Conjugation of porphyrin to nanohybrid cerasomes for photodynamic diagnosis and therapy of cancer

Article abstract of DOI:10.1002/anie.201103557

Shining light on cancer cells: A cerasomal photosensitizer of high stability and loading efficiency was developed by conjugation of a porphyrin molecule to an organoalkoxysilylated lipid followed by sol-gel and self-assembly processes (see picture). This

Full text of DOI:10.1002/anie.201103557

Communications
DOI: 10.1002/anie.201103557
Photochemistry
Conjugation of Porphyrin to Nanohybrid Cerasomes for
Photodynamic Diagnosis and Therapy of Cancer**
Xiaolong Liang, Xiaoda Li, Xiuli Yue, and Zhifei Dai*
Angewandte
Chemie
11622
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Angew. Chem. Int. Ed. 2011, 50, 11622 –11627

Photodynamic therapy (PDT) is an emerging treatment for
eradicating premalignant and early-stage cancer and reducing
the tumor size in end-stage cancers by photosensitizers
(PSs).[1–3] When PSs are exposed to light of appropriate
wavelength, they produce highly reactive oxygen species
(ROS) that cause an effective and selective destruction of
diseased tissues without damage to the surrounding healthy
tissues.[4–8] However, most photosensitizers that are used
clinically or in preclinical development are hydrophobic and
strongly aggregate in aqueous media.[9] This aggregation
significantly reduces their photosensitizing efficacy because
only monomeric species are appreciably photoactive.[9] Many
types of nanocarries, such as polymeric micelles, conjugated
polymer nanoparticles, and silica nanoparticles have been
extensively used to fabricate a stable dispersion of PDT drugs
in aqueous systems.[10–14] However, most of them suffer from
poor drug loading or increased self-aggregation of the drug in
the entrapped state.
Herein, we fabricated porphyrin bilayer cerasomes
(PBCs) for the first time by sol–gel and self-assembly
processes from a conjugate of porphyrin-organoalkoxysily-
lated lipids (PORSILs) with two triethoxysilyl groups, a
hydrophobic double-chain segment, a porphyrin moiety, and a
connecting unit (Figure 1). Compared with the reported
cerasomes,[17] the covalent linkage of porphyrin to cerasomes
may result in a drug loading content of 33.46% in the PBCs.
This loading is significantly higher than that of physically
entrapping cerasomes or liposomes (generally less than
10%). In addition, the premature release of PSs can be
avoided during systemic circulation.
With the aim of improving the efficacy and safety of PDT,
liposomes with their versatility in accommodating PSs of
different physicochemical properties, came into focus as a
valuable carrier and delivery system.[9,15] However, the
insufficient morphological stability of the conventional lip-
osomes may lead to rapid clearance of the vesicles from
circulation before reaching their target. For example, a lipid
exchange between the liposomes and lipoproteins leads to an
irreversible disintegration of the liposome.[15] The fast dis-
integration process releases PSs in the bloodstream. This
premature release results in a reduced efficacy of treatment
because the release of the PS drugs is not a prerequisite for
PDT action unlike conventional chemotherapy.[10,11] In addi-
tion, in liposomes the drug contents are generally less than
10%. Therefore, highly stable liposomes with a high PS
loading efficiency offer a better prospect in becoming carriers
of photosensitizers.
Figure 1. Formation of PBCs from a PORSIL lipid.
Porphyrins are the most popular PSs for PDT. Porphyrin-
loaded liposomes have been described for PDT applications,
but they can accommodate only a small fraction of porphyrin
molecules.[9] Recently, the so-called porphysomes, self-assem-
bled from phospholipid–porphyrin conjugates that exhibit a
liposome-like structure and high absorption of near-infrared
light, have been reported.[16] As porphysomes are highly self-
quenched, they show promise for diverse biophotonic imaging
applications and photothermal therapy. But porphysomes
exhibit almost no photosensitizing effect because the energy
that is normally released to fluorescence and singlet-oxygen
generation is dissipated thermally.
Our basic idea for the synthesis of a bilayer-forming lipid
is to couple both silica precursor and porphyrin units to a lipid
that contains several functional groups. In this respect, one of
the major issues is the multifunctional core. Therefore,
multivalent pentaerythritol is used for conjugation with
different target structures because it is readily available and
its four primary hydroxy groups are symmetric and highly
reactive. A protecting–deprotecting strategy is applied for the
synthesis of the PORSIL lipid, as shown in Scheme S1 in the
Supporting Information. Excess-protected pentaerythritol
2[18] was first coupled with compound 1[17c] to give 3 bearing
a hydroxy group, which reacted with succinic anhydride
followed by a deprotection step to obtain intermediate 5 with
two hydroxy and one carboxy groups. Then, the latter group
was selectively coupled with 5-(4-aminophenyl)-10,15,20-
triphenylporphyrin catalyzed by N-(3-dimethylamino-
propyl)-N-ethylcarbodiimide (EDC) to afford 7. Then, its
remaining two hydroxy arms were reacted with succinic
anhydride. Finally, the resultant scaffold 8 with two free
carboxy groups was coupled to 3-aminopropyltriethoxylsilane
(APTES) through simple amide linkages to obtain the
targeting PORSIL lipid. For the preparation and character-
ization of the above-mentioned compounds see the Support-
ing Information.
[*] X. Liang, X. Li, Prof. X. Yue, Prof. Z. Dai
Nanomedicine and Biosensor Laboratory, School of Sciences
State Key Laboratory of Urban Water Resources and Environment
Harbin Institute of Technology, Harbin, 150001 (P.R. China)
E-mail: zhifei.dai@hit.edu.cn
Homepage: http://nanobio.hit.edu.cn/
[**] This research was financially supported by the National Natural
Science Foundation of China (grant numbers NSFC-30970829 and
20977021).
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201103557.
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The PORSIL lipid was dispersed in water by ultrasonica-
tion, resulting in the formation of vesicles which self-rigidified
Supporting Information) were used as control. The control
cerasome, in which porphyrins were just physically encapsu-
lated, is eluted similarly to free porphyrin molecules in the
direction of the solvent front. In contrast, PBCs remain at the
bottom. The physically encapsulated porphyrin molecules can
be easily extracted from the cerasomes by organic solvent
while the porphyrins conjugated to PBCs are not able to be
washed off because of the covalent linkage.
ꢀ
ꢀ
through in situ sol–gel processes (Si OCH2CH3 + H2O!Si
ꢀ
ꢀ ꢀ
OH + CH3CH2OH followed by 2Si OH!Si O Si + H2O)
on the vesicular surface.[17] Transmission electron microscopic
(TEM) images clearly showed the formation of spherical
vesicles with a diameter range from 50 to 100 nm (Figure 2a),
which is in agreement with dynamic light scattering (DLS)
Compared with PORSIL lipids in chloroform, the UV/Vis
absorption spectra of PBCs broadened and the absorbance of
PBCs increased but the formation of cerasomes did not have a
major effect on the position of the Soret and Q bands of
porphyrin (Figure 3A). The shape of the absorption spectrum
of PBCs is quite similar to that of the solution-phase PORSIL
lipid. This similarity indicates that aggregation does not occur
Figure 2. a) TEM micrograph of PBCs. b) Dynamic light scattering
measurement of PBCs in H2O.
measurements that gave a sharp peak with a narrow
distribution at (70 ꢁ 13) nm (Figure 2b). The vesicular struc-
ture of PBCs was further confirmed by encapsulation of a
water-soluble fluorescent dye of calcein. The calcein-loaded
vesicles had brightly green-fluorescent cores and red-fluores-
cent shells, which we could clearly observe by confocal laser
scanning microscopy (CLSM; see Figure S1 in the Supporting
Information). The red emission resulted from the conjugated
porphyrin in the lipid bilayer of PBCs and the green emission
from calcein in the aqueous core of PBCs. Thus, the capability
to load water-soluble compounds into the core of PBCs
proved the existence of a vesicular structure. The formation of
siloxane bonds on the surface of PBCs was proved by Fourier
transform infrared (FT-IR) spectroscopy (see Figure S2 in the
Figure 3. A) Absorption spectra of a) PBCs in water and b) PORSIL in
chloroform. B) Fluorescence spectra under excitation with light of
419 nm: a) PORSIL in chloroform, b) PBCs in water, and c) free
porphyrin in DMF/water (0.2%). The excitation and emission slit
widths were both set at 10 nm, and all samples had the same
porphyrin concentration of 15 mm.
in the nanoparticles. Both Soret and Q bands display a
bathochromic shift of 3 nm because of the arrangement of the
porphyrin groups and alkyl chains in the lipid bilayer
membrane. The existence of double-alkyl chains sterically
hinders porphyrin moieties to approach each other, which can
greatly reduce the aggregation of the porphyrin moieties.
Therefore, we believe this method is highly effective for the
preparation of photofunctional cerasomes without aggrega-
tion or reduced photoluminescent quality.
ꢀ ꢀ
Supporting Information). Stretching bands assigned to Si O
ꢀ
Si and Si OH groups were observed around 1100 and
950 cmꢀ1, respectively. The former peak intensity was much
stronger than the latter, suggesting that PBCs had a silica-like
surface with siloxane frameworks and a high degree of
polymerization.
The morphological stability of PBCs was evaluated using
surfactant dissolution experiments. Although five equivalents
of nonionic surfactant Triton X-100 can completely destroy
the conventional liposomes from distearoyl phosphatidylcho-
line (DSPC), almost no change was observed for the hydro-
dynamic diameter of the PBCs, even in the presence of 35
equivalents of Triton X-100 after incubation for 24 h (see
Figure S3 in the Supporting Information). Such remarkable
morphological stability of the PBCs was attributed to the
development of a siloxane network on the vesicular surface.
To confirm that porphyrins are conjugated with PBCs,
both PORSIL lipids and PBCs have been applied onto silica
thin layer chromatography (silica-TLC) plates in developing
agent of ethyl acetate (see Figure S4 in the Supporting
Information).[11] Free porphyrin (see Figure S5 in the Sup-
porting Information) and the porphyrin-entrapped cerasomes
from an organoalkoxysilylated lipid (see Figure S6 in the
Figure 3B shows the fluorescence emission spectra of
PORSIL lipid in chloroform (Figure 3Ba) and the aqueous
dispersion of PBC vesicles (Figure 3Bb) at the same concen-
tration of porphyrin, which was determined by UV/Vis
absorption spectra (see Figure S7 in the Supporting Informa-
tion). The aqueous PBC vesicles were clearly fluorescent
upon light irradiation (see Figure S8 in the Supporting
Information). At the same concentration of porphyrin, the
fluorescence intensity of the aqueous PBCs is about 32%
lower than the PORSIL lipid in chloroform, but much higher
than free porphyrin (see Figure S5 in the Supporting Infor-
mation) in 0.2% DMF/water, which shows practically no
emission (Figure 3Bc). This absence of fluorescence may be
due to either porphyrin–solvent interactions promoting non-
radiative decay or self-aggregation of porphyrin molecules.
PBCs show a remarkably stronger fluorescence in aqueous
media than free porphyrin, which permits the use of PBCs as
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fluorescent nanoprobes for biological applications. PBCs are
very different from the reported porphysomes, which showed
almost complete fluorescent self-quenching because of an
energetically favorable supramolecular structure, in which the
orientation of the constituents of porphyrin and lysophos-
phatidylcholine facilitate extensive porphyrin interactions
and quenching.[16] The porphysome subunits consisted of a
hydrophobic porphyrin and a single alkyl chain. In contrast,
the PBCs subunits consisted of a hydrophobic porphyrin and
two alkyl chains and the presence of the two alkyl chains
prevented porphyrins from aggregating, avoiding significant
fluorescent self-quenching of the PBCs.
(TEMP) as a spin-trapping agent, which is selective for 1O2.[20]
Figure S10 in the Supporting Information shows that the ESR
signal intensity of both free porphyrin and PBCs increased
linearly as the irradiation time increased whereas the signal
intensity of PBCs increased more significantly than that of
free porphyrin. In contrast, no ESR signal was observed
without light irradiation, proving the inactivity of PSs in the
1
dark. After irradiation of cells incubated with PSs, the O2
generation was characterized by ESR spin trapping using a-
(4-pyridyl-1-oxide)-N-tert-butylnitrone (POBN) combined
with ethanol (2%).[21] As shown in Figures S11 and S12 in
the Supporting Information, upon light irradiation, the ESR
spectrum appeared to be a triple double-lined spectrum,
representing the presence of POBN-ethoxy adducts. The ESR
signal intensity was found to increase with increasing PSs
concentration and irradiation time (Figure 4C,D). Upon
addition of 1,4-diazabicyclo[2.2.2]octane (DABCO), a spe-
cific quencher of 1O2, the POBN-ethoxy adduct signal
intensity almost disappeared (see Figure S12Bf in the Sup-
porting Information), further confirming the generation of
1O2 in cells. The comparison of ESR spectra obtained with
free porphyrin and PBCs revealed that the conjugation of a
porphyrin molecule to cerasomes led to higher efficiency of
1O2 generation. These findings were in good agreement with
the results shown in Figure 4B.
The generation of singlet oxygen by PBCs was detected
chemically using the disodium salt of 9,10-anthracenedipro-
pionic acid (ADPA) as a detector, which was bleached to its
1
nonfluorescent endoperoxide in the presence of O2.[19] Fig-
ure 4A,B shows the decrease in optical density (OD) at
378 nm as a function of irradiation time. In the case of the
PBCs (Figure 4A and 4Ba), the sharp decrease of the ADPA
1
absorbance with irradiation time confirmed the O2 gener-
ation from PBCs. Nevertheless, the ADPA absorbance
decreased much slower in the presence of free porphyrin
(Figure 4Bb and Figure S9 in the Supporting Information),
indicating that the free porphyrin had a much lower capability
1
to generate O2 than PBCs. The conjugation of porphyrin to
1
cerasomes can significantly improve the O2 generation. In
Irradiation of the PBCs with light of suitable wavelength
results in the efficient generation of singlet oxygen, which is
possible because of the inherent porosity of the nanoparticles.
The siloxane network is not so highly developed on the PBCs
stark contrast, the cerasome without porphyrin unit (Fig-
ure 4Bc) produced no change in the OD of ADPAunder light
irradiation, further confirming that the bleaching of ADPA in
the presence of porphyrin is caused by singlet oxygen and not
by the irradiated light.
ꢀ ꢀ
surface because the length of the Si O Si bond is much
shorter than the diameter of the cross-section of the double-
chain segment and the porphyrin moiety of the amphiphiles,
which takes the molecular packing of the stable bilayer
structure into account. This effect perturbs the membrane
structure and may induce the formation of pores. Under
irradiation the photosensitizing porphyrin units within the
PBCs can interact with molecular oxygen which has diffused
through the pores. This can lead to the formation of singlet
oxygen by energy transfer from the excited photosensitizer to
molecular oxygen, which can then diffuse out of the PBCs to
produce a cytotoxic effect in the tumor cells.
1
The O2 generation in D2O solution was further detected
by ESR spectroscopy using 2,2,6,6-tetramethyl-4-piperidone
CLSM was used to verify the cellular localization of PBCs
(Figure 5). Fluorescent PBCs were clearly observed inside the
cells as red spots distributed in the cytoplasm and mainly
localized in the lysosomes. An almost complete co-local-
ization could be observed between red and green fluores-
cence as it was evident from the pale yellow fluorescence
arising from the overlap of the two fluorescence images. After
the nuclei of the tumor cells were stained with 4’,6-diamidino-
2-phenylindole (DAPI; blue in Figure 5c), PBCs were found
to be distributed throughout the entire cytoplasm (Figure 5d),
indicating that PBCs were indeed endocytosed as reported
previously on the cellular uptake of cerasomes.[22] After the
cells were treated by sucrose and K+-free buffer the uptake of
cerasomes by cells decreased to 73 and 75%, respectively,
suggesting that the uptake of PBCs occurred possibly through
a clathrin-dependent endocytosis pathway.[22] Silica nano-
particles could be endocytosed partially due to their strong
affinity to the head-groups of a variety of phospholipids and
Figure 4. A) Time-dependent bleaching of ADPA caused by singlet
oxygen generated by PBCs. B) The change in ADPA absorption at
378 nm as a function of the time of light exposure: a) PBCs, b) free
porphyrin (1%, DMSO/D2O), and c) cerasome without a porphyrin
unit. C) Concentration-dependent increase of ESR signal intensity in
HeLa cells (irradiation for 60 s): a) PBCs and b) free porphyrin.
D) Time-dependent increase of ESR signal intensity in HeLa cells:
a) PBCs (5 mm) and b) free porphyrin (5 mm).
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Figure 5. Subcellular localization of PBCs observed by fluorescent
CLSM. HeLa cells were incubated with PBCs at a concentration of
5 mm for 4 h at 378C. Observations of a) the PBCs channel, b) Lyso-
Tracker green (DND-26, to label lysosomes) channel, c) the nuclear
dye DAPI channel, and d) the overlap of the above images.
clathrin-coated vesicles. This suggests that the similar surface
properties of PBCs and silica nanoparticles may facilitate
their adhesion to the cell membrane and their subsequent
uptake. Therefore, the high affinity to cell surfaces, which
eventually leads to endocytosis of PBCs with silica surface, is
not surprising.[23]
Figure 6. A) Fluorescence microscopic observations of HeLa cells
treated with 0.2 mm PBCs and stained with calcein AM. a,b) Bright-field
images. c,d) Fluorescence images. a,c) Before irradiation. b,d) After
irradiation with light of 400–700 nm for 10 min followed by 24 h of
incubation at 378C in the dark. B) HeLa cell viability at different
concentrations of PBCs with or without irradiation for 10 min with
reference to the irradiated but untreated cells. The cell viability was
assayed by the MTT method (values: mean valueꢁstandard devia-
tion). C) Change in the blood fluorescence of a) PLLs and b) PBCs
(n=3). Fluorescence intensities F0 and Ft of the porphyrin molecule at
the initial and the given time, respectively.
In in vitro PDT tests bright field images and fluorescence
images of HeLa cells were taken to monitor cell viability.
Images of HeLa cells before and after photoirradiation using
a xenon lamp equipped with a VIS mirror module at
wavelengths of 400–700 nm for 10 min are shown in Fig-
ure 6A,a,c and Figure 6A,b,d, respectively. After treatment
with 0.2 mm PBCs followed by 10 min of irradiation and 24 h
of incubation, significant damage to the impregnated HeLa
cells could be clearly observed and the cells displayed
shrinkage and deformation (Figure 6A,b). Cell survival was
then determined with calcein AM, a fluorescence dye that
stains the cell green when the cell is still living. As shown in
Figure 6A,d, the number of living cells decreased greatly. In
contrast, the control experiments without light irradiation
showed that PBCs alone did not cause cell death (Fig-
ure 6A,c). These experiments indicated the significant photo-
toxicity of PBCs.
The cell phototoxicity study, represented in Figure 6B,
shows the percentage of cell survival after treatment of HeLa
cells at various concentrations of PBCs followed by subse-
quent irradiation with light (400–700 nm).[24] Significant
phototoxic effect on the cultured cells can be observed. In
addition, the uptake amount of PBCs increased with the
elevated concentration, further leading to a higher photo-
toxicity against HeLa cells. In contrast, after 24 h of incuba-
tion, no significant decrease in cell viability in the dark could
be observed at concentrations of PBCs below 1.0 mm,
indicating low dark toxicity of PBCs. These data were well
consistent with the results of cell images (Figure 6A). Never-
theless, free porphyrin molecules showed higher dark toxicity
and remarkably lower phototoxicity relative to that of PBCs.
As shown in Figure S13 in the Supporting Information, no
apparent phototoxicity was observed at concentrations below
1.0 mm. When the concentration was above 5.0 mm, the cell
viability decreased, mainly arising from the dark toxicity of
the free drug. These results were in good accordance with the
singlet oxygen generation data, further confirming the
potential of PBCs for destroying tumor cells by light
exposure.
The blood circulation dynamics of PBCs was investigated
and compared to that of the porphyrin-loaded DSPC lip-
osomes (PLLs). The blood fluorescence intensity of PLLs
decreased to 30% after 6 h and almost no fluorescence was
observed after 24 h, exhibiting rapid clearance kinetics (Fig-
ure 6Ca). On the contrary, the decrease in the blood
fluorescence intensity of PBCs was only 33.5% at 24 h,
showing dramatically prolonged and slow clearance kinetics
(Figure 6Cb). Values of elimination half-life (t1/2) and area
under the plasma concentration–time curve from 0 to 24 h
(AUC0-24) for PBCs were calculated to be (38.04 ꢁ 5.68) h and
(726.84 ꢁ 61.38) mghmLꢀ1, respectively. In contrast, the t1/2
and AUC0-24 of PLLs were (8.18 ꢁ 0.69) h and (264.53 ꢁ
32.96) mghmLꢀ1. Thus, evidence was provided that PBCs
can maintain a long circulation without PEG chains.
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In conclusion, we have successfully developed a cerasomal
photosensitizers by chemical conjugation of porphyrin to an
organoalkoxysilylated lipid. We showed that it is convenient
to regulate the optical density bands of the vesicles to the
desired operating wavelengths by inserting metal ions into the
porphyrin moiety or replacing porphyrin molecules with
other types of photosensitizers. The silica-like surface endows
the PBCs with a remarkably high stability whereas the
inherent porosity allows oxygen molecules to diffuse in and
out of the vesicles freely. In addition, the drug loading
efficiency of PBCs (33.46%) is significantly higher than that
of the physical entrapment of cerasomes. The covalent
linkage of porphyrins can prevent cerasomes to release PSs
during systemic circulation and thus enhances the outcome of
PDT. The proper arrangement of porphyrins in the lipid
bilayer ensures the efficacy of singlet oxygen production.
Even at an extremely high number of porphyrins, fluores-
cence loss of the porphyrins is effectively prevented, resulting
in a powerful tool for in vivo imaging and photodynamic
diagnostics. The capability to load chemotherapy drugs in the
internal aqueous core of PBCs makes it possible to develop
drug–carrier systems for the synergistic combination of
chemotherapy and PDT for the treatment of cancer. The
PBCs are avidly taken up by malignant cells and show cellular
phototoxicity under irradiation. There is no doubt that PBCs
will play an important role in clinical photodynamics.
Received: May 24, 2011
Revised: September 24, 2011
Published online: October 14, 2011
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Keywords: cancer · liposomes · photodynamic therapy ·
self-assembly · sol–gel processes
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