Nanoparticles based on glycyrrhetinic acid modified porphyrin for photodynamic therapy of cancer

Article abstract of DOI:10.1039/c7ob03108d

Self-assembled small molecules, as a novel form of drug presentation, have splendid capabilities for water stability and cell endocytosis. Photodynamic therapy (PDT) is regarded as a promising cancer treatment because it is less invasive and has fewer sid

Full text of DOI:10.1039/c7ob03108d

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Takeharu Haino et al.
Solvent-induced emission of organogels based on tris(phenylisoxazolyl)
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Nanoparticles based on glycyrrhetinic acid modified porphyrin for
photodynamic therapy of cancer
Received 00th January 20xx,
Accepted 00th January 20xx
Xin Wang,a,b Peisong Wang, Shuai Xue, Xiaohua Zheng, Zhigang Xie, Guang Chen* and
a
a
b,c
b
a
b,c
Tingting Sun*
DOI: 10.1039/x0xx00000x
Self‐assemble of small molecules, as novel form of drug presentation, have splendid capability of water stability and cell
endocytosis. Photodynamic therapy (PDT) is regarded as a promising cancer treatment because of its less invasion and
fewer side effects. In this work, an amphiphilic glycyrrhetinic acid‐porphyrin conjugate (TPP‐GA) was designed and
synthesized, which could self‐assemble into nanoparticles (TPP‐GA NPs) in water. The morphology of TPP‐GA NPs was
observed by transmission electron microscopy (TEM) and the stability in water was revealed by dynamic light scattering
www.rsc.org/
(
DLS). The PDT effect of TPP‐GA NPs was confirmed by means of in vitro experiment, after the photostability verification. It
could be conceived that applying TPP‐GA NPs into PDT is a feasible choice and shows progress in self‐assembly of
amphiphilic small organic molecules.
Introduction
Although medical technology has gained rapid development, cancer
1
is still one of the irresistible death causes in our age. Besides
chemotherapy, operation and radiation therapy, which are regarded
as three main therapies for cancer in clinic, photodynamic therapy
(
PDT) has increasingly been employed for cancer treatment.2, 3
A
myriad of researchers and clinical scientists display enthusiasm for
PDT on account of its excellent therapeutic effect with less invasion
4
and fewer side effects. Photosensitizers (PSs), which generate
cytotoxic reactive oxygen species (ROS) under the irradiation of light
5
at an appropriate wavelength, are of great importance in PDT. The
6
, 7
Scheme 1 Schematic illustration of the synthesis of TPP‐GA and
preparation of TPP‐GA NPs.
generated ROS can lead to the necrosis or apoptosis of tumor.
Porphyrin (TPP) and its derivatives, as one of the most widely used
3, 8, 9
photosensitizers, attracts much attention from researchers.
Our
group has also reported the application of porphyrin‐containing
nanomaterials, which could kill cancer cells effectively.
coworkers took advantage of polymerization to induce self‐
9
, 10
1
4
assembly of block copolymer. Zhang et al. applied disulfide bond
bridge for self‐assembly and successfully obtained stable
Comparing with using drug carriers, self‐assembly of small
organic molecules to prepare nanodrugs is a more convenient and
9
nanomedicines. Choosing hydrophilic molecules or a drug as the
1
1
efficient way to deliver drugs, which can realize high drug content
building component of amphiphilic organic molecules is also a
1
2
and fantastic water solubility simultaneously. There are many
1
5
wonderful path towards nanoparticles formation. In this work,
8β‐glycyrrhetinic acid (GA, 3β‐hydroxyl‐11‐oxo‐olean‐12‐ene‐29‐
oic acid), the hydrolysate of glycyrrhizic acid, which is water
1
3
different ways towards self‐assembly. For example, M. J. Derry and
1
1
6
soluble, was selected to be conjugated with TPP. TPP is
hydrophobic, so amphiphilic conjugate of TPP and GA (TPP‐GA) can
a. Department of Thyroid Surgery, The First Hospital of Jilin University, 71 Xinmin
self‐assemble into nanoparticles, enhancing the solubility of TPP in
water.17 TPP‐GA NPs show fantastic capability of endocytosis and
precise phototoxicity.
Street, Changchun, Jilin 130021, P. R. China. E‐mail: cg9293@sina.com
State Key Laboratory of Polymer Physics and Chemistry, Changchun Institute of
b.
Applied Chemistry, Chinese Academy of Sciences, 5625 Renmin Street,
Changchun, Jilin 130022, P. R. China. E‐mail: suntt@ciac.ac.cn
In this work, an amphiphilic glycyrrhetinic acid‐porphyrin
conjugate (TPP‐GA) was designed and synthesized, which could self‐
assemble into nanoparticles (TPP‐GA NPs) in water. TPP‐GA NPs
have better photodynamic effect than NPs from TPP alone (TPP
c.
University of Chinese Academy of Sciences, Beijing 100049, P. R. China
1
†
Electronic Supplementary Information (ESI) available: H NMR spectrum of TPP‐
NO
2
, TPP‐NH
2
and TPP‐GA; MALDI‐TOF MS of TPP‐NH
2
and TPP‐GA; Time‐
with
dependent changes of the diameter and PDI of TPP‐GA NPs; MTT of TPP‐NH
2
or without light; Live/dead staining of HeLa cells. See DOI: 10.1039/x0xx00000x
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Journal Name
NPs). TPP‐GA NPs could be endocytosed by various cancer cells as 7.4) containing 10% fetal bovine serum (FBS) for 24 h, which
revealed by confocal laser scanning microscopy (CLSM) and showed demonstrate that the stability is satisfactory.
favourable photodynamic therapy (PDT) effect toward cancer cells
as confirmed by in vitro experiment.
Optical Properties of TPP‐GA NPs
The optical properties of TPP‐GA NPs are detected by UV/vis
absorption and photoluminescence (PL) spectra. Porphyrin and its
derivatives feature two specific absorption regions, so‐called low‐
energy Q bands and high‐energy B bands.20 As shown in Fig. 1c, the
Results and discussion
Synthesis of TPP‐GA
absorption spectrum of TPP‐GA in N,N‐Dimethylformamide (DMF)
exhibits a typical B band at 418 nm and four less intense Q bands in
The main synthesis route of TPP‐GA is shown in Scheme 1. After
purification by silica gel column, the product was obtained and well
characterized by proton nuclear magnetic resonance ( H NMR) (Fig.
S5) and matrix‐assisted laser desorption/ionization time‐of‐light
mass spectrometry (MALDI‐TOF MS) (Fig. S6), which demonstrated
their molecular structures. The peak at m/z 1082.5 corresponds to
the theoretical molecular weight of TPP‐GA, which confirms the
2
1
1
the range of 500‐700 nm. Moreover, the maximum absorption of
TPP‐GA NPs was red‐shifted compared to the record of TPP‐GA in
DMF, which probably due to the formation of nanoscale
2
2
aggregation in aqueous solution. In Fig. 1d, TPP‐GA (10 μM) has
two obvious peaks of fluorescence wavelength at around 650 and
750 nm, but weak fluorescence could be detected in TPP‐GA NPs at
successful synthesis of the compound
.
the same concentration, because the Dexter‐type excitonic
2
3
migration between porphyrins results in fluorescence quenching.
Preparation and characterization of TPP‐GA NPs
Inset of Fig. 1d further validated this phenomenon visually, that is
TPP‐GA in DMF under 365 nm light irradiation showed strong red
fluorescence, while nearly no fluorescence of TPP‐GA NPs in
aqueous solution could be observed. The disassembly process of
TPP‐GA NPs in cells was also studied. It was simulated by adding
cell lysis solution into TPP‐GA NPs and the mixture was stored at 37
TPP‐GA could self‐assemble into nanoparticles through
nanoprecipitation method. The size distribution and morphology of
the nanoparticles were characterized by dynamic light scattering
(
DLS) and transmission electron microscopy (TEM) (Fig. 1a, b). The
2
4
TEM image demonstrates that TPP‐GA could self‐assemble into
spherical nanoparticles at a size of about 100 nm in aqueous
solution, while DLS result shows that the average diameter of TPP‐
GA NPs is about 150 nm, perhaps due to the dehydration of TPP‐GA
℃
. TPP‐GA NPs in Dulbecco's modified Eagle's medium (DMEM)
was used as the control. As shown in Fig. S8, the PL intensity of TPP‐
GA NPs (5 μM) in cell lysis solution increased with incubation time,
while that of TPP‐GA NPs (5 μM) in DMEM remained weak even
after 24 h. The results demonstrated that TPP‐GA NPs could
disassemble in cells, while keep stable in culture media.
1
9
NPs in preparation of TEM sample. As shown in Fig. S7, the
changes of average diameter and polydispersity index (PDI) suggest
that TPP‐GA NPs could keep stable in aqueous solution for two
weeks or after being incubated in phosphate buffer saline (PBS, pH
Fig.1 (a) TEM image and (b) DLS data of TPP‐GA NPs. (c) Normalized UV/vis absorption and (d) fluorescence spectra of TPP‐GA (10 μM) in
2
DMF and nanoparticles in aqueous solution. (e) Photostability of TPP‐GA NPs in aqueous solution under irradiation (620 nm, 12 mW/cm )
for 45 min. (f) Singlet oxygen generation of TPP‐GA (10 μM) in DMF, TPP‐GA NPs (10 μM) and ICG blank in water monitored by decrease
2
of normalized absorption of ICG (5 μM) at 789 nm upon irradiation (620 nm, 12 mW/cm ) for 20 min.
2
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Photostability and generation of ROS
Photostability of PS is essential for PDT, thus the absorption
spectrum of TPP‐GA NPs was tested every 15 minutes under 620 nm
2
irradiation (12 mW/cm ). The results show perfect stability of TPP‐
GA NPs under long‐time irradiation (Fig. 1e). The crucial factor of
1
PDT is the generation of singlet oxygen ( O
2
) upon irradiation, which
was tested by using indocyanine green (ICG) as the detector and
monitoring the change of absorption spectra with irradiation time.
ICG in DMF solution was irradiated by an LED lamp (620 nm, 12
2
mW/cm ) for 20 min and small change of the absorbance at 789 nm
(the maximum absorption of ICG) was observed. Furthermore, ICG
was added into TPP‐GA (10 μM) in DMF and TPP‐GA NPs (10 μM) in
water respectively, the obvious time‐dependent decrease of
1
absorbance at 789 nm showed the generation of O
2
(Fig. 1f). TPP‐
1
GA molecules possess better capability in generation of O
2
than
TPP‐GA NPs at the same concentration, because aggregation of
25
molecules could inhibit the generation of ROS. In addition, TPP‐
NH could also self‐assemble into nanoparticles (TPP NPs) via
previous method and were confirmed via DLS (Fig. S9a). To compare
2
Fig. 3 Cellular uptake of TPP‐GA NPs. Representative CLSM images
of HeLa, HepG2 and K1 cells incubated with TPP‐GA NPs for 6 h.
Different imaging channels are displayed horizontally for each
sample (from left to right): DAPI (405 nm excitation), TPP (488 nm
excitation) and merged images.
1
the ability between TPP‐GA NPs and TPP NPs in O
2
generation,
9,10‐anthracenediyl‐bis(methylene)dimalonic acid (ABMDMA, 20
μM) was employed as the ROS detector. The results in Fig. S9b
demonstrates that TPP‐GA NPs have better photodynamic effect
than TPP NPs.
The generation of ROS was also detected intracellularly through
confocal laser scanning microscopy (CLSM) and 2′,7′‐
dichlorofluorescin diacetate (DCFH‐DA) was used as a ROS probe. In
Fig. 2, strong green fluorescence could be observed in cells treated
with TPP‐GA NPs (10 μM) for 2 h and following with one‐hour
NPs could be endocytosed by these three kinds of cancer cells (Fig.
). The quantitative analysis of TPP‐GA uptake by HeLa cells was
3
monitored by flow cytometry and shown in Fig. S10. The cellular
uptake of free TPP, TPP‐GA, TPP NPs and TPP‐GA NPs were also
studied via CLSM, as shown in Fig. S11. There was no obvious
difference in fluorescence observed among the four formulations,
which might due to the weak fluorescence of porphyrin.
2
irradiation (620 nm, 12 mW/cm ). Meanwhile, green fluorescence
could hardly be observed in cells treated with TPP‐GA NPs in dark.
The contrast in Fig. 2 illustrated that TPP‐GA NPs could generate
ROS in cells after irradiation.
Cytotoxicities
Cellular uptake
In order to reveal the intracellular photodynamic effect, HeLa,
HepG2 and K1 cells were all separated into three groups, the GA
group, the dark group and the light group. All the groups were
treated for 72 h, and the light group was irradiated for 1 h at the
second day. GA group was simply treated with GA at the same
concentration as the other two groups. The cell viability was then
performed by thiazolyl blue tetrazolium bromide (MTT) assays (Fig.
The cellular uptake of TPP‐GA NPs was verified via CLSM. Human
cervical carcinoma (HeLa), liver hepatocellular carcinoma (HepG2)
and human thyroid papillary carcinoma (K1) cell lines were
incubated with TPP‐GA NPs (10 μM) for 6 h. The cell nuclei were
stained with 4,6‐diamidino‐2‐phenylindole (DAPI). Distinct green
fluorescence was shown in cell plasma, demonstrating that TPP‐GA
4). The cell viability of the light group was gradually decreased with
the increasing of concentration, while that of the dark group
maintained above 80%, indicating obvious photodynamic effect of
TPP‐GA NPs. The half‐maximal inhibitory concentrations (IC50) in
the light group (24.2 μM for HeLa cells, 26.5 μM for K1 cells, and
2
9.6 μM for HepG2 cells) further validated the excellent
photodynamic therapy effect of the nanoparticles. As shown in Fig.
S12, GA showed no photocytotoxicities. In addition, cell viabilities of
HeLa cells incubated with free TPP‐NH
irradiation for 72 h are shown in Fig. S13, which suggested than TPP
NPs had weaker PDT effect than TPP‐GA NPs and free TPP‐NH
2
or TPP NPs with or without
2
.
Live/dead staining of cells was applied to view the therapy effect
directly. Live cells turn to be green after staining with calcein‐AM
and dead/late apoptotic cells become red after staining with
propidium iodide (PI). As shown in Fig. S14, in the dark group
Fig. 2 Intracellular detection of ROS by CLSM. HeLa cells were
incubated with TPP‐GA NPs (10 μM) for 2 h and then under
2
irradiation (620 nm, 12 mW/cm ) for 1 h. Cells in dark were set as
the control group.
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Materials and measurements
MTT (3‐[4,5‐dimethylthiazol‐2‐yl]‐2,5‐diphenyltetrazolium bromide)
and Reactive Oxygen Species Assay Kit was purchased from
Beyotime Biotechnology Co., Ltd. (China). Dimethyl sulfoxide
(
DMSO), 4‐dimethylaminopyridine (DMAP) were purchased from
Sigma‐Aldrich (St. Louis, MO, U.S.A.). Live/Dead cells staining kit
was purchased from Nanjing KeyGen Biotech. Co., Ltd. (China). 18β‐
glycyrrhetinic acid (GA, 3β‐hydroxyl‐11‐oxo‐olean‐12‐ene‐29‐oic
acid) was purchased from Shanghai Sun Chemical Technology Co.,
Ltd. All of the other chemicals were obtained commercially and
used without further purification, unless otherwise noted. All the
solvents were purified according to the standard methods wherever
needed. Ultrapure water was prepared from a Milli‐Q system
(
Millipore, USA).
1H NMR spectra were recorded in chloroform‐d (CDCl
) on a
3
Bruker NMR‐400 DRX spectrometer at 400 MHz at room
temperature. The mass spectra (MS) of the samples were recorded
by the German company Bruker auto flex III smart beam MALDI‐
TOF/TOF mass spectrometer with smart beam laser at 355 nm
wavelength. UV‐vis absorption spectra were monitored with a
Shimadzu
UV‐2450
PC
UV/Vis
spectrophotometer.
Photoluminescence (PL) spectra were obtained by using
PerkinElmer LS‐55 Spectrofluorophotometer. Size and size
distribution of the nanoparticles were recorded by Malvern Zeta‐
sizer Nano for dynamic light scattering (DLS). TEM images were
recorded by JEOL JEM‐1011 electron microscope (acceleration
voltage of 100 kV). Confocal laser scanning microscopy (CLSM)
images were taken by a Zeiss LSM 700 (Zurich, Switzerland).
Fig. 4 Cell viabilities of HeLa (a), HepG2 (b) and K1 (c) cells
incubated with various concentration of GA and TPP‐GA NPs with
or without irradiation.
Synthesis of TPP‐GA.
TPP‐NH
2
:
4‐(10,15,20‐Triphenyl‐porphyrin‐5‐yl)‐phenylamine(TPP‐
NH ) was synthesized from tetraphenylporphyrin (TPP) by three
2
steps according to the previous reported methods (Fig. S1).
According H NMR spectra of intermediate products were exhibited
in the following.
treated with TPP‐GA NPs, bright green fluorescence could be
viewed while red fluorescence could hardly be seen, indicating the
perfect biocompatibility of TPP‐GA NPs. The light group treated
1
with TPP‐GA NPs shows strong red fluorescence and weak green TPP‐NO2:
fluorescence, which directly reveals the cytotoxicity of TPP‐GA NPs 2H), 8.66 (d, J = 8.1 Hz, 2H), 8.42 (d, J = 7.9 Hz, 2H), 8.24 (d, J = 6.3
1
3
H NMR (300 MHz, CDCl ) δ 8.90 (d, J = 7.0 Hz, 6H), 8.77 (s,
under light irradiation.
Hz, 6H), 7.80 (s, 10H), ‐2.77 (s, 2H) (Fig. S2).
TPP‐NH ) δ 8.94 (d, J = 4.5 Hz, 2H), 8.83 (s,
: 1H NMR (400 MHz, CDCl
H), 8.21 (d, J = 6.4 Hz, 6H), 7.99 (d, J = 7.6 Hz, 2H), 7.75 (d, J = 6.8
Hz, 9H), 7.05 (d, J = 7.6 Hz, 2H), 4.01 (s, 2H), ‐2.75 (s, 2H) (Fig. S3).
MALDI‐TOF‐MS of TPP‐NH , calcd for C44 : 629.26, found 629.3
Fig. S4).
TPP‐GA: TPP‐NH
2
3
6
Conclusions
In summary, we have designed a new kind of porphyrin based
nanomaterial which could be applied on photodynamic
therapy of cancer cells. The combination of hydrophilic
molecule and hydrophobic photodynamic agent shows
excellent capability of self‐assembly. The amphiphilic material
could self‐assemble into stable nanoparticles and be
endocytosed easily by cancer cells. By means of irradiation, the
photosensitive nanoparticles show efficient photodynamic
therapeutic effect of cancer. Prospectively, this work is a step
forward toward the development of nanomedicines based on
amphiphilic small molecules for photodynamic therapy of
cancer.
2
31 5
H N
(
2
(188.7 mg, 0.3 mmol) and GA (211.8 mg, 0.45
mmol) were dissolved in about 10 mL of anhydrous methylene
chloride. Then 4‐dimethylaminopyridine (DMAP) (5.5 mg, 0.045
mmol)
and
1‐(3‐Dimethylaminopropyl)‐3‐ethylcarbodiimide
hydrochloride(EDC∙HCl) (172.5 mg, 0.9 mmol) were added
sequentially at room temperature. After stirring for 1 h at room
temperature, additional EDC∙HCl (86.3 mg, 0.45 mmol) and DMAP
(5.5 mg, 0.045 mmol) were added and the solution was stirred for 2
days. Then the solvent was removed by evaporation, and the mixed
product was purified by column chromatography on silica, eluting
1
with dichloromethane/methanol (70:1, v/v) as the eluent. H NMR
3
(400 MHz, CDCl ) δ 8.92 (d, J = 4.7 Hz, 2H), 8.87 (d, J = 4.8 Hz, 6H),
Experimental
4
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8.23 (dd, J = 7.6, 1.6 Hz, 7H), 8.20 (s, 1H), 7.94 (t, J = 8.4 Hz, 2H), culture medium. After incubation at 37 °C for 15 h, the supernatant
7.83 – 7.72 (m, 10H), 7.69 (s, 1H), 5.85 (s, 1H), 3.17 (d, J = 7.3 Hz, was carefully removed and the cells were washed three times with
1H), 2.80 (dd, J = 10.3, 3.3 Hz, 1H), 2.44 (dd, J = 13.2, 3.2 Hz, 1H), phosphate buffered saline (PBS) and treated with trypsin. Then 1
2.38 (s, 1H), 2.23 – 2.11 (m, 1H), 2.05 (t, J = 15.9 Hz, 2H), 1.87 (ddd, mL of PBS was added to each well and the solutions were
J = 18.1, 17.6, 9.0 Hz, 2H), 1.73 – 1.64 (m, 4H), 1.53 (s, 1H), 1.42 (d, J centrifuged at 1000 rpm for 5 min. The cells were resuspended in
3.8 Hz, 7H), 1.38 (t, J = 6.7 Hz, 1H), 1.33 – 1.20 (m, 3H), 1.19 – 1.10 0.8 mL of PBS after removing the supernatants. Data for the 5,000
m, 7H), 1.07 (d, J = 13.9 Hz, 1H), 1.03 – 0.88 (m, 8H), 0.80 (s, 3H), gated events were collected and analyzed by Guava easyCyte 6‐2L
.69 (d, J = 11.4 Hz, 1H), ‐2.76 (s, 2H) (Fig. S5). MALDI‐TOF‐MS of Base System (Merck Millipore, USA).
TPP‐GA, calcd for C74 : 1082.42; Found:1082.5 (Fig. S6).
=
(
0
75 5 3
H N O
MTT assays
Preparation of TPP‐GA NPs.
Cells harvested in a logarithmic growth phase were seeded in 96‐
3
TPP‐GA NPs could be obtained by means of self‐assembly of TPP‐GA well plates at a density of 5 ×10 cells per well and incubated in
in aqueous solution. Briefly, TPP‐GA (1 mg) was dissolved in acetone DMEM for 24 h. The medium was then replaced by TPP‐GA NPs or
(
4 mL). After dissolving about 3 min by hyperacoustic method, the GA at various concentrations. The incubation was continued for 72
solution was added dropwise slowly to the deionized water (10 mL) h, but at the 24th hour, the light group was irradiated by a LED lamp
2
and stirred to evaporate acetone for 48 h.
at 12 mW/cm with a 620 nm wavelength for 1 h. Then, 20 µL of
MTT solution in PBS (5 mg/mL) was added and the plates were
incubated at 37 °C for another 4 h. After removing the culture
medium containing MTT, 150 µL of dimethyl sulfoxide (DMSO) was
added in each well to dissolve the formazan crystals formed. Finally,
the plates were shaken for 5 min, and the absorbance of formazan
product was measured at 490 nm by a microplate reader.
Cell lines and cell culture
Human cervical carcinoma (HeLa), liver hepatocellular carcinoma
(HepG2) and human thyroid papillary carcinoma (K1) cell lines were
grown in Dulbecco's modified Eagle's medium (DMEM, GIBCO)
supplemented with 10% heat‐inactivated fetal bovine serum (FBS,
GIBCO), and the culture medium was replaced once every two days.
Live/ dead cell staining assays.
Photostability and singlet oxygen detection
To visually confirm the phototoxicity and PDT efficacy of TPP‐GA
NPs, HeLa cells were stained with the calcein‐AM/propidium iodide
(PI) to determine their viabilities. Firstly, the cells were incubated
with TPP‐GA NPs (18 μM) for 24 h and then irradiated with 620 nm
In order to detect the photostability, TPP‐GA NPs (10 mM) were
2
irradiated by a LED lamp at 12 mW/cm with 620 nm wavelength for
4
5 min. To confirm the singlet oxygen generation ability of TPP‐GA
2
LED lamp at a power density of 12 mW/cm for 60 min. After that,
NPs, a chemical method using indocyanine green (ICG) was
employed with UV/vis spectroscopy. TPP‐GA dissolved in DMF (10
o
the cells were incubated at 37 C for another 48 h. The cells treated
with the same concentration of TPP‐GA NPs without irradiation and
blank group with or without irradiation were set as the controls at
the same time. The cells were stained with calcein‐AM (green for
living cells) and PI (red for dead cells) for 40 min at room
temperature, after washing with PBS, and finally imaged by a Nikon
C1si laser scanning confocal microscopy (Fig. S8).
mM) were mixed with ICG solution (5 μM) and then irradiated with
2
1
2 mW/cm of a 620 nm light source. The absorption intensity of
the ICG at 789 nm (maximum of ICG absorbance) was detected
every 2 min. The same experiments were done with single ICG (5
μM) as control.
Confocal laser scanning microscopy (CLSM)
Cellular uptake by HeLa, HepG2 and K1 cells was detected by CLSM.
Cells were seeded in 6‐well culture plates (a clean cover slip was put
in each well) at a density of 1 × 10 cells per well and incubated for
Conflicts of interest
There are no conflicts to declare.
5
2
4 h. The medium was then replaced by TPP‐GA NPs (10 μM) in
culture medium. After incubation at 37 °C for 6 h, the supernatant
was carefully removed and the cells were washed three times with
phosphate buffered saline (PBS). After that, the cells were fixed
with 1 mL of 4 % formaldehyde each well for 10 min at room
temperature and washed three times with PBS again. Finally, the
nuclei of cells were stained with 4’,6‐diamidino‐2‐phenylindole
Acknowledgements
This work was supported by the Foundation of Health Department
of Jilin Province (2010Z100) and National Natural Science
Foundation of China (Project No. 51522307).
(
DAPI) for 5 min and washed three times with PBS. Samples were
Notes and references
examined by CLSM using a Zeiss LSM 700 (Zurich, Switzerland).
1
(a) W. He, J. Frueh, Z. Wu and Q. He, Langmuir, 2016, 32, 3637;
(b) A. Mallick, P. More, S. Ghosh, R. Chippalkatti, B. A. Chopade,
M. Lahiri and S. Basu, ACS Appl. Mater. Interfaces, 2015, 7,
Flow cytometry
Quantitative analysis of cellular uptake was examined by Flow
Cytometry Scanning (FCS). HeLa cells were seeded in 6‐well culture
plates at a density of 1 × 10 cells per well and incubated for 24 h.
7
584.
5
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DOI: 10.1039/C7OB03108D
Nanoparticles from amphiphilic glycyrrhetinic acid-porphyrin conjugate (TPP-GA)
were prepared and applied to photodynamic therapy of cancer.