Folate chitosan conjugated doxorubicin and pyropheophorbide acid nanoparticles (FCDP-NPs) for enhance photodynamic therapy

Article abstract of DOI:10.1039/c7ra08757h

We prepared new folate chitosan conjugated doxorubicin (DOX) and pyropheophorbide acid (PPa) nanoparticles (FCDP-NPs) using an ionic gelation method with tripolyphosphate (TPP) to enhance photodynamic therapy activity, based on the considerations of the l

Full text of DOI:10.1039/c7ra08757h

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Folate chitosan conjugated doxorubicin and
pyropheophorbide acid nanoparticles (FCDP–NPs)
Cite this: RSC Adv., 2017, 7, 44426
for enhance photodynamic therapy
Wenting Li, Guanghui Tan, Hongyue Zhang, Zhiqiang Wang* and Yingxue Jin *a
a
b
a
a
We prepared new folate chitosan conjugated doxorubicin (DOX) and pyropheophorbide acid (PPa)
nanoparticles (FCDP–NPs) using an ionic gelation method with tripolyphosphate (TPP) to enhance
photodynamic therapy activity, based on the considerations of the long absorption wavelength (683 nm)
of pyropheophorbide acid (PPa) in water and the excellent chemotherapeutic characteristics of
doxorubicin (DOX) in cancer therapy. The obtained FCDP–NPs demonstrated a typical spherosome
structure, a strong near infrared (NIR) absorption (705 nm) and significantly improved stability and
dispersity in PBS (pH ¼ 5, 7, 9); as well as a high singlet oxygen quantum yield (F
free PPa (F
D
¼ 64%) compared to
D
¼ 59.1%). In addition, the in vitro cell experiments suggested that FCDP–NPs could be
uptaken by HepG2 cells quickly and were mainly located in the cell nucleus. FCDP–NPs showed
improved PDT efficiency over pure PPa and DOX at the same concentration after irradiation. Specifically,
ꢀ1
ꢀ1
FCDP–NPs could lead to a 92% inhibition rate on HepG2 cells at 40 mg mL (equal to 6 mg mL DOX).
ꢀ1
However, the pure DOX showed little cytotoxicity at 6 mg mL , which suggests that a small amount of
DOX could effectively enhance the PDT activities of PPa and lead to little “dark” cytotoxicity. Moreover,
cell morphological changes after PDT treatment further indicated that FCDP–NPs could induce damage
and apoptotic cell death efficiently. Finally, the photochemical mechanism of FCDP–NPs during PDT
process was investigated by using specific quenching agents sodium azide (SA, a single oxygen
quencher) and D-mannitol (DM, a hydroxyl radicals quencher), respectively. The results suggested that
Type I and Type II photodynamic reactions can occur simultaneously, yet Type I reaction (the generation
of hydroxyl radicals) might play a more important role. All these studies indicated that the FCDP–NPs
could be potential nanoparticles in photodynamic cancer treatment.
Received 8th August 2017
Accepted 7th September 2017
DOI: 10.1039/c7ra08757h
rsc.li/rsc-advances
6,7
photosensitizer by visible light.
Photosensitizers can be
1
. Introduction
excited from its ground state to a triplet excited state by
Photodynamic therapy (PDT) has been a potential therapeutic a momentary excited singlet state. The formed triplet states
method for the treatment of various cancers with signicant either undergo electron or hydrogen atom transfer reactions
1–3
efficacy. Traditional methods such as chemotherapy, radio- with a substrate to produce free radicals such as hydroxyl
therapy and surgery have limitations in clinical treatment. Take radical, or transfer its energy directly to oxygen to form excited
8
–10
the case of chemotherapy, it can successfully kill the cancer state singlet oxygen.
These reaction oxygen species (ROS)
cells, yet it can produce systemic toxicity and resistance, have a very strong activity to cause destruction through direct
severely affecting the physical and mental health of the cellular damage and activation of an immune response against
4,5
11
patients. Owing to the low side effects, special selectivity and cancer cells. Besides, the photosensitizers can quickly
being minimally invasive, PDT overcomes the limitations of enrichment in the tumour cells, thus PDT can reach a good
traditional treatments. PDT relies on selective accumulation of curative effect on cancer therapy. Therefore, many PSs have
a photosensitizing agent (photosensitizer) in tumor and been developed for PDT, such as Photofrin, hemoporn,
12,13
subsequent photodynamic effect induced by activation of the Chlorin e6 (Ce6).
Nevertheless, some of the disadvantages of
the photosensitizers (PSs) are not neglected. Most of the
photosensitizers possess porphyrin or porphine structures,
a
Key Laboratory of Photochemistry Biomaterials and Energy Storage Materials of which have larger hydrophobic groups, and the solubility in
Heilongjiang Province, College of Chemistry
&
Chemical Engineering, Harbin
14
water is not ideal. The larger aromatic conjugate system leads
Normal University, Harbin, 150025, China. E-mail: jyxprof@163.com; wzq70402@
to the molecular accumulation and reduces the yield of reactive
oxygen. Moreover, some PSs take longer time to metabolize in
the tumour site with the blood circulation; therefore the
1
63.com; Tel: +86-451-88060569
b
Key Laboratory of Molecular Cytogenetics and Genetic Breeding of Heilongjiang
Province, Harbin, 150025, China
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patients should stay in dark for a long period of time. So PDT pyropheophorbide acid nanoparticles (FCP–NPs) simulta-
needs to be studied deeply to overcome the limitation in clinic neously. Furthermore, the chemical and photo-stability of
therapy.
FCDP–NPs in phosphate buffered solution (pH ¼ 5, 7, 9) were
Generally, appropriate drug carrier with good biocompati- investigated to evaluate the potential in PDT. In addition, the
bility and target ability could reduce or overcome the disad- in vitro PDT activities, including the cellular uptake experi-
15
vantages of chemotherapy drugs and photosensitizers. Such ments, the cell photo-toxicity and dark toxicity experiments
carrier systems could not only control the rate of drug admin- were investigated on HepG2 cells, the FCDP–NPs could enter
istration that prolongs the duration of the therapeutic effect but the cells quickly and mainly located in cell nucleus in some
16–18
also deliver the drug to specic site.
Chitosan is a non-toxic degree. Besides, the formation of reactive oxygen species in
biodegradable polycationic polymer with low immunoge- HepG2 cells aer PDT treatment was also studied. All the
19
nicity.
Hydrophilic chitosan nanoparticles have shown studies indicated that the small amount of DOX in FCDP–NPs
favourable biocompatibility characteristics and could improve can enhance the photodynamic activities, and the FCDP–NPs
membrane permeability both in vitro and in vivo, and could be can be a great potential candidate in cancer therapy.
20
degraded by lysozyme in serum. Although chitosan nano-
particles are good substrate in drug delivery system, but most of
the nanoparticles are ingested by mononuclear phagocytic
systems (MPS), it has good effect on the treatment of MPS
2
. Results and discussion
2.1 Synthesis of FCDP–NPs
21
related diseases, yet it does little for other diseases. Folic acid PPa is a good photosensitizer with large molar extinction coef-
FA) is a kind of vitamin and is appealing as a ligand for tar- cient and long absorption wavelength (683 nm in water).
geting cell membrane, allowing nanoparticle endocytosis via Meanwhile, DOX was known as the widely used chemotherapy
(
22,23
the folate receptor (FR).
Folate receptor (FR) is a single-chain drug, so we selected PPa as PDT drug and DOX as chemotherapy
glycoprotein with high specic affinity for folic acid and drug to increase the effective of PDT. The prepared methods
methotrexate, and is largely shielded from the immune system were shown in Scheme 1, the folate chitosan (FC) was rst
in normal tissue but highly expressed on a variety of malignant
24–26
tumour tissues.
More importantly, the high affinity of folic
acid to its receptor and the small size allows its use for specic
cell targeting. Moreover, the ability of FA to bind its receptor to
allow endocytosis is not altered by covalent conjugation of small
molecules. Therefore, many researchers have used FA as
a ligand with liposome and other polymers for target therapy in
27,28
many elds.
Doxorubicin and its bioactive derivatives are
among the most widely used anticancer drugs in chemotherapy
treatment. For example, doxorubicin (DOX) used as a prodrug to
formulated nanoparticles for NIR-triggered high-efficient
29,30
photodynamic and chemocascade therapy.
Considering the advantages of FA, CS and DOX mentioned
above, in this paper, we designed and prepared folate chitosan
conjugated doxorubicin and pyropheophorbide acid nano-
particles (FCDP–NPs) to enhance photodynamic therapy of PPa,
which is the second generation photosensitizer with large molar
extinction coefficient to transfer the energy for sufficient ROS
generation, and a larger absorption wavelength (683 nm,
located in therapeutic window) ensuring the deep penetration
of light into tissues, being suitable for the photodynamic
therapy. In our strategy, we used folic acid as a specic
substance for tumor target, chitosan as the matrix of nano-
particles, PPa as a photosensitizer and DOX as a chemotherapy
drug to improve the PDT activities. Firstly, folate chitosan was
synthesized by dehydration condensation reaction with the use
of dicyclohexylcarbodiimide (DCC) and N-hydroxysuccinimide
(NHS). Secondly, the obtained folate chitosan were blended
with doxorubicin (DOX) and pyropheophorbide acid (PPa).
Then we got the multifunction nanoparticles (FCDP–NPs) by
inotropic gelation methods with tripolyphosphate (TPP). The
singlet oxygen yield of FCDP–NPs was then tested by using the
1,3-diphenylisobenzofuran (DPBF) as a detector, and compared
Scheme 1 The preparation of folate chitosan conjugated doxorubicin
with pure PPa and folate chitosan
conjugated and pyropheophorbide acid nanoparticles (FCDP–NPs).
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prepared throw dehydration condensation reaction by DCC and peaks were observed and the separation was complete. The rst
NHS using folic acid and chitosan as the starting materials. The one corresponded to folate chitosan with a high molecular
FC was puried from unreacted folic acid and chitosan by weight and the second one corresponded to the unreacted folic
31
Sephadex G-10 gel chromatography, using 2% acetic acid buffer acid with a low molecular weight. The UV spectra of folic acid,
as eluent. Secondly, the obtained FC blended with DOX and PPa chitosan and folate chitosan were showed in Fig. 1b. The folic
under stirring to generate folate chitosan conjugated doxoru- acid had two absorption, a weak peak at 363 nm and a strong at
bicin and pyropheophorbide acid (FCDP), which was trans- 280 nm, chitosan have no absorption from 300 to 800 nm and
formed
tripolyphosphate (TPP) by ionic gelation method. The FCDP– evaluate the extent of folate conjugation in folate chitosan, the
NPs were shown good stability under test conditions. quantitative UV analysis was performed, and it was found that
into
nanoparticles
(FCDP–NPs)
through the absorption of folate chitosan was similar to the folic acid. To
8.23% folic acid was linked to chitosan molecules.
In order to clarify the size and morphology of FCDP–NPs,
2
.2 Characterization of FCDP–NPs
transmission electron microscope (TEM) were adopted. Fig. 2a
showed the TEM image of FCDP–NPs, which clearly displayed
that the nanoparticles presented a typical spherosome structure
with a diameter of 8 ꢁ 1.23 nm. Meanwhile, the hydrodynamic
diameters of FCDP–NPs were measured using dynamic light
scattering (DLS) analysis showed in Fig. 2d, and the mean
diameters of FCDP–NPs in water was approximately 69.5 ꢁ
Via inotropic gelation methods, the FCDP–NPs were formed
successfully, which was comprehensively conrmed by the
following characterization analysis.
The folate–chitosan (FC) was prepared through dehydration
condensation reaction by DCC and NHS using folic acid and
chitosan as the starting materials. The folate chitosan were
puried by Sephadex G-10 gel chromatography, during which
the fractions with high molecular weight would be eluted rstly
followed by the fractions with small molecular weight. Due to
that the chitosan has no absorption from 300 to 800 nm, the
absorbance of folic acid at 363 nm was used to monitor the
different collected fractions. As shown in Fig. 1a, two distinct
4.47 nm, noted that the size of FCDP–NPs characterized by TEM
was much smaller than the ones obtained by DLS, which is
mainly due to that DLS gives a hydrodynamic size that corre-
sponds to the core and the swollen corona of the micelles,
whereas TEM oen gives a size of the core for micelles in a dried
state as the corona with low electronic density is not visible.
Therefore, the nanoparticles size from TEM oen stands for the
actual size of the nanoparticles. In addition, the functional
groups on the surface of the nanoparticles in water solution,
such as the hydroxyl group and carboxyl group, will make the
nanoparticles absorb together to form a larger agglomeration,
which will make DLS more larger than the actual size. Mean-
while, the hydrodynamic sizes of the FCDP–NPs at different
storage time periods were also recorded to evaluate their long-
term colloidal stability, and the hydrodynamic size does not
have any appreciable changes within a time period of 24 h.
What's more, the zeta potential of the FCDP–NPs, free chitosan–
NPs, FCD–NPs (folate chitosan conjugated DOX nanoparticles)
and FCP–NPs (folate chitosan conjugated PPa nanoparticles) at
pH 7.0 in water were all investigated. As shown in Fig. 2c, the
zeta potential of free chitosan–NPs was 12.45 ꢁ 0.55 mV, FCD–
NPs was 11.19 ꢁ 0.9 mV, FCP–NPs was 4.97 ꢁ 0.24 mV, and
FCDP–NPs was 7.43 ꢁ 0.67 mV. Based on the above investiga-
tion, it was clear that FCDP–NPs had been successfully
synthesized, which was also conrmed by the UV spectra.
Fig. 2b displayed the UV spectra of folate chitosan, PPa, DOX
and FDCP–NPs. PPa presented the typical absorptions of chlo-
rophyll, that is, a strong absorption soret band at 413 nm and
y
a shoulder Q (0,0) band at 683 nm in water; the DOX showed
a strong absorption at 490 nm, and the folate chitosan showed
a strong absorption peaks at 280 nm and a shoulder peak at
363 nm. The obtained FCDP–NPs exhibits a broader soret peak
(
415 nm) and Q (0,0) bands at 705 nm. It can be easily
y
y
concluded that the largest absorption wavelength Q (0,0) of
FCDP–NPs showed signicant red-shi from 683 nm to 705 nm
comparing with PPa, yet it still maintains the normal absorp-
tion characteristics of free PPa.
Fig. 1 (a) The Sephadex G-10 gel chromatography of folate chitosan
eluted with 2% acetic acid. The absorbance at 363 nm was used to
monitor the different collected fractions. (b) The UV spectra of folic
acid, chitosan and folate chitosan in water solution.
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Fig. 2 (a) TEM images of FCDP–NPs (b) the UV spectra of folate chitosan, PPa, DOX and FCDP-NPs in water solution (c) zeta potential of CS–
NPs, FCD–NPs, FCP–NPs and FCDP–NPs (d) the hydrodynamic size distribution of FCDP–NPs measured by dynamic light scattering (DLS).
The loading capacity (LC) of the nanoparticles was also and alkaline environment than acidic environment. Therefore,
determined by the change of absorption of the samples before it is reasonable to believe that FCDP–NPs has better stability in
and aer encapsulation. Firstly, pure PPa was determined by PBS medium and at physiological pH, which could be served as
measuring its Q absorbance (A) at 683 nm, the correlation a potential photosensitizer for in vivo photodynamic therapy.
y
between absorption and the concentrations of PPa was
normalized by linear regression, and the standard curve has 2.3 Singlet oxygen quantum yield
2
a well correlation coefficient (R ¼ 0.9976), described by the
Energy transfer between the triplet state of a PS and ground
state molecular oxygen leads to the production of singlet
following equation: Y ¼ 0.0066X + 0.0812. Pure DOX was
determined by the same methods by measuring its absorbance
oxygen. It is essential to generate large amounts of singlet
(
A) at 490 nm, and the standard curve also has a well correlation
1
oxygen. Generally speaking, single oxygen ( O ) is the mainly
2
2
coefficient (R ¼ 0.9984), described by the following equation:
Y ¼ 0.0048X + 0.0771. According to the formula as describe in
the experimental part, the nal loading capacity of PPa was
reactive agent of photosensitizer induced photodynamic
therapy (PDT), and it is necessary for PDT. In order to measure
the generation of singlet oxygen, DPBF (1,3-diphenylisobenzo-
1
0.24 ꢁ 0.92% and DOX was 15.67 ꢁ 1.2%. The loading capacity
1
furan), a quencher of singlet oxygen ( O
2
) with maximum
was also used to calculate the concentration of PPa and DOX in
FCDP–NPs.
32,33
absorption peak at 415 nm was used in this experiment.
Fig. 4a showed the UV absorption spectra of DPBF aer reacting
Next, we studied the drug release of FCDP–NPs in Dulbecco's
Modied Eagle Medium (DMEM). As shown in Fig. 3a, the
FCDP–NPs showed a burst release of 45% at 2 h, followed by an
additional release of 36% over 24 h, indicating that the FCDP–
NPs could release half of the PPa in a short time, and aer 24 h,
about 80% of the PPa could be released from FCDP–NPs.
Moreover, the absorption FCDP–NPs at 705 nm in PBS (5 ꢂ
1
with
O
2
produced by FCDP–NPs under different irradiation
ꢀ2
times (675 ꢁ 10 nm, 10 J cm ). The gradually decreased
absorbance with increasing irradiation time indicated that
1
FCDP–NPs could efficiently produce O
decomposition of DPBF. Meanwhile, the
2
, thus leading to the
1
2
O quantum yield
(
F
D
2
) of FCDP–PNs was obtained as 64% (correlation coefficient
R > 0.9936) according to the calculation by using methylene
blue as the reference compound (F
However, the O
ꢀ
4
1
0
M, pH ¼ 5, 7, 9, respectively) was measured in evaluate the
34
D
¼ 49.1% in DMF).
stability of FCDP–NPs. The results were shown in Fig. 3b–d. It
was found that FCDP–NPs was degraded in different pH
conditions, yet aer 24 h, the degradation rate of FCDP–NPs in
PBS (pH ¼ 5) was higher than that in PBS (pH ¼ 7) and PBS
1
2
quantum yield of pure PPa was 59.1% and
FCP–NPs was 61.2%, there were little lower than that of FCDP–
NPs. Thus, FCDP–NPs may cause excellent cell toxicity in vitro
because of the high generation of singlet oxygen in the experi-
mental conditions.
(
pH ¼ 9), indicated that FCDP–NPs were more stable in neutral
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Fig. 3 (a) Release of PPa from FCDP–NPs in DMEM cell culture medium (b) the UV spectra of FCDP–NPs in pH ¼ 5 PBS at different times. (c) The
UV spectra of FCDP–NPs in pH ¼ 7 PBS at different times. (d) The UV spectra of FCDP–NPs in pH ¼ 9 PBS at different times.
2
.4 Photobleaching
irradiation time. According to the curve in Fig. 5, despite the
slightly decreased absorption of FCDP–NPs and FCP–NPs with
increasing time, yet the declining trend was slow. Specially,
aer irradiation of 10 min, there was only 3% photobleaching
rate for FCDP–NPs. Considering that the PS could achieve good
PDT effect aer a 10 min irradiation, the photobleaching of
FCDP–NPs has a little inuence on PDT effect. Moreover, it is
interesting that FCDP–NPs showed lower photobleaching rate
than those of FCP–NPs in spite of a not signicant different, yet
it still suggested that the introduction of DOX would improve
the photostability to some extent. We suspect the multicyclic
structure of DOX with potential chromophore (multi-
Photobleaching is an important property of a photosensitizer,
and it is oen completely or partially coupled to the photody-
namic reaction. Excellent anti-photobleaching ability is very
important for an ideal PS. Photobleaching is usually considered
as a disadvantage because it can reduce the yield of reactive
oxygen substance (ROS) and decreases the ability to destroy
tumours, so the photostability of a PS is great importance for
PDT. In this paper, the absorptions of FCDP–NPs and FCP–NPs
ꢀ
4
in PBS (2 ꢂ 10 M) at 705 nm were recorded to test the pho-
tostability aer different irradiation time. The photobleaching
curves were drawn according to the absorption at 705 nm and
1
Fig. 4 Ultraviolet-visible absorption spectra (a) of photodecomposition of DPBF by O
2
after irradiation of FCDP–NPs, and first-order plots (b) for
the photodecomposition of DPBF photosensitized by PPa, FCP–NPs and FCDP–NPs respectively, monitoring the maximum absorption of DPBF
at 415 nm.
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
uorescence, and PPa in the FCDP–NPs would emit red uo-
rescence, which could act as an indicator of the uptake behav-
iour of FCDP–NPs in cells. The cell uptaking experiments of PPa
was also conducted. Fluorescence inverted microscopy was
adopted to analyse the uptake behaviour. In our study, the
ꢀ1
concentration of FCDP–NPs was 50 mg mL , and the concen-
ꢀ
1
tration of PPa was 5 mg mL . The amounts of PPa in the two
samples were consistent for reasonable comparison. Fig. 6
showed the experimental results, the spots with uorescence
stand for the cell nuclear stained by DAPI, the ‘PPa’ column and
‘FCDP–NPs’ column stand for the location of PPa with red

uorescence, and the ‘Overlay’ column stands for the merge of
the DAPI column and the FCDP–NPs or PPa column. The
images clearly showed that both FCDP–NPs and PPa can enter
the cells quickly, and the uorescence were observed to be
increasing over the cell culture time for all samples. PPa and
Fig.
5 The photobleaching plots based on the absorption of
maximum at 705 nm in PBS.
conjugated p bonds) could protect the PPA from being FCDP–NPs showed similar trend of uptaking for all time. But
photobleached.
some remarkable differences of uorescence intensity can be
observed. The red uorescence in PPa were a little stronger than
that in FCDP–NPs aer incubated with PSs for 1 h, the reason
for which may be that it need time for FCDP–NPs to release free
2.5 Cell uptake
A better therapeutic therapy needs efficient cellular uptaking for PPa from the nanoparticles. As described earlier, about 46% of
photosensitizer. In order to study the cell uptake of FCDP–NPs PPa would be resealed from FCDP–NPs aer 1 h, at this time,
and efficient cellular location in HepG2 cells, the cells were part of PPa was still encapsulated in the nanoparticles, so the
treated with FCDP–NPs at various incubation times (0.5 h, 1 h, 3 uorescence of FCDP–NPs groups were lower than that of the
h) were stained using DAPI, which is a uorescence probe for pure PPa. Moreover, the location of nanoparticles in cells could
cell nucleus. Aer staining the nucleus would display blue also be observed from the uorescence signal. We have
Fig. 6 Intracellular uptake of FCDP–NPs and PPa. Fluorescence inverted microscopic images of HepG2 cells incubated with FCDP–NPs and
ꢀ1
ꢃ
pure PPa at equivalent PPa concentration (1 mL, 4 mg mL ) at 37 C for 0.5 h, 1 h, 3 h respectively. The nucleus was stained with DAPI showing
blue fluorescence, and the red fluorescence was free PPa and PPa in FCDP–NPs. (a) Image of cells incubated with PPa for 3 h (b) the fluorescence
distribution in image a (c) image of cells incubated with FCDP–NPs for 3 h (d) the fluorescence distribution in image (c).
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that the FCDP–NPs was also mainly located in cell nucleus. This
interesting phenomenon may be attributed to the target group
folic acid and the existence of the DOX. It has been widely re-
29,30
ported that DOX mainly acted in the site of cell nucleus.
Therefore, the introduction of DOX to FCDP–NPs could improve
the nucleus targetability to some degree.
2.6 Dark toxicity and photodynamic activities toxicity
In our previous report, pure PPa showed little dark cytotoxicity.
However, due to the cytotoxicity of DOX, the cell dark toxicity of
the FCDP–NPs should be evaluated for biomedical applications.
Here, the dark toxicity and photodynamic toxicity were
measured by MTT assay. As shown in Fig. 7a, the FCDP–NPs at
ꢀ
1
the concentration ranging from 1 to 40 mg mL did not cause
any obvious cell toxicity effect compared with the negative
control groups (cells treated with DMEM). For cell toxicity
experiments, the tests were divided into three groups, that is,
ꢀ
1
FCDP–NPs (1, 5, 10, 20, 30 and 40 mg mL ) groups, pure DOX
ꢀ
1
groups (0.15, 0.75, 1.5, 3, 4.5, and 6 mg mL ) and pure PPa
ꢀ
1
groups (0.1, 0.5, 1, 2, 3 and 4 mg mL ). The concentrations of
PPa were equal to that in FCDP–NPs, and the concentrations of
DOX were equal to that in FCDP–NPs. The cell toxicity was
shown in Fig. 7b, both FCDP–NPs and PPa groups showed
a drug concentration-dependent cytotoxicity to HepG2 cells
aer 5 min irradiation, while FCDP–NPs remarkably led higher
cell mortality rate compared with free PPa, indicating that the
PDT activity of FCDP–NPs has been effectively improved
compared with free PPa. Meanwhile, at the concentration of 40
ꢀ
1
mg mL , FCDP–NPs caused 92% cell mortality rate as shown in
ꢀ
1
Fig. 7c, DOX (whose concentration was 6 mg mL in FCDP–NPs)
only cause 20% cell mortality rate. Based on the above analysis,
the FCDP–NPs presented higher cell toxicity than pure DOX and
pure PPa, and introduction of DOX could effectively improve the
PDF effect, with little dark cytotoxicity.
2.7 Morphology
We also assess the cell morphological changes of HepG2 cells by
FIM in bright eld aer FCDP–NPs (20 mg mL ) PDT treatment
Fig. 7 The In vitro dark toxicity and PDT toxicity of PPa, DOX and
FCDP–NPs on HepG2 cells incubated with different concentrations
ꢀ1
followed with or without irradiation for 5 min. Cell viability was and subsequently incubation for 0 h, 3 h, 6 h, 12 h respectively.
determined by MTT assay. Data are expressed as mean ꢁ SD (n ¼ 3). From Fig. 8a–d, it is clear that the cells treated with FCDP–NPs
Statistical significant between groups were performed by t-test, and
p < 0.05 showed the difference was significant.
at the studied concentrations displayed remarkable morpho-
logical changes when compared with the control groups treated
with PBS. With the increasing of time, the cells were damaged.
These results corroborated with the cell toxicity, conrmed the
displayed the enlarged HepG2 cells aer incubated with PPa
excellent photodynamic activities of the FCDP NPs. In order to
and FCDP–NPs for 3 h in Fig. 6a and c respectively. Meanwhile
further investigate the cell morphology aer PDT. Acridine
the uorescence distribution images of HepG2 cells were dis-
orange (AO) and ethidium bromide (EB) double uorescent
played in Fig. 6b and d, in which the blue lines stand the
staining were used, then cell imaging was analysed by uores-

uorescence signal of DAPI, red lines stand for the uorescence
cent inverted microscope and shown in Fig. 8e–h. Generally
speaking, aer incubation with AO and EB dyes, the dyes can
bind to nucleus DNA of normal cell and necrotic cell, showing
yellow-green uorescence and reddish-orange uorescence,
respectively. Specially, the nucleus of early apoptotic cells would
exhibit yellow-green uorescence and appeared karyopyknosis;
late apoptotic cell nucleus would show concentrated and
asymmetrical reddish-orange uorescence and appeared
signal of PPa, and the green lines stand for the background
absorption. As showed in Fig. 6b, the blue uorescence mainly
distributed in cell nucleus, yet the red uorescence distributed
in a broad district suggesting that PPa was distributed
throughout the cell. For FCDP–NPs shown in Fig. 6d, the blue
and red uorescence signal showed similar distribution. Due to
DAPI was mainly located in cell nucleus, thus we can conclude
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ꢀ1
Fig. 8 Morphological changes of HepG2 cells incubated with FCDP–NPs (20 mg mL ) after PDT treatment 0 h (a), 3 h (b), 6 h (c), 12 h (d) in bright
field, respectively. FCDP–NPs induced damage and apoptotic death on HepG2 cells. Acridine orange (AO) and ethidium bromide (EB) double
fluorescent staining were utilized as nuclear DNA marker to further observe the nuclear changes after FCDP–NPs PDT treatment. HepG2 cells
were not treated (e) or treated 3 h (f), 6 h (g), 12 h (h) with FCDP–NPs with light irradiation for 5 min, and then monitored by fluorescence inverted
microscopic.
karyopyknosis as well; necrotic cells would show increased oxygen, producing oxygen centred radicals such as hydroxyl
nucleus in volume and exhibit uneven reddish-orange uores- radicals (HR), superoxide anion as well as hydrogen peroxide
cence at their periphery. Based on the above investigation, the (Type I PDT). Alternatively, the excitation energy can be directly
1
cell and nucleus morphological changes aer FCDP–NPs transferred to nearby tissue oxygen to form O (Type II PDT).
2
treatment clearly indicated that FCDP–NPs had a higher PDT The above ROS has a strong activity to induce tissue damage. In
activity to induce damage and apoptotic cell death in HepG
2
order to visualize the photochemical mechanisms of PDT,
cells, which were also in good agreement of the signicantly sodium azide (SA) and D-mannitol (DM), which has of relative
1
improved PDT efficiency in in vitro PDT testing.
.8 Type I and Type II mechanism during PDT progress
2
specicity to O and hydroxyl radicals (HR) respectively, were
used to quench corresponding ROS of Type II and Type I orig-
inated from photodynamic reaction, and meanwhile conrm
the two photodynamic types.
Aer adding quencher SA and DM into cells incubated with
various concentration of FCDP–NPs, the cell viability of FCDP–
NPs–PDT–SA groups and FCDP–NPs–PDT–DM groups were
2
35,36
The result were shown in Fig. 9.
The triplet state photosensitizer can directly interact with
a substrate, and then passing an electron to nearby tissue
signicantly higher than that of FCDP–NPs–PDT groups, indi-
1
cating that the ROS ( O
2
and hydroxyl radicals) generated from
FCDP–NPs during PDT reaction have been quenched effectively.
From the results, the Type I and Type II occurred simulta-
neously in this PDT process. Besides, the cell viability of FCDP–
NPs–PDT–DM groups was obviously higher than that of FCDP–
NPs–PDT–SA groups, indicating that Type I signicantly greater
effect on photodynamic effects than Type II.
3
. Experimental
3
.1 Materials
Chitosan (M > 100 kDa), tripolyphosphate (TPP), dicyclohex-
ylcarbodiimide (DCC), doxorubicin and N-hydroxysuccinimide
NHS) were all purchased from Sigma. Pyropheophorbide acid
w
Fig. 9 The effects of different ROS (Type I and Type II photodynamic
reaction) on HepG2 cells after PDT for 24 h. FCDP–NPs–PDT group:
ꢀ1
(
different concentrations of FCDP–NPs (1, 5, 10, 20, 30 and 40 mg mL
)
and exposed to light; FCDP–NPs–PDT–SA group: with FCDP–NPs was prepared according to the previous report. Dulbecco's
and SA (singlet oxygen quencher, 20 mM) and exposed to light. FCDP–
Modied Eagle Medium (DMEM), dimethyl sulfoxide (DMSO),
phosphate buffered saline (PBS), trypsin, 4 ,6-diamidino-2-
phenylindole (DAPI), 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-
NPs–PDT–DM group: with FCDP–NPs and DM (a hydroxyl radical
quencher, 40 mM) and exposed to light. Cell viability was determined
by MTT. Data represent mean ꢁ SD (n ¼ 3). The significant difference
between two groups was determined by P values, which were
calculated by student's t-test (p < 0.05).
0
2-H-tetrazolium bromide (MTT), D-mannitol (DM), sodium
azide (SA), 1,3-diphenylisobenzofuran (DPBF), acridine orange
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(AO) and ethidium bromide (EB) were purchased from sigma, observed by a Tecnai G2 F20S-TWIN transmission electron
and other reagent were of analytical grade and used without any microscope (TEM FEI, America) operating at 200 kV. TEM grids
purication. Deionized water with a resistivity of 18.2 M cm was were prepared by depositing a drop of the diluted nanoparticle
obtained from a Milli-Q Gnadient System and used in all suspension on a carbon-coated copper support lms and drying
40
experiments. Human hepatoma cell line (HepG2) was obtained the grids under vacuum for 2 h. The stabilities of FCDP–NPs in
from ATCC.
PBS (PH ¼ 5, 7, 9), were studied by an ultraviolet visible spec-
trophotometer (UV-2000, SHIMADZU, Japan). All the measure-
ments were carried out at room temperature in a quartz cuvette
with a path length of 1 cm. The same concentration of FCDP–
NPs was dissolved in 3 mL PBS (PH ¼ 5, 7, 9) to got a nally
3
.2 Synthesis of folate–chitosan conjugated with
doxorubicin and pyropheophorbide acid nanoparticles
FCDP–NPs)
The folic acid conjugated chitosan was prepared according to
(
ꢀ
4
ꢃ
concentration of 1 ꢂ 10 M respectively, then stored at 4 C for
different times. Then the absorbance at 705 nm was monitored
at various time points. The loading capacity of FCDP–NPs was
determined by the separation of nanoparticles from the water
medium containing non-associated doxorubicin and pyro-
31,37,38
the previous reports.
conjugated chitosan, at rst, 300 mg folic acid was dissolved in
Briey, for the synthesis of folic acid
1
7
0 mL DMSO, then 93 mg dicyclohexylcarbodiimide (DCC),
7 mg N-hydroxysuccinimide (NHS) and 0.5 mL triethylamine
ꢃ
pheophorbide acid by ultracentrifugation at 15 000 rpm, 4 C
ꢃ
were added, 30 C stirring overnight. Flitted to remove the by-
products dicyclohexyl urea, then mixed by driping into cold
anhydrous diethyl ether containing 30% acetone, nally got
yellow precipitate, ltered, anhydrous diethyl ether washing
several times aer the vacuum drying, got NHS-ester of folic
acid. Second, a DMSO solution of NHS-ester of folic acid was
prepared and stirred at room temperature until the NHS-ester of
folic acid was well dissolved, then added to a solution of chi-
tosan in acetate buffer (PH ¼ 5.5), the mixture were stirred at
room temperature in the dark for 16 h. Then it was brought to
PH ¼ 9 by dropping 10 M NaOH, ltrated to get the precipita-
tion, wash by water for several times and resolved in 2% acetic
acid aqueous solution, last separated by SepHadex G – 10 gel
column ion, removed the free folic acid not reaction. The
moving phase 2% acetic acid solution and the velocity was
for 40 min. The absorbance of 490 nm (for doxorubicin) and
683 nm (for pyropheophorbide acid) were measured by UV-2000
spectrometry (SHIMADZU, Japan), using a calibration curve of
41
doxorubicin and pyropheophorbide acid in water. Loading
capacity (LC) of the FCDP–NPs was calculated as follows: LC% ¼
(
total drug ꢀ free drug)/(NPs weight) ꢂ 100. The drug release of
PPa was studied in DMEM cell culture medium, in brief, 2 mg
FCDP–NPs was dispersed in 3 mL DMEM cell culture medium at
ꢃ
3
7 C. At varying time points, the medium were measured by
UV-vis spectra. Following supernatant extraction, FCDP–NPs
were discarded. Calibration curves were made with the incu-
bation medium, and all the experiments were performed three
times, and the results were depressed as mean ꢁ SD.
ꢀ
1
3.4 Singlet oxygen quantum yield
1.5 mL min . A UV-visible spectrophotometer (UV-2000 SHI-
MADZU, Japan) was used to monitor the cleaning process at The singlet oxygen quantum yield of FCDP–NPs, FCP–NPs and
63 nm, collected the rst effluent peak and freeze drying PPa aer light irradiation were measured by the singlet oxygen
reserve to get the folate chitosan. Next a solution of the obtained induced bleaching of 1,3-diphenylisobenzofuran (DPBF) using
3
4
2,43
folate chitosan was prepared in the 3 mL 2% acetic acid methylene blue as the reference compound.
In a typical
experiment, 20 mL of solutions of pure PPa, FCP–NPs and
00 mg doxorubicin and 300 mg pyropheophorbide acid were FCDP–NPs (containing the same PPa concentration of 2.8 ꢂ
ꢀ1
aqueous solution to get a nal concentration of 2 mg mL
,
3
ꢀ5
added into the solution, it was brought to PH ¼ 5.5 by dropping 10 M) in DMF respectively, and then added into 3 mL of
ꢀ
5
1
0 M NaOH, then stirred at room in the dark for 24 h to get a stock solution of DPBF (6 ꢂ 10 M) in DMF which had been
folate chitosan conjugated doxorubicin and pyropheophorbide transferred to a sealed quartz cuvette. Then irradiation by
ꢀ2
acid (FCDP). Finally, the FCDP was prepared in the 1.5 mL 2% a calibrated visible light 675 ꢁ 10 nm (10 J cm
NBET
acetic acid aqueous solution to get a nal concentration of HS-UV300), the absorbance of the solution at 415 nm was
ꢀ1
4
mg mL , then it was brought to PH ¼ 5 by dropping 10 M measured every 10
s
for
a
110
s
period with an
ꢀ1
NaOH, aer that, 0.5 mL 2 mg mL tripolyphosphate (TPP) was UV-spectrophotometer (UV 2000, SHIMADZU, Japan). The
slowly dropped into the solution under magnetic stirring singlet oxygen quantum yield (F
conditions, keeping stirring for 30 min to get the FCDP–NPs. following equations:
D
) was calculated using
S
R
F (S) ¼ F (R)k I (R)/k IaT(S)
(1)
(2)
D
D
aT
3.3 Physicochemical characterization of FCDP–NPs
(1 ꢀ eꢀ
2.3A
)
UV-vis spectra was collected with a UV-2000 (SHIMADZU, Japan)
at room temperature in a quartz cuvette with a path length of
I
a
¼ I
0
39
1
cm. The hydrodynamic size and zeta potential of composite
D
F was calculated on the basis of eqn (1) and (2). Superscript S
was carried out with NanoBrook Zeta PALS Zeta Potential and R represent the sample and reference compound, respec-
Analyzer (Brookhaven) at room temperature in water, the tively. Where k is a slope of the liner model. The rst-order
samples were diluted by ultrapure water and the pH value and linear least squares model to get the singlet oxygen quantum
concentration of the NPs dispersion were xed before yield of PPa, FCP–NPs and FCDP–NPs in DMF, obtained by
measurement. The shape and size of the FCDP–NPs were plotting ꢀln([DPBF]
t 0
/[DPBF] ) as a function of the irradiation
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time t, where [DPBF]
DPBF at time t and at time 0, respectively. IaT is dened as the
t
and [DPBF]
0
represent the absorbance of
Cell viability (%) ¼ (A ꢀ A )/(A ꢀ A ) ꢂ 100%
s
b
c
b
total amount of light absorbed by photosensitizers. A and I0 where A
stands the corresponding absorbance at irradiation wavelength various concentrations of FCSP–NPs, A
s
is the average absorbance of the wells treated with
is the average absor-
and transmittance of lter at given wavelength, respectively. bance of the wells treated with DMEM containing 10% fetal
The rate constant was then converted into singlet oxygen bovine serum (FBS), and A is the average absorbance of the
c
b
quantum yield by comparison with the similar rate of methy- wells treated with PBS buffer. For each samples mean and
lene blue mediated photo oxidation to DPBF. Methylene blue standard deviation of ve parallel wells were recorded and the
(
F
D
¼ 49.1%) in DMF was used as the reference compound.
.5 Photobleaching of FCDP NPs
For the photobleaching experiments,
experiments were performed three times.
3
3.8 Cell uptake
44,45
FCDP–NPs was dis- Fluorescence Inverted Microscope (FIM) was used to investi-
ꢀ
4
6
solved in PBS to get a nal concentration of 5 ꢂ 10 M, and gated the cell uptake on HepG2 cell. HepG2 cells (1 ꢂ 10 cells
transfer the solution to 1 ꢂ 1 cm glass cuvettes, then irradiation per well) were seeded in 6-well plates and incubated overnight at
ꢃ
with a calibrated visible light every 5 min (675 ꢁ 10 nm, 37 C in a humidied CO atmosphere. Aer being rinsed with
2
ꢀ
2
ꢀ1
1
0 J cm NBET HS-UV300), the absorption at 705 nm were PBS, the cells were incubated with 1 mL, 50 mg mL of FCDP–
ꢀ1
recorded using a UV-spectrophotometer at interval of 5 min NPs or pure 5 mg mL PPa for 0.5 h, 1 h and 3 h respectively at
ꢃ
before irradiation and during the irradiation, aer irradiation 37 C in the dark under the same conditions. For observed, the
for 0 min, 5 min, 10 min, 15 min, 20 min, 25 min, 30 min, cells were rinsed with PBS, then xed with glutaraldehyde for
ꢀ
1
3
5 min, 40 min, 45 min, 50 min, the absorptions at 705 nm had 10 min and stained with DAPI (1 mg mL in PBS) for 10 min,
been tested respectively. All the experiments were carried out at observed via a Fluorescence Inverted Microscope (Leica DM IL
47,48
room temperature and in dark. The experiments were per- LED, Leica Microsystems, Germany).
formed three times; the results were showed as mean ꢁ SD.
3
.9 Cytotoxicity and cell morphology observation
3
.6 Cell culture
MTT assay was used to quantify the viability of the cells aer
treated with FCDP–NPs, pure DOX and pure PPa at different
concentration. Briey, HepG2 cells were seeded into a 96-well
Human hepatica cell line (HepG2) were cultured in Dulbecco's
modied Eagle's medium (DMEM medium) supplemented with
10% FBS, 100 units per mL of penicillin and 100 units per mL of
4
plate at an initial density of 1 ꢂ 10 cells per well in 100 mL
ꢃ
streptomycin at 37 C in a humid 5% CO
2
incubator. Cells were
DMEM medium. Aer overnight incubation to bring the cell to
conuence, the medium was replaced with 100 mL fresh
2
grown in 75 cm culture acks with 7 mL of medium that was
changed every second days until a sufficient number of cells
were obtained. Cells were than washed three times with PBS and
incubated with trypsin–EDTA solution (0.25% trypsin, 1 mM
EDTA) for 2 min at 37 C to detach them from the ask. The
cells were then suspended in medium in preparation for
reseeding. Cells density was determined by counting cells using
medium containing pure PBS buffer (set as blank), FCDP–NPs
ꢀ1
(1, 5, 10, 20, 30 and 40 mg mL ), pure DOX (0.15, 0.75, 1.5, 3, 4.5
ꢀ1
ꢀ1
and 6 mg mL ), pure PPa (0.1, 0.5, 1, 2, 3 and 4 mg mL ) were
added into the 96 cell plates respectively. Aer 4 h incubation,
exposed the cells to visible light for 5 min (675 ꢁ 10 nm,
ꢃ
ꢀ2
ꢃ
10 J cm NBET HS-UV300), incubation at 37 C and 5% CO
2
for
3
46
a haemocytometer with 0.9 mm counting chamber. And cell
viability were determined by MTT (3-(4,5-dimethylthiazol-2-yl)-
ꢀ1
another 24 h. Then 20 mL MTT (0.5 mg mL in PBS buffer) was
added to each well to detect the metabolically active cells. Aer
further incubation for 4 h in an incubator at 37 C, 150 mL
DMSO was added to each well to replace the culture medium
and dissolve the insoluble formazan crystals. The assays were
carried out according to the manufacturer's instructions and
2,5-diphenyltetrazolium bromide) assay and a Biotek ELx800
ꢃ
absorbance microplate reader was used in MTT assay.
3.7 Dark toxicity assay
47
The dark toxicity was tested by MTT colorimetric assay, in the absorbance of each well was measured using a (a Biotek
brief, HepG2 cells were seeded into 96-well plates with 100 mL ELx800 absorbance microplate reader) at 570 nm. The results
4
fresh medium at a density of 1 ꢂ 10 cells per well. Aer incu- were shown mean ꢁ SD, all the experiments were performed
bation for 12 h to bring the cells to conuence, the medium was three times. For morphology observation, HepG2 cells were
6
replaced with 200 mL fresh medium containing FCDP–NPs with seeded into a 6-well plate at an initial density of 1 ꢂ 10 cells per
ꢀ
1
different nal concentrations (1, 5, 10, 20, 30, and 40 mg mL
,
well in 1000 mL DMEM medium. Aer overnight incubation to
ꢃ
respectively) and the cells were incubated for 24 h at 37 C and bring the cell to conuence, the medium was replaced with
% CO . Thereaer, MTT solution (20 mL, 5 mg mL in PBS 1000 mL FCDP–NPs (20 mg mL ), aer 4 h, the cells were
2
ꢀ
1
ꢀ1
5
ꢀ2
buffer) was added into each well and the cells were incubated exposed under light (675 ꢁ 10 nm, 10 J cm NBET HS-UV300),
for another 4 h. The assays were carried out according to the then continue incubated for 0 h, 3 h, 6 h, 12 h respectively, then
ꢀ1
manufacturer's instruction. The absorbance at 570 nm of each stained by acridine orange (AO, 100 mg mL ) and ethidium
well was measured using a Biotek ELx800 absorbance micro- bromide (EB, 100 mg mL ), nally, the cells morphology were
ꢀ
1
plate reader. Cell viability (%) was then calculated by the observed by a Fluorescence Inverted Microscope (FIM, Leica DM
equation:
IL LED, Leica Microsystems, Germany).
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.10 Type I and Type II mechanism during PDT progress
Paper
3
Acknowledgements
Type I and Type II mechanism of PDT on HepG2 cells was inves-
tigated by MTT colorimetric assay and cell viability were moni-
tored. The quenchers sodium azide (SA, Sigma-Aldrich) and D-
mannitol (DM, Sigma-Aldrich), which have relative specicity for
Financial support of this research was provided by the National
Natural Science Foundation of China (No. 21272048), the
Natural Science Youth Foundation of Heilongjiang Province
(No. QC2016011) and the Scientic Research Fund of Hei-
longjiang Provincial Education Department (No. 12531194,
1
49
2
singlet oxygen ( O ) and hydroxyl radicals respectively, can
quench the corresponding ROS generated from photodynamic
reaction. The experiments were divided into three groups: FCDP–
NPs photo-toxicity experiment groups, FCDP–NPs–SA photo-
toxicity experiment groups, FCDP–NPs–DM photo-toxicity experi-
1
2541234) and the Graduate Innovation Foundation of Harbin
Normal University.
ment groups. HepG2 cells were seeded into 96-well plates at References
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