Peroxynitrite (ONOO-) generation from the HA-TPP@NORM nanoparticles based on synergistic interactions between nitric oxide and photodynamic therapies for elevating anticancer efficiency

Article abstract of DOI:10.1039/c9nj04763h

Due to biological safety and negligible toxicity, nitric oxide (NO) therapy has gained increasing interest in the field of cancer therapy during the past few years. However, individual NO cancer therapy normally exhibited limited therapeutic efficiency. In order to acquire satisfactory therapeutic outcomes, the NO therapy is usually combined with other therapeutic treatments, mostly chemotherapy. Herein, we constructed HA-TPP@NORM nanoparticles based on the co-assembly of an NO donor (NORM) and tetraphenylporphyrin (TPP)-modified hyaluronic acid, which can efficiently generate a highly biocidal molecule of peroxynitrite (ONOO^-) via the synergistic interactions of the nitric oxide (NO) therapy and photodynamic therapy (PDT) to enhance the cancer therapeutic efficiency. In addition, MTT results exhibited that without light irradiation, the HA-TPP@NORM nanoparticles have favourable biocompatibility with the cell viability above 95% at the maximum TPP concentration (20 μg mL^-1). Under simultaneous irradiation with 365 nm and 650 nm light, ONOO^- can be efficiently produced in cancer cells via the direct coupling reaction of the generated NO and superoxide anion radical (O₂^-), which significantly enhanced the anticancer effect, when compared with individual NO therapy or PDT therapy. Therefore, the HA-TPP@NORM nanoparticles may provide a new insight into the design of efficiently NO-related cancer therapeutic systems.

Full text of DOI:10.1039/c9nj04763h

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Peroxynitrite (ONOOꢀ) generation from the
HA-TPP@NORM nanoparticles based on
synergistic interactions between nitric oxide and
photodynamic therapies for elevating anticancer
efficiency†
Cite this: New J. Chem., 2020,
44, 162
b
Dawei Jiang,a Tao Yue,a Guichen Wang,a Chaochao Wang,a Chao Chen,
Hongliang Cao *a and Yun Gao*a
Due to biological safety and negligible toxicity, nitric oxide (NO) therapy has gained increasing interest in
the field of cancer therapy during the past few years. However, individual NO cancer therapy normally
exhibited limited therapeutic efficiency. In order to acquire satisfactory therapeutic outcomes, the NO
therapy is usually combined with other therapeutic treatments, mostly chemotherapy. Herein, we
constructed HA-TPP@NORM nanoparticles based on the co-assembly of an NO donor (NORM) and
tetraphenylporphyrin (TPP)-modified hyaluronic acid, which can efficiently generate a highly biocidal
molecule of peroxynitrite (ONOOꢀ) via the synergistic interactions of the nitric oxide (NO) therapy and
photodynamic therapy (PDT) to enhance the cancer therapeutic efficiency. In addition, MTT results
exhibited that without light irradiation, the HA-TPP@NORM nanoparticles have favourable
biocompatibility with the cell viability above 95% at the maximum TPP concentration (20 mg mLꢀ1).
Under simultaneous irradiation with 365 nm and 650 nm light, ONOOꢀ can be efficiently produced in
Received 18th September 2019,
Accepted 19th November 2019
cancer cells via the direct coupling reaction of the generated NO and superoxide anion radical (O2 ꢀ),
ꢁ
which significantly enhanced the anticancer effect, when compared with individual NO therapy or PDT
therapy. Therefore, the HA-TPP@NORM nanoparticles may provide a new insight into the design of
efficiently NO-related cancer therapeutic systems.
DOI: 10.1039/c9nj04763h
rsc.li/njc
of the NO donor in the tumour site. At low concentrations, nitric
oxide (NO) can not only accelerate tumour angiogenesis,14 but it
Introduction
As a ‘‘star’’ molecule, nitric oxide (NO) is involved in various can also overcome the multidrug resistance of chemotherapy by
physiological and pathological processes, such as immune suppressing the expression of P-glycoprotein.15 Thus, NO is usually
responses, apoptosis, angiogenesis, cardiovascular homeo- combined with chemotherapeutic drugs (DOX, etc.) to elevate the
stasis and neurotransmission.1–5 Due to its biological safety anticancer efficiency.16–18 However, inevitable nonspecific systemic
and negligible toxicity, NO has gained increasing interest toxicity could be caused by the early release of chemotherapy drugs,
during the past few years for applications in cancer therapy.6–12 which severely limits its clinical applications.
However, individual NO therapy normally exhibited a limited
Peroxynitrite (ONOOꢀ), as a derivative of NO, is also an
cancer therapeutic efficiency. High concentrations of NO release endogenous signalling molecule and plays a key role in various
(41 mM) in cancer cells are the prerequisite to induce limited physiological and pathological processes.19–22 As a highly reac-
cancer cell death,13 which need to efficiently supply a large amount tive nitrogen species, ONOOꢀ shows significant oxidization or
nitration capabilities, which can considerably cause cell death
a Shanghai Key Laboratory of Functional Materials Chemistry, School of Materials
Science and Engineering, East China University of Science and Technology,
Shanghai 200237, People’s Republic of China. E-mail: caohl@ecust.edu.cn,
yungao127@163.com
b State Key Laboratory of Bioreactor Engineering, Biomedical Nanotechnology
Center, School of Biotechnology, East China University of Science and Technology,
Shanghai 200237, People’s Republic of China
† Electronic supplementary information (ESI) available. See DOI: 10.1039/c9nj04763h (O2 ꢀ).25 One feasible way to produce O2 can be realised by
by destroying proteins, lipids and DNA. In addition, its cyto-
toxicity is higher than most free radicals, such as NO and
hydroxyl radical (ꢁOH).23,24 Thus, we assume that the introduc-
tion of ONOOꢀ can enhance the cancer therapeutic efficiency.
In living systems, ONOOꢀ can be produced by a direct
coupling reaction between NO and superoxide anion radical
ꢀ
ꢁ
ꢁ
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the type I photodynamic therapy (PDT).26 Due to the minimal irradiation, NORM can rapidly release NO. Moreover, the
invasiveness, spatiotemporal controllability and excellent anti- quenched fluorescence can be recovered, which can be used to
cancer effect, PDT has attracted a great deal of interest in monitor the released NO. First, we have checked the as-prepared
cancer therapy.27–36 Porphyrins and their derivatives, as typical HA-TPP@NORM nanoparticles, and subsequently evaluated for
photosensitizers (PSs), have been widely used during PDT the capability of generating NO, 1O2 and ONOOꢀ. Finally, the MTT
processes due to their high molar absorption coefficients, high assay was utilized to evaluate the dark- and photo-cytotoxicity of
singlet oxygen quantum yields and excellent biocompatibility.37–42 HA-TPP@NORM nanoparticles.
Based on the different photochemical reaction processes, PDT can
be divided into two types: type I PDT and type II PDT. In particular,
PSs (in the excited triplet state) convert triplet oxygen (3O2) into
singlet oxygen (1O2) via direct energy transfer (type II). Simulta-
neoꢀusly, the excited PS can also directly react with 3O2 to produce
Materials and methods
Materials
Benzaldehyde, propionic acid, pyrrole, trichloroacetic acid
(CCl3COOH), trifluoroacetic acid (CF3COOH), acetic acid
(CH3COOH), sodium nitrite (NaNO2), hydrochloric acid, tin(II)
dichloride dehydrate, ammonium hydroxide, 40,6-diamidino-
2-phenylindole (DAPI), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-
tetrazolium bromide (MTT), 1-(3-dimethylaminopropyl)-3-ethyl-
carbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS),
1,3-diphenylisobenzofuran (DPBF), 4-bromo-1,8-naphthalic
anhydride, n-butylamine, ethanol and sodium bicarbonate
were purchased from Aladdin and used as received. A nitric
oxide assay kit (Griess assay) was obtained from Beyotime
Institute of Biotechnology. Sodium hyaluronate (7500 Dalton)
was purchased from BLOOMAGE FREDA BIOPHARM CO., LTD.
All other reagents were of analytical grade and used directly.
O2 (type I).26
ꢁ
Herein, the intelligent photo-responsive HA-TPP@NORM
nanoparticles based on the co-assembly of tetraphenylporphyrin
(TPP) modified hyaluronic acid (HA) and NO donor (NORM) have
been successfully constructed for generating ONOOꢀ via syner-
gistic interactions between PDT and NO therapy in order to
elevate the anticancer efficiency (Scheme 1). With a 650 nm light
irradiation, the excited TPP can react with surrounding 3O2 to
produce 1O2 and O2 ꢀ. Simultaneously, the released NO from
ꢁ
NORM irradiated with 365 nm light can immediately react with
ꢀ
ꢁ
the produced O2
to generate the highly biocidal molecule
ONOOꢀ. It is noteworthy to mention that a lot of NO donors,43
such as bis-N-nitroso compounds (BNNs),44,45 Roussin’s black salt
(RBS),46,47 S-nitrosothiols,48,49 and N-diazeniumdiolates,50,51 have
been widely used for curing cancer. In this study, N-nitrosated
napthalimide (NORM)52 was used as an NO donor. Under UV light
Characterisation
An AV 400 Spectrophotometer (400 MHz) was used to record the
1H NMR spectra. Tetramethylsilane (TMS) was used as an
internal reference, and CDCl3 or DMSO-d6 were used as the
solvent. A UV-2450 UV-visible spectrophotometer and F-4500
fluorescence spectrophotometer were used to obtain the UV-vis
spectra and fluorescence spectra, respectively. Dynamic light
scattering (DLS) measurements were performed using a BECKMAN
COULTER Delasa Nano C particle analyzer at room temperature.
A transmission electron microscopy (TEM) image was recorded on
a JEOL JEM1400 electron microscope at an accelerating voltage of
100 kV. Confocal laser scanning microscopy (CLSM) was performed
on a Nikon A1R, and flow cytometry was performed using a BD
FACS Calibur flow cytometer. The MTT assay was conducted using
a spectrophotometric microplate reader (Thermo Multiskan MK3
spectrometer) at a wavelength of 560 nm.
Synthesis of TPP
TPP was synthesized according to the previous literature.53
Benzaldehyde (8.0 g, 75 mmol) and propionic acid (220 mL)
were added into a 500 mL three-necked round-bottom flask
fitted with a constant pressure drop funnel and a reflux con-
denser. Under nitrogen protection, the mixture was heated to
140 1C, then pyrrole (5.2 mL) was added dropwise into the
mixture. After reacting for 4 h, the reaction mixture was slowly
cooled to room temperature. Propionic acid was removed with
reduced pressure distillation. 400 mL methanol was subse-
quently added into the mixture, and it was placed in ꢀ20 1C
for 24 h. The resulting precipitate was obtained by filtration
Scheme 1 Schematic of peroxynitrite (ONOOꢀ) generation from
HA-TPP@NORM nanoparticles. (a) The structure and preparation of
HA-TPP@NORM nanoparticles. (b) Illustration of the mechanism for intra-
cellular generating ONOOꢀ from HA-TPP@NORM nanoparticles.
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and washed with cold methanol several times. Finally, the Synthesis of M3
M3 was synthesized also according to same previous literature.52
M2 (10 g, 30.29 mmol), n-butylamine (2.66 g, 36.35 mmol) and
NaHCO3 (3.05 g, 36.35 mmol) were added into 300 mL of DMSO.
The mixture was stirred and heated to 110 1C for 12 h and then
cooled to room temperature. Then, DMSO was evaporated under a
reduced pressure. The resulting residue was transferred into
300 mL of H2O, and then extracted by CH2Cl2 three times. The
organic layer was dried with Na2SO4, filtered and rotary-
evaporated to obtain the crude product, which was further
purified with a silica gel column with CH2Cl2 and PE as the
eluent to afford the M3 (7.96 g) in 81% yield.
crude product was further purified with column chromato-
graphy to obtain 2.1 g TPP with 18.4% yield.
Synthesis of TPP-NH2
TPP (2.0 g, 3.25 mmol), trichloroacetic acid (26.6 g, 163 mmol),
acetic acid (9.3 mL, 163 mmol) and trifluoroacetic acid (7.2 mL,
98 mmol) were added into a 250 mL round-bottom flask. After
the mixture was stirred at 0 1C for 30 min, NaNO2 (561.2 mg,
8.13 mmol) was added into the reaction mixture and stirred for
another 24 h. 100 mL of H2O was added into the reaction
mixture to quench the reaction, and then CH2Cl2 (3 ꢂ 100 mL)
was used to extract the crude product. The organic layer was
dried with Na2SO4, filtered and rotary-evaporated to obtain the
crude product of nitration (TPP-NO2).
Synthesis of NORM
M3 (1 g, 3.08 mmol) was dissolved in a co-solvent of AcOH,
HCl, CH2Cl2 (10 : 0.5 : 7, v/v) (40 mL) and cooled to 0 1C.
NaNO2 (478.17 mg, 4.62 mmol) was added into the mixture,
stirred for 30 min. Then, the ice bath was removed and the
reaction mixture was stirred at room temperature for another
2 h. A saturated NaHCO3 solution was added to the flask until
the solution pH was adjusted to neutral. The crude product
was extracted with CH2Cl2 three times. Then, the organic
layer was dried with Na2SO4, filtered and rotary-evaporated to
obtain the crude product. The crude product was further
purified using a silica gel column using the co-solvent of
CH2Cl2 and CH3OH as eluent to afford the NORM (674 mg,
62% yield).
The crude product was added to 200 mL concentrated
hydrochloric acid, and then tin(II) dichloride dehydrate (2.7 g,
12.13 mmol) was added into the mixture. After the mixture was
heated to 80 1C for 2 h, 100 mL of H2O was added into the
mixture to terminate the reaction and kept at room tempera-
ture for another 12 h. The green precipitate was obtained by
filtration and subsequently dissolved in 150 mL of CH2Cl2.
Afterwards, 100 mL of H2O was added into the solution, and
then ammonium hydroxide was used to neutralize the mixture
until the color changed to violet. The organic layer was collected
and dried with Na2SO4, subsequently filtered and rotary-
evaporated to obtain the crude product, which was further
purified with a silica gel column using CH2Cl2 and PE as the
eluents to afford 0.9 g TPP-NH2 in 43.6% yield.
Preparation of the HA-TPP@NORM nanoparticles
10 mg HA-TPP and 5 mg NORM were dissolved in 1 mL of
DMSO, and then the solution was dropwise added into 4 mL of
H2O at room temperature. The obtained solution was dialyzed
against deionized water using a dialysis membrane (MWCO =
3500) for 24 h in order to remove DMSO. In order to measure
the NORM content in the nanoparticles, the solution was
lyophilized and then dissolved in DMSO and irradiated with
UV light for 20 min to make the encapsulated NORM totally
decomposed to M3. The content of NORM was then determined
by UV-vis absorption spectrum at 445 nm. The NORM loading and
entrapment efficiency of the HA-TPP@NORM nanoparticles were
separately calculated using the following equations:
Synthesis of the TPP-modified hyaluronic acid (HA-TPP)
Sodium hyaluronate was transformed into hyaluronic acid
(HA), according to the procedure mentioned in the previous
work.54 Sodium hyaluronate (3.75 g, 0.5 mmol) was dialyzed
against 0.01 N HCl, followed by dialysis against deionized water
several times. After the solution was lyophilized, the acidic
form of HA (3.5 g, 0.47 mmol) was obtained. HA (1 g, 0.13 mmol)
and TPP-NH2 (500 mg, 0.79 mmol) were dissolved in the
co-solvent of water and DMF, then NHS (115 mg, 1 mmol)
was added into the reaction mixture. HCl was used to adjust
the pH of the solution to 4.5. After stirring for 30 min, EDC
(191.7 mg, 1 mmol) was added into the reaction mixture. The
reaction was set to proceed for 48 h, and then the reaction
mixture was dialyzed using a dialysis tube (MWCO = 3500)
using the co-solvent of DMF and deionized water, followed by
dialysis against deionized water to remove DMF. Finally, the
solution was lyophilized to get HA-TPP (1.2 g).
NORM loading (%) = NORM amount in the NPs/mass of NPs
ꢂ 100%
Entrapment efficiency (%) = NORM amount in NPs/amount
of NORM used ꢂ 100%
Synthesis of M2
M2 was synthesized according to the previous literature.52 M1
1
Detection of O2 with DPBF
(20 g, 72.18 mmol) and n-butylamine (6.34 g, 86.616 mmol) 1,3-Diphenylisobenzofuran (DPBF) was used to detect the gene-
were added into 500 mL of ethanol. Under reflux conditions, rated singlet oxygen (1O2) in an aqueous solution with 1% DMSO
the mixture was stirred for 8 h, and then filtered and washed by to improve the solubility of DPBF. The HA-TPP@NORM nano-
ethanol to afford a crude product. The crude product was particles were irradiated with a 650 nm laser with 1 W cmꢀ2, then
purified by recrystallization in the co-solvent of ethanol and the decay of DPBF at 420 nm was monitored every 5 s using a
water to obtain M2 (20.6 g) in 86% yield.
UV-vis spectrophotometer.
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Quantitative detection of the NO release from the
HA-TPP@NORM nanoparticles by the Griess assay
Detection of the intracellular NO release from HA-TPP@NORM
nanoparticles by self-calibration with CLSM and flow cytometry
A typical Griess assay was used to quantitatively detect the NO The behaviour of the NO release in HeLa cells was detected
release from the HA-TPP@NORM nanoparticles. The detection by self-calibration with CLSM. HeLa cells were seeded onto
principle of the method is that the released NO molecules, after glass-bottom Petri dishes with 1 ꢂ 105 cells per well and then
contact with water, could be converted into nitrate or nitrite cultured for 24 h. After the addition of HA-TPP@NORM nano-
then react with a Griess agent and finally converted into an azo particles and incubation for another 24 h, the cells were washed
dye, which could be quantitatively determined using a micro- with PBS 3 times to remove residual nanoparticles, and then
plate reader or via UV-vis absorption spectroscopy (l = 540 nm). stained with DAPI and fixed with 4% paraformaldehyde
Before testing, a standard curve was established by the NaNO2 solution for 30 min. Then, the cells were irradiated with UV
standard samples (0–100 mM) for the calculation of NO light for 0 s, 15 s or 45 s. Finally, confocal luminescent imaging
concentrations (Fig. S12, ESI†). Detailed detection steps are experiments were performed on a confocal laser scanning
described below. 250 mL of the sample solution was added into microscope (Nikon AIR). The excitation wavelength was
250 mL of Griess reagent I and shaken for 1 min. Then, 250 mL 488 nm, and the emission wavelength was 525 nm.
of Griess reagent II was added into the mixture solution and
The behaviour of the NO release in HeLa cells was also
shaken for another 2 min in dark. At last, 1.25 mL of water was detected via flow cytometry. HeLa cells were seeded into a
added into the mixture before the mixture was measured using 6-well plate at 2 ꢂ 105 per well and then cultured at 37 1C for
a UV-visible spectrophotometer.
24 h. After the addition of HA-TPP@NORM nanoparticles and
incubation for another 24 h, the cells were washed with PBS
3 times to remove residual nanoparticles. Then, the cells were
irradiated with UV light for 0 s, 15 s or 45 s, respectively.
Subsequently, the cells were harvested and washed with PBS
three times and re-suspended in PBS. Finally, the fluorescence
intensity was detected using a BD FACS Calibur flow cytometer.
Determination of ONOOꢀ
L-Tyrosine was used as a probe molecule in the presence of CO2
in a weak alkaline environment. Dityr was generated by the
oxidation of ONOOꢀ. In a typical experiment, 10 mL of an
aqueous solution containing PBS (0.10 M, pH = 8.2), NaHCO3
(0.015 M), and L-tyrosine (5.0 ꢂ 10ꢀ4 M) was prepared at room
temperature. Then, 1 mg of HA-TPP@NORM nanoparticles
was added into the solution and illuminated with 365 nm
(130 mW cmꢀ2) and 650 nm (1 W cmꢀ2) lights for 1 min.
Subsequently, a fluorescence spectrophotometer was used to
detect the ONOOꢀ at an excitation wavelength at 313 nm.
Intracellular detection of the generated ONOOꢀ
To detect intracellular ONOOꢀ, a Cell Meter Assay Kit (AAT
Bioquest) for peroxynitrite assay was used. The cells with
concentration of 5 ꢂ 103 cells per well were treated with a
medium containing 100 mL of HA-TPP or HA-TPP@NORM
nanoparticles for 24 h. Then, DAX-J2 PON Green working
solution (10%) was added into the cells and incubated for
another 30 min. After washing with PBS, the cells were irra-
diated with 365 nm, 650 nm or 365 nm and 650 nm light for
10 min and incubated for another 30 min. The absorbance of
each well was monitored at Ex/Em = 490/530 nm.
Cell culture
HeLa cells kindly provided by the Institute of Biochemistry and
Cell Biology, SIBS, Chinese Academy of Sciences were cultured
in Dulbecco’s modified Eagle medium (DMEM), containing
10% fetal bovine serum (FBS) and antibiotics (50 units per mL
streptomycin and 50 units per mL penicillin) under a humidified
atmosphere with 5% CO2 at 37 1C.
In vitro cytotoxicity assay
Dark toxicity. HeLa cells were seeded into a 24-well plate at
5000 per well and then cultured at 37 1C for 24 h. HA-TPP or
HA-TPP@NORM nanoparticles with different concentrations
Intracellular ROS detection
Intracellular ROS production was measured via 20,70-dichloro- dispersed in a culture medium were added into the wells and
dihydrofluorescein diacetate (DCFH-DA) via confocal laser scan- incubated for another 24 h. Subsequently, the cells were
ning microscopy (CLSM). After taken up by the cancer cells, washed using sterilized PBS three times, and the culture
DCFH-DA will be deacetylated by the intracellular esterases to medium was replaced with 200 mL of fresh DMEM included
form dichlorodihydrofluorescein (DCFH). DCFH can transform to 20 mL of MTT (5 mg mLꢀ1). The cells were incubated for another
fluorescent dichlorofluorescein (DCF) in the presence of ROS. 4 h. After that, 150 mL of DMSO was added into the wells with
HeLa cells were plated onto glass-bottom Petri dishes with gentle shaking to extract the formazan products. Then, the
1 ꢂ 105 cells per well. After 24 h of incubation, the cells were absorbance was measured at 560 nm using a microplate reader.
treated with the HA-TPP@NORM nanoparticles. After washing Cell viability (%) was calculated according to the equation: cell
with PBS to remove the residual nanomaterials, the cells were viability (%) = (ODtest ꢀ ODbackground)/(ODcontrol ꢀ ODbackground) ꢂ
incubated with DCFH-DA (10 mM) for 30 min. Then, the cells were 100, where ODtest is the absorbance in the presence of sample
thoroughly washed with PBS then irradiated with 650 nm laser solutions, and ODcontrol is the absorbance of the control group.
(1 W cmꢀ2, 10 min) or not. Finally, the fluorescence of DCF was
observed by CLSM for each group.
Photo-toxicity. HeLa cells were seeded into a 24-well plate at
5000 per well and then cultured at 37 1C for 24 h. HA-TPP or
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HA-TPP@NORM nanoparticles with different concentrations absorption peak at 450 nm has increased. Accompanying
dispersed in a culture medium were added into the wells and the NO releasing processes, the fluorescence at 555 nm was
incubated for another 24 h. Subsequently, the cells were recovered steadily (Fig. S8, ESI†). This behaviour has also been
washed with sterilized PBS 3 times to remove residual nano- found in previous literatures,52–55 which can be used to con-
particles, and fresh DMEM was added into the plate. Then, the veniently detect intracellular NO release.
cells were irradiated using a 365 nm light (130 mW cmꢀ2) or
Finally, the HA-TPP@NORM nanoparticles were prepared by
650 nm light (1 W cmꢀ2) or 365 nm plus 650 nm light for the co-assembly of HA-TPP and NORM in an aqueous solution
10 min, and put back into an incubator for another 24 h. via a nanoprecipitation method. TEM and dynamic light
Finally, cell viability was evaluated using the MTT assay.
scattering (DLS) were utilized to characterize the obtained
HA-TPP@NORM nanoparticles. According to the TEM result
(Fig. 1b), the diameter of the nanoparticles is about 95.3 nm.
As shown in Fig. 1c, the diameter detected by DLS is about
108.6 nm, and polydispersity index (PDI) is 0.187. The size
calculated by DLS is bigger than that determined by TEM,
which may be due to the extending state of a hydrophilic segment
in an aqueous solution. In addition, the HA-TPP@NORM nano-
particles have favourable stability because the diameter of the
nanoparticles remained almost unchanged (when stored at room
temperature in the dark for two weeks), which is beneficial for
biomedical applications (Fig. S9, ESI†).
Statistical analysis
All experiments were performed at least three times under the
same experimental conditions. The results are expressed as
means ꢃ standard errors. A statistical analysis was performed
by one way (ANOVA), followed by the Bonferroni test using the
SPSS 19.0 software, the p-value o 0.05 was considered to be
statistically significant in all cases.
Results and discussion
The UV-vis absorption spectra (Fig. 1d) indicated that
NORM was successfully loaded into HA-TPP, according to the
simultaneous appearance of the characteristic absorption
peaks of TPP (about 420 nm) and NORM (about 360 nm). After
the HA-TPP@NORM nanoparticles were irradiated with UV
light, the absorption peak at 360 nm decreased but the absorp-
tion peak at 485 nm increased. This phenomenon is similar
to that of NORM under an UV light irradiation (Fig. S7, ESI†).
The encapsulation and loading efficiencies of NORM were
determined to be 50.2% and 26.7%, respectively, which were
quantified by the standard curve of the UV-vis absorption
spectrometry of M3, which is the degradation product of NORM
(Fig. S10, ESI†).
1,3-Diphenylisobenzofuran (DPBF) was widely used to deter-
mine the produced 1O2. The structure of DPBF can be destroyed
by 1O2, resulting in a decrease in the absorption peak at
420 nm. As shown in Fig. 2a, the absorption of DPBF sharply
reduced after the HA-TPP@NORM nanoparticles were irradiated
with a 650 nm light (1 W cmꢀ2) to excite TPP for only several
seconds. This result indicated that TPP in the nanoparticles can
efficiently produce 1O2 during the PDT process.
HA-TPP was synthesized via amidation reaction between
TPP-NH2 and hyaluronic acid (HA), catalysed with EDC and
NHS. A detailed synthetic route has been shown in Scheme S1
(ESI†). According to the 1H NMR spectrum (Fig. 1a), the TPP
was successfully grafted onto HA due to the appearance of
hydrogen proton signals (d = 9–7 ppm and ꢀ3 ppm), which all
belong to TPP-NH2. The successful conjugation of TPP was
further confirmed via Fourier transform infrared spectroscopy
(FTIR) (Fig. S3, ESI†). NORM, as a self-calibrating NO donor was
selected, and the detailed synthetic route has been shown in
supporting information (Scheme S2, ESI†). Under UV light
irradiation, the characteristic absorption peak at 345 nm has
disappeared rapidly (Fig. S7, ESI†), while the characteristic
The behaviour of the NO release was quantitatively detected
using a typical Griess assay. As shown in Fig. 2b, a large amount
of NO was released (440 mM) from the HA-TPP-NORM nano-
particles under the excitation of UV light within 100 seconds,
which is beneficial for cancer therapy due to only high con-
centrations of NO with tumoricidal effect. Moreover, the ability
of the controllable NO release from the HA-TPP@NORM nano-
particles was also studied. Under UV light irradiation, a NO
burst was released, but the enhancement in the NO level
immediately stopped after turning off the UV light (Fig. 2c).
It can be concluded that NO can be quickly and controllably
released from the HA-TPP@NORM nanoparticles. With the NO
release, the fluorescence signal at 558 nm can also be recovered
(Fig. 2d), exhibiting a similar change in small NORM molecules
(Fig. S8, ESI†). This ability of self-calibration for the NO release
Fig. 1 Preparation and characterization of the HA-TPP@NORM nanoparticles.
(a) 1H NMR spectrum of HA-TPP. (b) TEM image of HA-TPP@NORM nano-
particles. (c) DLS of HA-TPP@NORM nanoparticles. (d) UV-vis absorption
spectra of HA-TPP@NORM nanoparticles in aqueous solution.
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signal appeared in this system, meaning that still no ONOOꢀ
was produced. Without synergistic interaction, the groups
(HA-TPP@NORM (365 nm), HA-TPP@NORM (650 nm), L-try +
HA-TPP@NORM (365 nm) and L-try
+ HA-TPP@NORM
(650 nm)) also could not produce ONOOꢀ. It was thrilling to
find that the fluorescence intensity at 400 nm of Dityr was very
strong after HA-TPP@NORM nanoparticles and L-try were
mixed together and then irradiated with 365 and 650 nm light.
Accordingꢀto these results, it can be concluded that the gene-
ꢁ
rated O2
from type I PDT can efficiently react with the
released NO to produce ONOOꢀ.
The cellular uptake of the HA-TPP@NORM nanoparticles
is shown in Fig. S11 (ESI†). Without HA-TPP@NORM nano-
particles, no red fluorescence of TPP has appeared in the HeLa
cells. However, incubated with the HA-TPP@NORM nano-
particles for 8 h, red fluorescence appeared. By extending
incubation time to 24 h, the red fluorescence was significantly
enhanced. These results indicated that the HeLa cells can
uptake the HA-TPP@NORM nanoparticles in a time-dependent
manner.
Fig. 2 The abilities of the HA-TPP@NORM nanoparticles for generating of
1O2 and NO. (a) Singlet oxygen (1O2) generation as determined by the
decay of DPBF. (b) NO release from HA-TPP@NORM nanoparticles under
irradiation with 365 nm light. (c) On demand NO release from
HA-TPP@NORM under UV light irradiation. (d) The fluorescence spectra
of HA-TPP@NORM nanoparticles excited with 445 nm under irradiation
with 365 nm light.
Intracellular 1O2 production was evaluated using 20,70-dichloro-
dihydrofluorescein diacetate (DCFH-DA), which exhibited green
1
fluorescence after having reacted with O2. As shown in Fig. S12
(ESI†), the HeLa cells incubated with the HA-TPP@NORM nano-
particles and irradiated with 650 nm light exhibited strong green
fluorescence, but negligible green fluorescence was found in the
HeLa cells without 650 nm laser irradiation, indicating that a large
amount of O2 was generated and an efficient PDT process was
conducted in cancer cells.
The intracellular NO release was investigated via CLSM
and flow cytometry. After HA-TPP@NORM nanoparticles were
incubated with HeLa cells for 24 h, these cells were irradiated
with 365 nm light for different durations. As depicted in Fig. 4a,
green fluorescence was not present in the group without
irradiation. After the group was irradiated for 15 s, green
fluorescence immediately appeared. However, by extending
illumination time to 45 s, enhanced green fluorescence was
found. According to these results, it can be concluded that the
intracellular controllable NO release could be realized by
manipulating irradiation durations. The data from the flow
cytometry (Fig. S13, ESI†) also exhibited a similar outcome
for the group irradiated for 45 s exhibited the strongest
fluorescence.
is convenient and advantageous for investigating the intra-
cellular NO release; thus, external NO detection reagents are
not needed during the intracellular detection process.
Peroxynitrite (ONOOꢀ) can be detected by L-tyrosine (L-try) in
the presence of CO2 in a weak alkaline environment.56 After
being oxidized by ONOOꢀ, the dimerization of L-tyrosine (Dityr)
will be generated. The fluorescence spectrophotometer was
used to detect Dityr. The excitation wavelength (lex) is located
at 313 nm, and the corresponding emission spectrum is around
400 nm. As revealed in Fig. 3, without light irradiation,
no fluorescence signal was detected in the following groups,
such as L-try, L-try + HA-TPP, HA-TPP@NORM and L-try +
HA-TPP@NORM, which mean that none ONOOꢀ was generated
in these groups. After HA-TPP was added into L-try and then
irradiated with 365 nm and 650 nm lights, still no fluorescence
1
As a highly selective reagent, DAX-J2 PON Green57 was utilized
to determine intracellular ONOOꢀ. As shown in Fig. 4b, the HeLa
cells treated with HA-TPP or HA-TPP@NORM nanoparticles and
irradiated with 650 nm laser (1 W cmꢀ2) slightly enhanced the
level of ONOOꢀ, which may have resulted from the reaction
ꢀ
ꢁ
between O2
from type I PDT and endogenous intracellular
NO. The cells treated with HA-TPP@NORM but irradiated only
with the 365 nm laser also slightly increased the ONOOꢀ level,
which may be ascribed to the generated NO from the nano-
particles reacted with endogenous intracellular O2 ꢀ. It should
ꢁ
be noted that, compared with treatments of HA-TPP (650 nm),
HA-TPP@NORM (650 nm) and HA-TPP@NORM (365 nm), the
intracellular ONOOꢀ level has significantly increased in the HeLa
Fig. 3 Detection of the produced ONOOꢀ with L-tyrosine by a fluorescence
spectrophotometer (lex = 313 nm).
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Fig. 5 (a) Dark- and (b) photo-toxicity of HA-TPP@NORM nanoparticles
against HeLa cells.
Fig. 4 (a) Intracellular NO release from the HA-TPP@NORM nano-
particles detected with CLSM. The blue signal is assigned to the nucleus
of HeLa cells stained with DAPI; the green signal is assigned to NO. Scale
bar: 50 mm. (b) Intracellular ONOOꢀ quantitative detection with DAX-J2
PON Green reagent.
from the nanoparticles, indicating that NO therapy alone has a
limited anticancer effect. In addition, the PDT treatment of the
HA-TPP (650 nm) group also showed weak cytotoxicity with cell
viability exceeded 65%, which may be ascribed to the low
therapeutic TPP concentration (2 mg mLꢀ1), resulting in limited
ROS production in the HeLa cells. It was surprising to find that
the HeLa cells incubated with HA-TPP@NORM nanoparticles
and irradiated with 365 and 650 nm light exhibited the lowest
cell viability compared with the other two groups, suggesting
that the synergistic interaction of PDT and NO therapy can
obviously elevate the anticancer efficiency by generating the
highly biocidal ONOOꢀ molecules.
cells treated with HA-TPP@NORM nanoparticles and irradiated
with the 365 nm and 650 nm lights, which further verified that the
synergistic interactions of PDT and NO can generate ONOOꢀ in
the cancer cells.
Encouraged by the controllable NO release and an efficient
1
generation of O2 and ONOOꢀ, the cancer therapeutic effects
of the HA-TPP@NORM nanoparticles were subsequently
evaluated by the virtue of the 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyl-tetrazolium bromide (MTT) assay. As shown in Fig. 5a,
no obvious dark toxicity against cancer cells was observed in
HA-TPP and HA-TPP@NORM groups because the cell viability
Conclusions
was still high above 95% with the highest TPP concentration In conclusion, we have successfully constructed multifunctional
(20 mg mLꢀ1). It can be concluded that HA-TPP and HA-TPP@ HA-TPP@NORM nanoparticles, which can efficiently produce
NORM have favourable biocompatibility. The photo-toxicity of 1O2 and NO. In addition, a highly biocidal molecule of ONOOꢀ
the HA-TPP@NORM nanoparticles was evaluated. As depicted can also be generated in cancer cells by synergistic interaction
in Fig. 5b, the cells treated with HA-TPP@NORM nanoparticles of the PDT and the NO therapy. According to MTT results,
and irradiated only with the 365 nm light exhibited slightly without light irradiation, the HA-TPP@NORM nanoparticles
cytotoxicity, probably due to a small number of NO released have favourable biocompatibility. However, the nanoparticles
168 | New J. Chem., 2020, 44, 162--170
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NJC
17 D. E. Kim, C. W. Kim, H. J. Lee, K. H. Min, K. H. Kwack,
H. W. Lee, J. Bang, K. Chang and S. C. Lee, ACS Appl. Mater.
Interfaces, 2018, 10, 26870–26881.
18 C. Hu, X. L. Cun, S. B. Ruan, R. Liu, W. Xiao, X. T. Yang,
Y. Y. Yang, C. Y. Yang and H. L. Gao, Biomaterials, 2018, 168,
64–75.
exhibited a remarkable effect of killing HeLa cells compared
with individual PDT or NO therapy when simultaneously irra-
diated with 365 and 650 nm light. As a result, the proposed
ONOOꢀ molecule generating the HA-TPP@NORM nano-
particles may have profound therapeutic implications for cancer
therapies, and it may provide new insights with regard to NO
based therapy.
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Conflicts of interest
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