, 4H, 3,5-
+
MHz, DMSO) 9.34 (d, J ¼ 6.0 Hz, 8H, 2,6-pyridinium), 9.06 (d, J phenyl), 4.72 (s, 6H, N –Me), 2.56 (s, 6H, –CH
2
), 1.26 (d, J ¼
¼
7.75 Hz, 6H, 3,5-pyridinium), 8.99 (s, 8H, pyrrole), 4.72 (s, 9H, 7.71 Hz, 9H, –CH
3
), ꢁ2.88 (s, 2H, NH). Anal. calcd for C53
$H O: C, 64.18; H, 4.67; N, 7.06. Found: C, 65.33; H, 4.75;
6 2
44
H -
+
N –Me), ꢁ3.01 (s, 2H, NH pyrrole). Anal. calcd for C44
H
38
N
8
I
4
-
N
5
IO
+
$
4H
2
O: C, 41.99; H, 3.68; N, 8.90. Found: C, 42.14; H, 2.15; N, N, 7.18. ES-MS EtOH, m/z: 846 (M ). UV-Vis (10 mM in Tris
.99. ES-MS EtOH, m/z: 678 (M ), 169 (M ). UV-Vis (10 mM in buffer), lmax (nm) (log 3): 424(5.01), 521(3.97), 558(3.72),
+
4+
8
Tris buffer), lmax (nm) (log 3): 423(4.63), 517(3.39), 562(3.48), 582(3.41), 646(3.21).
1
585(3.00), 643(2.99).
The H NMR spectra of these compounds are given as ESI in
5
,10,15-Tri(1-methylpyridinium-4-yl)-20-(4-methoxyphenyl) Fig. S1.†
porphyrin (2). Similar method as 1 was employed to synthesize
For the hemolysis testing experiment, 4 mL fresh anti-
2, with 4-pyridinecarboxaldehyde was replaced by 2.1 mL (0.03 coagulation human blood was diluted with 5 mL physiological
mol) 4-pyridinecarboxaldehyde and 1.2 g (0.01 mol) p-hydrox- saline. 1 mL SPION–COOH nano suspension (concentration is
ꢁ
1
ꢀ
ybenzaldehyde, and the eluent was replaced by CH
1
2
Cl
2
/EtOH ¼ 1.0 mg mL , drug free) was incubated in the 37 C water bath
5 : 1. The purple powder obtained from the rst band was then for 30 min, and then 1 mL diluted blood was added to the nano
methylated by an excess amount of methyl iodide afforded suspensions, following by shocking for 60 min in the water
1
target compounds. Yield: 94.2%. H NMR (500 MHz, DMSO): bath. The mixture was then centrifuged at 1000 ꢂ g centrifugal
chemical shi d: 9.34 (d, J ¼ 7.75 Hz, 6H, 2,6-pyridinium), 9.06 for 5 min, and the absorbance of the supernatantat at 545 nm
(
d, J ¼ 7.75 Hz, 6H, 3,5-pyridinium), 9.01 (s, 2H, pyrrole), 8.99 (s, was measured. Physiological saline and distilled water were
6
5
7
H, pyrrole), 8.27 (d, J ¼ 8.41 Hz, 2H, 2,6-phenyl), 6.96 (d, J ¼ used as negative control and positive control, respectively.
+
.55 Hz, 2H, 3,5-phenyl), 4.72 (s, 9H, N –Me), 1.27 (d, J ¼
.71 Hz, 3H, –CH
), ꢁ2.93 (s, 2H, NH). Anal. calcd for C47
$3H O: C, 48.26; H, 3.96; N, 8.38. Found: C, 49.74; H,
.15; N, 8.29. ES-MS EtOH, m/z: 734 (M ), 244 (M ). UV-Vis (10
mM in Tris buffer), lmax (nm) (log 3): 424(5.09), 528(3.92),
59(3.70), 584(3.56), 640(3.18).
,15-Di(1-methylpyridinium-4-yl)-10,20-di(4-methoxyphenyl)
porphyrin (3). Similar method as 1 was employed to synthesize to the absorbance of the supernatantat for the studied material
The hemolysis rates of materials were calculated using the
following equation:
3
40
H -
N
4
7
I
3
O
2
2
A
m
ꢁ A
n
+
3+
H% ¼
ꢂ 100%
A
p
ꢁ A
n
5
5
m
Here, H% refers to the hemolysis rates of materials, A refers
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at 545 nm, A
n
refers to the absorbance of the supernatantat for and incubated for 24 h. The medium was removed and replaced
ꢁ
1
the negative control at 545 nm, A refers to the absorbance of with fresh medium containing the 0.1 mg mL porphyrin
p
the supernatantat for the positive control at 545 nm.
3@SPION. Aer 4 h incubation, the cells were washed with PBS,
To investigate the biocompatibility of the SPION–COOH, trypsinized, and collected. The cells were counted and digested
human umbilical vein endothelial (HUVE) and Hela cells were with HNO
3
(65%, 0.5 mL).
5
placed in 96-well plates at a cell density of 1 ꢂ 10 cells per well.
Aer incubation for 24 h, the medium was replaced with Magnetic targeting study
SPION–COOH nanosphere solutions (dissolved in fresh
HeLa cells were seeded in a 60 mm Petri dish and incubated
overnight, and then a solution of the magnetic nanospheres
with a concentration of 0.1 mg mL was added. For magnetic
targeting study, a magnet (about 4 T) was placed beside the Petri
dish. Aer incubation for 24 h, the Petri dish was photographed
and the cells in the position of magnet (targeting area) and the
medium) at different concentrations. Aer incubation for
a further 48 h, standard MTT assay was carried out to determine
the cell viabilities relative to the control untreated cells.
ꢁ1
The adsorption and release of porphyrin@SPION
The schematic diagram of loading cationic porphyrins to SPION area with a much weaker magnetic eld strength (control area)
was given in Fig. 2. Porphyrin@SPION nanospheres were ob- were observed using a light microscope. For photodynamic
tained by incubating the porphyrins with the SPION–COOH PBS combination therapy, aer incubated nanospheres with cells
buffer at pH 7.40 and gently shaking for 24 h, the drug-loaded for 12 h, the plates were irradiated with UV-A (310–390 nm,
SPION were harvested by centrifugation. Finally, the nano- 4 mW) or visible (400–800 nm, 2 mW) light for 10 min. Then the
particles were washed with the mixture of cold ethanol and wells were incubated for another 12 h and treated with the same
dichloromethane (1 : 1) to remove the residual porphyrins and procedure as described above.
used immediately aerwards. The porphyrin concentration in
the supernatant was measured by means of UV-vis spectro- Detection of singlet oxygen
photometry, using a calibration curve established previously.
Singlet oxygen in aqueous was determined following the Kralji´c
procedure. Solution of p-nitrosodimethylaniline (RNO, 30 mM)
The porphyrin loading (%) was dened was thus obtained by
method of subtractive using loading porphyrin vs. SPION (w/w).
Each determination was performed in quadruplicate. The
SPION nanospheres loading porphyrins 1, 2, 3 and 4 were
named as porphyrin 1@SPION, porphyrin 2@SPION, porphyrin
20
and imidazole (0.5 mM) in PBS (10 mM, pH ¼ 5.86 or 7.40) was
ꢁ1
added into porphyrin@SPION (0.1 mg mL ) and then irradi-
ated with visible (400–800 nm, 2 mW) light. The absorbance
spectra of the solution were recorded at 30 s intervals.
BEL-7402 cells were seeded into six-well plates and incu-
bated for 24 h. The cells were cultured in RPMI 1640 supple-
3@SPION, porphyrin 4@SPION, respectively.
To start the drug release, small aliquots of the drug loaded
porphyrin@SPION nanospheres were added to equal volumes of
mented with 10% of fetal bovine serum (FBS) and incubated at
ꢀ
PBS buffer pH 5.86 or 7.40, thermostated at 37 C and contin-
ꢀ
3
7 C and 5% CO
2
. The medium was removed and replaced with
uously shaken. At given time intervals, each tube was centri-
fuged, and the released drug concentration in the supernatant
was determined from the intensity of the drug absorption. The
release ratio was calculated from the porphyrin in the solution
vs. the original porphyrin loaded on the nanospheres (w/w).
medium containing porphyrin@SPION nanospheres (0.1 mg
mL ) for 24 h. The cells were washed with RPMI 1640 medium
ꢁ1
and then incubated with 10 mM DCFH-DA in RPMI 1640
ꢀ
medium at 37 C for 20 min. Aer washing twice with RPMI
1
5
640 medium, cells were irradiated with 660 nm LED light for
min. The cells were then harvested and the cell pellets were
Prussian blue staining
suspended in PBS-EDTA, which were then imaged by uores-
5
HeLa cells (1 ꢂ 10 ) were seeded on 24-well plates and incubated cence microscope. The uorescence intensity was determined
ꢀ
overnight at 37 C with 5% CO in a humidied chamber. A by microplate analyzer (Innite M200, TECAN, Switzerland)
2
solution of the magnetic nanospheres with a concentration of with excitation at 488 nm and emission at 525 nm. The uo-
ꢁ
1
0
.1 mg mL was then added and the cells were cultured for 4 h. rescence intensity was calculated by the determined uores-
Aer the end of the culture period, each well was washed three cence intensity minus the uorescence intensity of the
times with phosphate buffered saline (PBS), treated with 4% complexes in the corresponding concentrations of porphyrin
ꢀ
paraformaldehyde solution at 4 C for 30 min to x the cells, and @SPION nanospheres.
washed three times with PBS again. A 1 : 1 mixture of 5%
potassium ferrocyanide(II) trihydrate solution and 5% HCl was Cell cytotoxicity
added to each well and the cells were incubated at room
temperature for 1 h before being counterstained with neutral red.
Each well was then washed three times with PBS and the cells
were immediately examined and analyzed by light microscopy.
Cell viability is measured by WST assay to evaluate the effects of
porphyrin@SPION-initiated photodynamic therapy. The HeLa
ꢀ
cells were seeded for 48 h in standard 96-well plates at 37 C in
a 5% CO /95% air humidied incubator. The cells were
2
5
collected and re-suspended in PBS buffer (at 1 ꢂ 10 cells per
ICP-MS
well), then seeded in 96-well plates (well diameter: 6.4 mm) to
To quantitatively measure the cellular uptake of the nano- attach for 24 hours before treatment with porphyrin@SPION.
spheres, Hela cells were seeded in 10 cm tissue culture dishes The medium at pH 5.86 was prepared from commercially
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available RPMI 1640 medium of pH 7.4 by addition of an information of protein from http://www.rcsb.org/pdb/
appropriate amount of HCl (0.2 M). Porphyrin@SPION was (ID:1AO6). The 3D structure of daunomycin was generated in
added to pH 7.40 or pH 5.86 medium (containing 0.5% DMF) to Discovery Studio 4.5 Client. The grid center coordinates of box
ꢁ
1
˚
˚
˚
produce a nal concentration of 0.1 mg mL . The above por- along the x-, y-, z-axes are set to 126 A, 126 A and 126 A. The
phyrin@SPION solution was then subjected to vortex or soni- Lamarckian genetic algorithm is employed to search the dock-
cation or a combination of both. A solution (10.00 mL) of ing conformation.
porphyrin@SPION or free porphyrins at the same concentration
was added to the HeLa cells. The mixture was then exposed to
visible simulated solar light for 20 minutes irradiated with UV-A
Results and discussion
Characterization and drug release of porphyrin@SPION
(310–390 nm, 4 mW) or visible (400–800 nm, 2 mW) light for
nanospheres
WST tests. To perform WST assay, WST kit was added to each
well and incubated for 30 minutes. The cells were centrifuged,
Characterization. All the porphyrin@SPION nanospheres
and the suspension was discarded. The absorbance was read at have the similar morphology and porphyrin 1@SPION was
50 nm using a Multiskan Ascent Plate Reader. The IC50 indi- taken as an example to characterize these nanospheres. SEM
cates the concentration of free porphyrins or porphyrins in the image of the porphyrin 1@SPION given in Fig. 3a exhibit the
4
nanospheres.
uniform roughly spherical shape of these nanospheres. TEM
image in Fig. 3b clearly shows that the SPION core is well
embodied in the particle.
Statistical analysis
The chemical composition of the organic layers, namely the
presence of the porphyrins, was conrmed by solid state UV-Vis
spectra. Fig. 3c exhibits the solid-state UV-Vis spectra of SPION
and porphyrin 1@SPION, and the UV-Vis spectra for porphyrin
All of the data were expressed as the mean ꢃ SD. Differences
between two groups were analyzed by a two-tailed Student's t
test. Differences with *P < 0.05 were considered statistically
signicant.
1
was given as inserted. It found that SPION has no substantial
absorption while the porphyrin 1@SPION has the characteristic
absorption peak (Soret band around 420 nm) of porphyrin.
Fluorescence microscopy of apoptosis assays
Fourty eight hours aer exposure to porphyrin@SPION nano- Meanwhile, the FTIR spectra (Fig. S2 in ESI†) also conrmed the
spheres, the cancer cells were washed twice with phosphate existing of the porphyrins on the nanospheres. It is found that
ꢁ
1
buffer solution, and were then stained with 0.01 mg mL
compared with that of pure SPION, the intensity of the pyrrole
ꢀ
ꢁ1
ꢁ1
Hoechst 33258 staining solution at 37 C for 30 min according ring's vibration bands at 887 cm and 1446 cm , which are
to the manufacturer's instructions. Finally, the cells were the characteristic FTIR signals of porphyrin, increases remark-
observed under a uorescence microscope.
ably. Moreover, thermostability of the porphyrin@SPION was
HeLa cells in the logarithmic growth phase were seeded in tested and further proves the existence of SPION and porphyrin,
ꢀ
ꢀ
2
4-well plates and allowed to attach overnight. Cells were with two degrade degrees at 274 C for organic shell and 488 C
ꢁ
1
incubated with 0.1 mg mL porphyrin 3@SPION nanospheres for inorganic core, and respectively. These results evidenced the
for 12 h under dark or exposed to 660 nm light for 5 min, then successful loading of porphyrins on SPION nanospheres.
stained with an annexin V–FITC apoptosis detection kit (Beyo-
time Institute of Biotechnology, China) as described in the
manufacturer's instructions, and observed by uorescence
microscopy.
Spectral experiments
The absorption and uorescence titrations were carried out by
the stepwise addition of porphyrin@SPION solution to a cell
containing 10 mM HSA solutions. Aer preparation, the reaction
systems were incubated for 5 min at room temperature before
measurement. Synchronous uorescence spectra were obtained
by using synchronous excitation and emission scanning with
right-angle geometry. The reaction solutions for CD spectral
experiments were placed in a 0.1 cm path length cell, and each
spectrum was the average of three scans.
Molecular model
In order to nd out the exact binding mode of porphyrins to
Fig. 3 The characterization of cationic porphyrin 1@SPION nano-
HSA, we perform docking simulations based on the AutoDock
program (downloaded from the http://autodock.scripps.edu/)
spheres: SEM image (a), TEM image (b), solid UV-Vis spectra of
porphyrin (insert), SPION ( ) and porphyrin@SPION ( ) (c) and
and DS Visualizer soware. The three-dimensional structural magnetic hysteresis loop (d).
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Porphyrin@SPION nanospheres are superparamagnetic and
have excellent magnetic responsibility, which is the key factor
for their targeting. Magnetic hysteresis loop (Fig. 3d) shows that
magnetic nanospheres porphyrin 1@SPION exhibit super-
paramagnetic behavior with zero coercivity and remanence. The
saturation magnetizations of these magnetic nanospheres are
ꢁ
1
almost the same, about 2.75 emu g , which is lower than that
of SPION nanoparticles. It could be explained by the porphyrins
19–21
coating of the SPION nanoparticles in nanospheres.
It was
experimentally observed that these nanospheres dispersed in
water were rapidly attracted by a conventional magnet placed
close to the reaction vessel (Fig. S4†), further demonstrating the
efficacy of magnetic separation.
The hemolysis rate for the SPION–COOH nanospheres is
0.51%, which is far lower than the maximum hemolysis rate
(5%) for the medical devise requested by FDA. This result
suggests that the studied material is biocompatible for the
circulating blood.
Meanwhile, it is well known that the cell toxicity is a very
important part for the biocompatibility of the nanoparticles. To
more deeply assess the toxicity of SPION–COOH nanospheres
Fig. 5 (a) In vitro porphyrin 1 ( ), 2 ( ), 3 ( ), 4 ( ) loading on SPION–
COOH vs. time; (b) release of porphyrins from porphyrin@SPION at pH
ꢀ
(
drug-free), two different cell lines, human umbilical vein 5.86 ( ), 7.40 ( ) (37 C).
endothelial (HUVE) cells and HeLa cells were employed and
incubated with nanospheres for 48 h. The cell viability of these
cell lines with nanospheres at concentrations ranging from 0.03
ꢀ
and 5.86 (pH of tumor cells) at 37 C, respectively. The releases
ꢁ
1
were monitored with UV and the release ratio results were given
in Fig. 5b. From Fig. 5b, one can easily nd that the tumor cell
condition (pH 5.86) is much more favored by all the nano-
spheres than the blood plasma condition (pH 7.40). When
considering the porphyrin 3@SPION as an example, at pH 5.86,
the drug released continuously during 4 h then reaches
a plateau equivalent to ꢄ78% from the loaded drug. However,
the release at pH 7.40 is relatively slow and sustained, with only
about 8% of original porphyrin releases from porphyrin
to 1.0 mg mL was given in Fig. 4.
From Fig. 4, it is found that no appreciable deduction in cell
viabilities were observed, indicating that the SPION–COOH
nanospheres are highly biocompatible for Hela and HUVE cells.
Drug loading and release. To optimize the protocols of
porphyrin loading on the ferrouids, we studied loading effi-
ciencies of these porphyrins to the SPION–COOH surface
during incubation at pH 7.40 (Fig. 5a). The loading process of
these porphyrins reaches equilibrium in less than 2 h. Mean-
while, for initial drug/SPION (w/w) ratio at 0.05, the extents of
porphyrin loading reach up to 36.9%, 30.2%, 21.5% and 12.4%
for porphyrins 1, 2, 3 and 4, respectively. As far as we know,
these drug loading exceed the maximal loading values for
3
@SPION when achieving the equilibrium. This indicates that
porphyrin@SPION has smart pH-controlled release and poten-
tial pH targeting to tumor cells. The observed pH sensitivity is
hypothesized to facilitate drug release from SPION once inside
the acidic endosomal compartment and increase the intracel-
13
porphyrins reported before. Thus, the proposed novel method
in this work was proved to be particularly efficient. It is notable
that more positive charges are favored by the loading process,
indicating that the electrostatic interaction is the main driving
force for the adsorption.
22
lular bioavailability of the drug.
Meanwhile, Fig. 5b also shows that the release ratio for the
porphyrins from porphyrin@SPION follows the order of 3 > 4 > 2
1. Generally, less positive charges on the porphyrin molecules
>
are more favored by the release process. This result could be
best understood by the fact that higher positively charged
porphyrins have stronger electronic attraction with the modi-
The in vitro release of porphyrins from loaded SPION ferro-
uids was studied in Tris buffer pH 7.40 (pH of blood plasma)


ed SPION surface and thus are more difficult to release into the
solution. It is interesting that the result for porphyrins 3 and 4 is
somewhat controversial to the conclusion above, with dica-
tionic porphyrin 3 releasing more efficiently than monocationic
porphyrin 4. This could be mainly attributed to the different
molecular solubility in the solution. Porphyrin 3 with two
positive charges is more soluble than monocationic porphyrin 4
in the release buffer. Positive charge(s) on the porphyrin
molecule seems to be a double-edged sword in loading and
release processes, and dicationic porphyrin 3 is more favored
than other studied porphyrins in this research.
Fig. 4 Hela (a) and HUVE (b) cell viability under different concentra-
tions of SPION–COOH nanospheres.
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Magnetic targeting and cell cytotoxicity research
Cellular uptake. It is well accepted that the biochemical
processes that control the complexity of life occur in the cell
cytosol and different subcellular organelles. Thus, the cellular
uptake efficiency of the magnetic nanospheres was evaluated in
order to assess its potential for efficient delivery of drug mole-
23,24
cules into cells.
The presence of the magnetic nanospheres in HeLa cells was
detected though determining the intracellular “Fe” in HeLa
cells, which had been incubated with the magnetic nanospheres
by Prussian blue staining method. Since porphyrin 3@SPION
exhibits the smartest pH release ability, it is chosen as the
model to elucidate the cellular uptake, as well as the anticancer
mechanism research. In the absence of porphyrin 3@SPION
nanospheres, we did not observe any blue staining of the
control cells (Fig. 6a). However, as shown in Fig. 6b, aer the
incubation with the porphyrin 3@SPION nanospheres,
substantial blue spots were observed in most of the cells. The
majority of the blue spots are localized around the cell
membrane and throughout the cytoplasm, indicating high
Fig. 7 Optical micrographs of HeLa cells at the targeting area (red
24,25
cellular uptake of the magnetic nanospheres.
Moreover, the intracellular Fe content can be easily and magnetic nanospheres (0.1 mg mL ) under dark, UV-A and visible
precisely determined by ICP-MS. Aer incubation of Hela cells
circle) and the control area (blue circle) after incubating with the
ꢁ1
light at pH 5.86.
with porphyrin 3@SPION nanospheres for 4 h, the cells were
harvested and digested and the intracellular Fe contents were
6
determined to be 73 ng/10 cells by ICP-MS.
Compared with the large change in the magnetic region, the
Magnetic targeting study. An magnetic targeting experiment
cells in the weak magnetic region (blue circle) suffered much
was carried out to examine the magnetic targeting property of the
fewer death than the magnetic position. The majority of the
magnetic nanospheres in vitro. A magnet (about 4 T) was placed
cells remained their long shape and uniform morphology,
beside the Petri dish, and the cells incubated with porphyrin
under dark or irradiation. This indicates that the magnetic
3@SPION in two locations, which referred to as the targeting area
nanospheres could efficiently accumulate to the targeted area
under a magnetic guidance. The results here allowed the
conclusion that these porphyrin@SPION nanospheres have
photodynamic and magnetic targeting multifunction.
(red circle) and the control area (blue circle), were photographed
and compared (Fig. 7). Red circle points to the position closest to
the magnet and thus is in the strongest magnetic eld, while the
magnetic eld applied to blue circle is much weaker.
As shown in Fig. 7, aer incubation under UV-A or Vis light,
clear morphological changes were observed for the cells in red
circles. Most of the adhered HeLa cells appeared to be round
and bright, with oating in the cell culture media. This is the
signicant signal of cell death. It is found that Vis light leads to
more cell death in the magnetic eld. However, under incuba-
tion in the dark, the morphology of the HeLa cell changed
negligibly. This observation clearly proved the photodynamic
potential of the porphyrin@SPION nanospheres.
Induction of apoptosis as evidenced by Hoechst 33258
Since live/dead cell staining was used to visualize the cell
viability more directly by uorescence microscope aer
different treatments, we identied the cell change in the pres-
ence of porphyrin@SPION by Hoechst 33258 staining.
Fig. 8 shows representative Hoechst 33258 uorescence
photomicrographs of cultured HeLa cells treated with
porphyrin 3@SPION under dark, UV-A or visible light. In
control cultures (Fig. 8a), nuclei of HeLa cells appeared with
regular contours and were round and large. Under dark, the
cells have no signicant change. By contrast, the condensa-
tion of nuclei characteristic of apoptotic cells was evident.
Most nuclei of HeLa cells treated under UV-A or visible light
appeared hypercondensed (brightly stained), and the typical
apoptotic bodies were observed, which was different from
what was observed in the control cells. This may imply that
these nanospheres can cause proliferative suppression of
cancer cells via the induction of apoptosis. Further investi-
gations are required to fully understand the mechanism
involved in the induction of apoptosis by the nanospheres.
Fig. 6 Prussian blue staining of HeLa cells treated in the absence (a)
and presence the porphyrin 3@SPION (b) magnetic nanosphere
ꢁ
1
(
0.1 mg mL ) for 4 h.
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Fig.
9
(a) Annexin V–FITC apoptosis detection Fluorescence
microscopy images of HeLa cells treated with control cell media; (b)
HeLa cells treated with porphyrin 3@SPION under dark; (c) HeLa cells
treated with porphyrin 3@SPION under dark for 12 h and then irradi-
ated by 660 nm light for 5 min.
detected in the nucleus, indicating that the HeLa cells under-
went early apoptosis induced by porphyrin 3@SPION
nanospheres.
Fig. 8 Effects of porphyrin 3@SPION on the morphology of HeLa cells
were assayed by Hoechst 33258 staining with fluorescence micros-
copy (original magnification 400ꢂ): control (a) and incubation in the
dark (b), UV-A (c) and visible light (d) for 48 h at pH 5.86.
Reactive oxygen species studies
The role played by ROS in apoptosis is becoming increasingly
recognized. It is widely accepted that porphyrins induce
apoptosis through ROS generation. ROS levels of these por-
Annexin V–FITC apoptosis detection
6,7
It is widely accepted that the loss of plasma-membrane asym-
metry and attachment is one of the striking morphological
features of the apoptotic process. In apoptotic cells, the
membrane phospholipid phosphatidylserine (PS) is trans-
located from the interior to the exterior of the plasma
membrane, thereby exposing PS to the external cellular envi-
ronment. Annexin V is a protein that has a high affinity for PS
and thus could binds to cells with exposed PS. By conjugating
with uorochromes including uorescein isothiocyanate
phyrin@SPION nanospheres were investigated with a uores-
cence microscope and microplate analyzer using H DCFDA as
2
a probe. H DCFDA is a uorescent dye that diffuses through cell
2
membranes and is hydrolyzed by intracellular esterases to
DCFH. In the presence of ROS, DCFH could be oxidized to DCF,
whose uorescent level corresponds to the level of generated
ROS. Fluorescent images and intensities were given in Fig. 10.
As shown in Fig. 10, in the control (a), no uorescent spots
are found. Aer BEL-7402 cells were incubated with 0.1 mg
(

FITC), Annexin V can serve as a sensitive uorescent probe for
uorescence-microscopy analysis of cells that are undergoing
apoptosis.
FITC–annexin V staining precedes the loss of membrane
ꢁ1
mL porphyrin 3@SPION nanospheres for 24 h without irra-
diation, negligible orescent spots were observed (Fig. 10b).
However, aer irradiation at 660 nm for 5 min, clear uores-
cence can be observed under a uorescence microscope
integrity that accompanies the last stages of cell death resulting
from either apoptotic or necrotic processes. Therefore, staining
with FITC–annexin V is typically used in conjunction with
a nuclear dye such as propidium iodide (PI) to identify early
apoptotic cells. Viable and early-apoptotic cells with intact
membranes exclude PI, whereas the membranes of dead and
late-apoptotic cells are permeable to PI, and thus the nucleus
can be labeled.
(Fig. 10c). To determine quantitatively the uorescence inten-
sity of DCF, the ROS levels were also evaluated by with micro-
plate analyzer. The results indicated that the mean uorescence
intensity (MFI) of the cells treated with porphyrin 3@SPION
under light irradiation is almost 5-times higher than that ob-
tained in the dark (Fig. 10d). It is well known that these highly
toxic ROS could damage the cellular proteins, lipids and nucleic
acids. Therefore, elevated intracellular ROS level causes oxida-
tive stress and a series of signal transduction pathway changes,
which are highly related to transcription factors, cell cycle
regulation, inammation, and so on. The cellular metabolism
changes following PDT treatment make the cells more fragile to
chemotherapeutic treatment and result in apoptosis or
necrosis.
We studied the mode of cell death induced by porphyrin
3@SPION nanospheres by uorescence staining with an FITC–
annexin V apoptosis detection kit for the morphological
assessment of cell apoptosis. The cytological changes were
classied into three types: (1) viable cells are FITC–annexin V
and PI negative; (2) cells that are in early apoptosis are FITC–
annexin V positive and PI negative; and (3) cells that are in late
apoptosis or already dead are both FITC–annexin V and PI
positive. From Fig. 9a, no obvious uorescence was observed in
1
The O
2
production of the porphyrin@SPION nanospheres
the control cells. Meanwhile, there were negligible uorescent The object of this research is to further understand the mech-
signals when HeLa cells treated with porphyrin 3@SPION anism of the photodynamic cell cytotoxicity of the porphyr-
nanospheres under dark (Fig. 9b). However, aer exposed to the in@SPION nanospheres. The basic principle of PDT is the
light, HeLa cells treated with porphyrin 3@SPION exhibited generation of highly cytotoxic ROS, especially singlet oxygen
1
signicant morphological changes. We can nd from Fig. 9c ( O ), under light activation. The capacity of porphyrin
2
1
that, the cell membranes of all of the treated cells were FITC– 3@SPION to generate O in aqueous solution under UV-A or
2
annexin V positive (green uorescence) but no PI signal was visible light at pH 5.86 or 7.40 was comparatively assessed
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physiological pH are desirable. This pH-triggered therapeutic
selectivity was further conrmed by WST cell cytotoxicity tests.
Cell cytotoxicity research
It has been demonstrated previously that drug-free SPION
28
produce negligible cytotoxicity on cancer cells. The IC50 values
for the porphyrins and corresponding porphyrin@SPION
nanospheres are given in Table 1.
From Table 1, porphyrin 1 (TMPyP), which is a well-known
anticancer research model, exhibits the best cell photo-
cytotoxicity under both UV and Vis light. However, because of its
1
poor release which further leads to a low
2
O production,
Fig. 10 Fluorescent images of BEL-7402 cells alone (a) and BEL-7402
cells with 0.1 mg mL porphyrin 3@SPION in the dark (b) or irradiated
ꢁ1
porphyrin 1@SPION has weak antitumor ability, with IC50
with 660 nm light (c). (d) gives the fluorescent intensity values for the values reach up 27.3 mM and 47.2 mM under Vis light at pH 5.86
images.
and 7.40, respectively. Porphyrin 4@SPION also has low anti-
cancer efficiency, which may result from the high IC50 value of
porphyrin 4 itself as well as its low release efficiency. However,
following the Kralji ´c procedure. Fig. 11 gives the absorption porphyrin 3@SPION could efficiently inhibit the HeLa cells
change of RNO in the presence of porphyrin 3@SPION under under both UV and Vis light. It seems that the anticancer ability
visible light irradiation. The absorbance of RNO at 440 nm for porphyrin@SPION controlled the synergy of two factors: the
decreased remarkably owning to the photooxidation bleaching IC50 of porphyrin molecule itself and release efficiency of
effect aer irradiation for 180 s with porphyrin 3@SPION, porphyrin from the SPION surface. Both lower IC50 and higher
1
which indicated the signicant production of
O
2
from release efficiency are important for porphyrin@SPION nano-
porphyrin 3@SPION when exposed to visible light.
spheres. For porphyrin molecules, lower IC50 always related to
1
The O
2
production at pH 5.86 was observed higher than that higher positive charges, which are however disfavored by the
at pH 7.40 under UV-A or visible irradiation. This could be release process. Dicationic porphyrin 3 has an good equilibrium
explained by the fact that, at an acidic solution, the attached between these two factors has thus porphyrin 3@SPION has
porphyrin molecules could be separated from the surface of the a relative high anticancer efficiency.
SPION–COOH nanoparticles because the surface charge
However, it is notable that it is impossible to put all the
became less negative via the protonation of carboxyl groups porphyrin@SPION nanospheres into the well that were inter-
with decreasing pH value. Since molecular aggregation is nalized by the cell or all the porphyrin released completely from
assumed to be absent or minimized with cationic porphyrins in the nanospheres. The major mechanism of action of free
26
the solution, the increased release of porphyrins leads to porphyrin is an intercalation in the DNA and an inhibition of
enhanced singlet oxygen generation, thus an improvement in the topoisomerase II.8–10,29 This hypothesis does not exclude the
cell apoptosis under visible light in the magnetic targeting study participation of other supplementary mechanisms of action. To
above. This result is highly consistent with our conclusion on further explore the mechanisms of this, more efforts are
1
27
the O PDT mechanism of cationic porphyrins before. These currently undergoing.
2
features make SPION-attached cationic porphyrin a promising
candidate for use in PDT for cancer treatment in which efficient Probing the interaction of porphyrin@SPION with HSA
1
2
O production at acidic pH and sensitizer deactivation at
Numerous exogenous and endogenous drugs were carried by
human serum albumins (HSA) when they are delivered in the
blood plasma of human body. It is understandable that the
signicant changes at secondary structure of HAS is a hallmark
of the drug bio-safety in the delivery process. The object of
following research is to elucidate the HSA binding behaviors
and mechanism of these porphyrin@SPION, which could be
used to evaluate the delivery bio-safety of these nanospheres.
Absorption spectroscopy
The structural changes of proteins are oen explored using by
30
the UV-visible absorption spectroscopy. Fig. 12 exhibits the
UV-visible absorption of HSA in the presence of porphyrin
Fig. 11 Singlet oxygen detection by photooxidation of RNO under
light irradiation. To the solution of p-nitrosodimethylaniline (RNO, 30
mM), imidazole (0.5 mM) in PBS (10 mM, pH ¼ 7.4) was added to
porphyrin 3@SPION, then the solution was irradiated with visible light
3@SPION.
As Fig. 12 shows, the characteristic peak at 198 nm, which
could be mainly attributed to the peptide bonds in the protein
at pH 5.86 (solid lines) or 7.40 (dot lines) for different periods of time. and reects the framework conformation of HSA, has no
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Table 1 (Photo)cytotoxicity of the porphyrins and porphyrin@SPION towards HeLa cells
IC50 (mM)
1@SPION (1)
2@SPION (2)
3@SPION (3)
4@SPION (4)
pH
5.86
7.40
5.86
7.40
5.86
7.40
5.86
7.40
Dark
UV-A
>100
25.2 ꢃ 2.8
43.1 ꢃ 3.7
(1.5 ꢃ 0.4)
47.4 ꢃ 4.1
(0.9 ꢃ 0.2)
19.3 ꢃ 1.7
(3.2 ꢃ 0.8)
16.8 ꢃ 1.4
(2.8 ꢃ 0.6)
35.2 ꢃ 4.8
(5.5 ꢃ 0.4)
37.3 ꢃ 5.1
(4.9 ꢃ 0.2)
13.5 ꢃ 1.1
(5.7 ꢃ 0.6)
12.7 ꢃ 1.0
(5.4 ꢃ 0.3)
26.2 ꢃ 1.8
(1.5 ꢃ 0.4)
28.3 ꢃ 2.1
(0.9 ꢃ 0.2)
24.5 ꢃ 1.6
(11.5 ꢃ 1.1)
28.2 ꢃ 2.3
(10.2 ꢃ 2.3)
37.2 ꢃ 2.6
(14.5 ꢃ 0.9)
39.5 ꢃ 3.2
(12.9 ꢃ 1.2)
(
1.2 ꢃ 0.3)
Visible
27.3 ꢃ 2.1
(0.7 ꢃ 0.2)
signicant change with the gradual addition of porphyr- where F and F
in@SPION. Meanwhile, with the increase of porphyrin@SPION and absence of a quencher, respectively. Ksv, K
solution, the absorption at 280 nm, which the characteristic Stern–Volmer constant, quenching rate constant, the original
0
are the uorescence intensities in the presence
q
0
, z and Q denote
ꢁ
8
37
absorption peak of the aromatic ring amino acids, has no lifetime of HSA (10 s) and the concentration of quencher,
obvious change (see Fig. 12 inserted). Since it is widely accepted respectively. The plot of the uorescence intensity ratio of
that the enhancement around 280 nm is the signal of unfolding protein at 340 nm in the absence and presence of quencher as
the skeletal structure of HSA, the negligible change at this a function of the quencher concentration shows a linear
region, as well the non-obvious change at 198 nm, indicate the dependence (Fig. S5†). We thus can obtain the bimolecular
structure of HSA is not affected with the addition of quenching constants (K
q
) of the porphyrin 3@SPION is 3 ꢂ
31
12
porphyrin@SPION.
10 , which is much larger than the largest dynamic quenching
10
37
constant for biological molecules, 2.0 ꢂ 10 . Therefore, we
Fluorescence quenching
suppose that it is the static rather the dynamic quenching
37,38
mechanism resulting in the uorescence quenching of HSA.
The binding constant K and binding site number n can be
obtained from the site binding model
The uorescence emission of HSA in the presence of porphyr-
in@SPION was monitored to further reveal the interaction
between these nanospheres and protein. As is shown in Fig. 13,
the uorescence intensity decreased slightly with the titration of
porphyrin 3@SPION, indicating that the drug delivery alters the
spatial structure of HSA and leads to a less hydrophobic envi-
b
39,40
which assumes the
existence of n independent binding sites is expressed in eqn (2):
ꢀ
ꢁ
F ꢁ F
0
lg
¼ lg K
b
þ n lg½Qꢅ
(2)
F
32–34
ronment the uorescence chromophore placed.
The newly
where F and F
and absence of a quencher, respectively. A plot of log(F
versus log Q (Fig. 13, insert) yielded the binding constant K
0
are the uorescence intensities in the presence
ꢁ F)/F
as
appeared shoulder peak located at 310 nm could be attributed
35
0
to the new state of porphyrin@SPION bound HSA. This new
state represents efficient binding of drugs with HSA, which is
very signicant for the drug delivery by the protein.
b
3
1
.2 ꢂ 10 . This binding constant is moderate and suitable for
the protein delivery of drugs. The binding site number (n) is
calculated to be 0.91, which is approximately equal to 1, indi-
cating that there is one type of binding site for porphyrin
Stern–Volmer equation is widely employed to analyze the
36
quenching mechanism:
3@SPION in HSA.
F
0
/F ¼ 1 + K
q
z
0
Q ¼ 1 + Ksv
Q
(1)
Fig. 12 Absorption spectra of HSA with different concentration of Fig. 13 Fluorescence emission spectra of HSA with different
porphyrin@SPION in PBS solution. Insert: the absorption change from concentration of SPION-DNM in PBS solution. Insert: plot of log(F
0
ꢁ
ꢁ6
ꢁ1
ꢁ6
ꢁ1
2
60 nm to 320 nm. Conditions: HSA: 1 ꢂ 10 mol L ; porphyr- F)/F versus log Q. Conditions: HSA: 1 ꢂ 10 mol L ; SPION-DNM:
in@SPION: 0 ( ); 2 ( ); 4 ( ); 6 ( ); 8 ( ); 10 ( ) ꢂ 10ꢁ mg 0 ( ); 2 ( ); 4 ( ); 6 ( ); 8 ( ); 10 ( ); 12 ( ) ꢂ 10ꢁ4 mg mL
4
ꢁ1
ꢁ1
mL (pH ¼ 7.40).
(pH ¼ 7.40).
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Synchronous uorescence spectroscopy
It has been thoroughly emphasized that, the uorescence of Trp
and Tyr residues are highly sensitive to the polarity of its envi-
ronment, which usually presents a red shi in a more polar
41
surrounding. Thus, the emission shis of the amino acid
residues could be employed to examine the conformational
changes of protein. To further probe the binding mechanism of
the porphyrin@SPION nanospheres and HSA, synchronous

uorescence spectroscopy experiment was carried out to
investigate the conformational changes of protein. The results
are shown in Fig. S6,† in which Dl for Trp and Tyr residues are
Fig. 15 Docking results of porphyrin and HSA system. Insert: detailed
illustration of the binding pocket for porphyrin within 8 A˚ distance. The
hydrogen bonds are marked with green blue lines.
60 and 15 nm, respectively.
From Fig. S6,† it is apparent that the emission peak had
a slight shi, indicating that a less hydrophobic environment of
tryptophan residues. The maximum emission wavelength kept
its position over the investigated concentration range, which
suggests that porphyrin@SPION has negligible effect on the
microenvironment of the tyrosine residues. The increasing
exposure of amino acid residues is not signicant, which is
consistent with the conclusion above.
6
9.48 to 68.75%, 70.12%, 68.24%, 67.95% when porphyrin
1@SPION, porphyrin 2@SPION, porphyrin 3@SPION,
porphyrin 4@SPION were added, respectively. It seems that
these porphyrin@SPION nanospheres have negligible changes
to the secondary structure and undetectable effects on the
frame conformation of the HSA. The porphyrin@SPION was
thus considered relatively safe in their delivery process, without
signicant inuence to the structure of protein. This result was
further evidenced by the theoretical study below.
Circular dichroism spectra
CD spectra of HSA were measured in the absence and presence
of porphyrin@SPION to quanticationally ascertain the
secondary structure changes of HSA molecules (Fig. 14).
For free HSA, there are two negative bands in the far-UV
region at 208 and 222 nm, which are characteristic of
Molecular modeling study
AutoDock-based blind docking has been employed to substan-
tiate the spectral experimental results.
42
As Fig. 15 shows vividly for porphyrin 3 binding with HSA,
instead of entering the pocket of HSA structures, porphyrin is
bond at the external region of domains, which indicates that the
interaction of porphyrin is relatively weak. The chemistry of
porphyrin plays a major role in determining the location of
bonding site. Moreover, van der Waals interactions also exist,
but the hydrogen bonds play a major role in the binding of
a-helical structure of protein. The a-helix contents of HSA
could be evaluated from the MRE value at 208 nm, using the
43
following eqn (3) and (4):
CD208
MRE208 ¼ 10C
(3)
p
nl
ꢁ
MRE208 ꢁ 4000
3 000 ꢁ 4000
a% ¼
(4) porphyrin to HSA. The calculation indicates that porphyrin
prefers a polar binding site. Thus, compared with the non-
specic binding of iron oxide nanoparticles to protein, the
3
The a-helix content of HSA was calculated quantitatively interactions of porphyrin@SPION with HSA prefer to occur
44
from the eqn (3) and (4). The content of a-helix changed from between porphyrin and protein. The similar HSA binding
behaviors of other porphyrins are given in Fig. S7.† The
monoporphyrin will not enter the pocket of HSA structures and
is thus safe to the protein.
Conclusions
In the present contribution, nano-supporting, magnetic effect
as well as pH controlling factors were integrated in designing
porphyrin@SPION systems in which a series of cationic
porphyrins are efficiently loaded on SPION–COOH surface.
These pH-controllable porphyrin@SPION nanospheres exhibi-
ted multifunction of both photodynamic potential and smart
magnetic targeting. Among these newly prepared nanospheres,
dicationic porphyrin@SPION nanospheres have smart pH-
responsive release, high cell uptake activity, targeted cell accu-
Fig. 14 CD spectra of HSA in the absence ( ) and presence of
porphyrin 1@SPION
(
), porphyrin 2@SPION
(
), porphyrin
mulation as well relatively high photodynamic cytotoxicity to
HeLa cells. The drug delivery for porphyrin@SPION was
3
@SPION ( ), porphyrin 4@SPION ( ) in PBS solution. Conditions:
ꢁ3
ꢁ1
HSA 2.5 mM; porphyrin@SPION: 2.5 ꢂ 10 mg mL
.
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103148 | RSC Adv., 2016, 6, 103137–103148
This journal is © The Royal Society of Chemistry 2016