Two-photon excited organic nanoparticles for chemo-photodynamic therapy

Article abstract of DOI:10.1016/j.dyepig.2019.04.038

Drug molecules assembling nanomedicines possess several advantages, including precise molecular structure, various combinations of therapeutic agents and high content of drugs. In this work, paclitaxel dimer and two-photo photosensitizer were devised and synthesized, which could coassemble into nanoparticles (Co-NPs) in aqueous medium through nanoprecipitation method. As-synthesized Co-NPs possess the uniform size of about 80 nm and great stability in physiological condition, and could produce the singlet oxygen upon near-infrared light irradiation. The Co-NPs indicate enhanced cellular uptake and endosomal escape upon irradiation, which result in the synergistic enhancement of cytotoxicity towards cancer cells and growth inhibition of human cervical cancer tumors. We believed this combination therapy based on organic nanoparticles represent a new and important development in the cancer therapy.

Full text of DOI:10.1016/j.dyepig.2019.04.038

Accepted Manuscript
Two-photon excited organic nanoparticles for chemo-photodynamic therapy
Tao Zhang, Li Fu, Xiaohua Zheng, Ming Liu, Qing Pei, Xue Wang, Shi Liu
PII:
S0143-7208(19)30240-2
https://doi.org/10.1016/j.dyepig.2019.04.038
DYPI 7493
DOI:
Reference:
To appear in: Dyes and Pigments
Received Date: 29 January 2019
Revised Date: 9 April 2019
Accepted Date: 15 April 2019
Please cite this article as: Zhang T, Fu L, Zheng X, Liu M, Pei Q, Wang X, Liu S, Two-photon excited
organic nanoparticles for chemo-photodynamic therapy, Dyes and Pigments (2019), doi: https://
doi.org/10.1016/j.dyepig.2019.04.038.
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ACCEPTED MANUSCRIPT
Two-Photon
Excited
Organic
Nanoparticles
for
Chemo-Photodynamic Therapy
Tao Zhanga, Li Fuc, Xiaohua Zhengb, Ming Liu b, Qing Pei b, Xue Wanga* Shi Liu b*
,
a
China-Japan Union Hospital of Jilin University, 126 Xiantai Street, Changchun, Jilin 130033, P. R.
China. E-mail: wangxue2263@163.com
b
c
State Key Laboratory of Polymer Physics and Chemistry, Changchun Institute of Applied
Chemistry, Chinese Academy of Sciences, Changchun 130022 (China). E-mail: liushi@ciac.ac.cn
Department of Implantology, Stomatological Hospital, Jilin University, Changchun 130021,
P.R.China
Keywords: paclitaxel; two-photon; photodynamic therapy; chemotherapy.
Abstract
Drug molecules assembling nanomedicines possess several advantages, including
precise molecular structure, various combinations of theraputic agents and high
content of drugs. In this work, paclitaxel dimer and two-photo photosensitizer were
devised and synthesized, which could coassemble into nanoparticles (Co-NPs) in
aqueous medium through nanopreicipitation method. As-synthesized Co-NPs possess
the uniform size of about 80 nm and great stability in physiological condition, and
could produce the singlet oxygen upon near-infrared light irradiation. The Co-NPs
indicate enhanced cellular uptake and endosomal escape upon irradiation, which
result in the synergistic enhancement of cytotoxicity towards cancer cells and growth
inhibition of human cervical cancer tumors. We believed this combination therapy
based on organic nanoparticles represent a new and important development in the
cancer therapy.
Introduction
It is highly desired to develop efficient cancer therapies due to the complicated and
heterogeneous solid tumor.[1, 2] Traditional single tumor treatments, including
chemotherapy, radiotherapy and phototherapy, usually are hard to completely inhibit
the tumor growth and recurrence.[3, 4] Combination of different therapy in one
system is valuable and versatile for enhancing the therapeutic index and
response.[5-15] Merging two and more therapies could be realized by encapsulating
or conjugating therapeutic agents with polymer or other nanoparticles.[16, 17]
Although great advance has been made in nanoformulation of antitumor drugs, the
drug loading and loading efficiency is suboptimal. A large amount of drug carrier was

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used for stabilizing the formulations, which result in the potential long term toxicity
and the difficulties in enlarged production. Design of optimized drug carriers is
imperative to improve the therapeutic outcomes and minimize undesirable side
effects.[18]
Recently, the self-assembly of drug molecules provide a straightforward way for
preparing nanoformulations of drugs.[19] Except of amphiphilic drug
molecules,[20-22] some hydrophobic molecules could also assemble into nanoscale
aggregates in aqueous medium through different mechanism, including
disulfide-induced assembly,[23, 24] symmetrical dimer assembly[25-27] and
conjugated molecules assembly. In our previous work, a number of paclitaxel (PTX)
dimers was synthesized and could form the nanomedicines in the absence of
surfactants or adjuvants.[28-30] Although the PTX dimers formed nanomedicines
possess effective cellular uptake and cytotoxicity, and significant antitumor efficacy,
the suboptimal tumor treatment could be improved through the combination therapy.
Chemotherapy drugs and photosensitizers were widely combined for overcoming
drug resistance and enhancing the therapeutic response.[31, 32] As well as we know,
the combination chemo and phototherapy in one organic nanoparticle isn’t reported
yet. We hypothesized the assembling PTX dimers and photosensitizers in one
formulation could produce synergistic effect for tumor treatment. In order to increase
the treatment depth, the photosensitizer possessed big absorption wavelength is
indispensible for efficient antitumor efficacy.[29, 33-36] Herein, the two-photo
excited photosensitizer (2PE-PS) is employed for the study in vitro and in vivo.[37-40]
In this work, the PTX dimer with disulfide bond as linker (PTX-s-s-PTX) and 2PE-PS
was used to construct a nanoprodrug in the absence of surfactants (Fig 1A), and their
morphologies, photophysical properties, cellular uptake and cytotoxicity, and tumor
inhibition was investigated in detail (Fig 1B).

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Fig 1. (A) Formation of Co-NPs. (B) Schematic of the synergistic chemotherapy and
PDT. (1) Enhanced cell uptake by a short light irradiation for PCI effect. (2)
Enhanced escape of Co-NPs from lysosome to the cytosol based on photochemical
rupture of lysosome membranes. (3) Anticancer efficacy from photodynamic therapy.
(4) Conversion of the PTX-SS prodrug into free PTX by GSH trigger. (5) Free PTX
diffused to the cytosol for chemotherapy and induced cell death.
Experimental sections
Materials
1, 3-dicyclohexylcarbodiimide (DCC), 4-dimethylaminopyridine (DMAP) and
paclitaxel were purchased from Sigma-Aldrich (St. Louis, MO, U.S.A.).
3,3-dithiodipropionic acid，sodium hydroxide and cyclopentanone were purchased
from Shanghai Sun Chemical Technology Co., Ltd.. MTT and Lyso-Tracker Red
were purchased from Beyotime Biotechnology Co., Ltd. (China). Live-Dead Cell
Staining Kit was purchased from KeyGEN BioTECH Co., Ltd.. All of the other
chemical substances were prepared commercially and were used without further
purification, unless otherwise noted. All the solvents were purified according to the
standard methods whenever needed.
Instruments

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1H NMR spectra were tested on a Bruker NMR-400 DRX spectrometer at room
temperature. The mass spectra (MS) of all the samples were recorded by the German
company Bruker autoflex III smartbeam MALDI-TOF/TOF mass spectrometer with a
smart beam laser with a wavelength at 355 nm. UV-vis absorption spectra were
recorded with a Shimadzu UV-2450 PC UV/Vis spectrophotometer. The fluorescence
intensity tests were obtained using PerkinElmer LS-55 Spectrofluorophotometer. Zeta
potential and size distribution of the nanoparticles were characterized by Malvern
Zeta-sizer Nano for dynamic light scattering (DLS). The morphology of all the
nanoparticles was measured by transmission electron microscopy (TEM),
characterized by a JEOL JEM-1011 electron microscope operating at an acceleration
voltage of 100 kV. Confocal laser scanning microscopy (CLSM) images were taken
by using a Zeiss LSM 700 (Zurich, Switzerland).
Synthesis of PTX-s-s-PTX
PTX-s-s-PTX was synthesized by the method described in the previous literature.
Synthesis of 2, 5-Bis(4-(diethylamino)benzylidene) cyclopentanone (2PE-PS)
2,5-Bis(4-(diethylamino)benzylidene)cyclopentanone was synthesized according
to the previous literature. Briefly, 0.2 g of sodium hydroxide (5 mmol) was added to a
stirred solution of 1.68
g
of cyclopentanone (20 mmol) and
4-diethylaminobenzaldehyde (7.08 g, 40 mmol) in 50 mL of ethanol, and the reaction
mixture was refluxed for 12 h. After cooling to room temperature, the precipitate was
filtered out and washed with water. The crude product was recrystallized using
ethanol to give 2PE-PS (93%). 1H NMR (400 MHz CDCl3): d (ppm) 1.20 (t, 12 H, J =
7.08 Hz), 3.06 (s, 4 H), 3.42 (q, 8 H, J = 7.08 Hz), 6.69 (d, 4 H, J = 8.92 Hz),
7.50–7.52 (m, 6 H).30 MALDI-TOF-MS: m/z Calcd for C27H35N2O [M + H]+
403.27439; found 403.3.
Nanoparticles preparation
A solution of PTX-SS (1 mg) and 2PE-PS (1 mg) in 4 mL tetrahydrofuran was
added dropwise into the 10 mL of deionized water under a constant stirring, and NPs
were formed after evaporation of 4 mL tetrahydrofuran and dialysis in water for 2
days. Different molar ratios of PTX-SS and 2PE-PS could form the corresponding
PTX/2PE-PS0, PTX/2PE-PS2, PTX/2PE-PS5 and PTX/2PE-PS10 NPs by the same
precipitation method.
Singlet oxygen detection, photostability and ROS stability of Co-NPs
To evaluate the reactive oxygen species generation ability of 2PE-PS, a chemical
method by using DPBF was employed with UV-vis spectroscopy.[41] 2PE-PS
dissolved in 3 mL of DMF were blended with DPBF (15 µg/mL) and then irradiated

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with an 808 nm laser at an intensity of 0.1 W/cm2. The absorption intensity of the
DPBF at the maximum wavelength of 414 nm was detected every 1 min. DPBF
solution alone was irradiated as control. As for the photostability, the Co-NPs
containing 2PE-PS was irradiated by an 808 nm laser resource at an intensity of 1
W/cm2 for 30 min. The absorption spectrum of 2PE-PS was recorded every 5 min. In
order to detect the ROS stability of PTX-SS, 1 mg PTX-SS and 1 mg 2PE-PS was
1
dissolved in DMSO, the H NMR spectrum was detected before and after irradiation
with the 808 nm laser for 20 min.
Intracellular ROS detection
Confocal laser scanning microscopy (CLSM) observation method was employed
to detect the ROS generation in cells. Firstly, HeLa cells were incubated with Co-NPs
for 4 h, and then, the culture medium was washed four times. Then, the DMEM
solution containing DCFH-DA (10 × 10−6 M) was added to the six well plates and
further incubated for 35 min. An 808 nm light irradiation was used subsequently (1
W/cm2 for 5 min). The outcomes were observed with CLSM as soon as possible
(excitation wavelength, 488 nm; emission band-pass, 500−550 nm)
Intracellular uptake studies, lysosome colocalization and lysosome permeability
measurements by AO staining
To verify the cellular endocytosis, HeLa cells were seeded in 6-well plates at
about 200,000 cells per well in 2 mL culture medium (Dulbecco Modified Eagle
Medium containing 10% fetal bovine serum), supplemented with 100 U/mL penicillin
and 100 U/mL streptomycin, and incubated at 37 ℃ in 5 % CO2 atmosphere for 20 h.
Then the cells were treated with 2PE-PS and incubated at 37 ℃ for another 0.5, 2, 4,
6 h. The different well plates were washed three times with PBS and tested with
CLSM method and quantified by flow cytometry analysis. The cells were incubated at
4 oC for 0.5, 2 h at the same condition to study the effect of temperature on the uptake
of Co-NPs by cells.
In order to track the Co-NPs in cells, lysosome colocalization was conducted.
Briefly, the lysosomal compartments of the cultured HeLa cells were stained with the
probe of Lyso-Tracker red DND-99. One group was irradiated by the 808 nm laser.
Thereafter, the cells were fixed with 4% formaldehyde for 10 min at room
temperature, and the cell nuclei were stained with 4, 6-diamidino-2-phenylindole
(DAPI, excited by 405 nm). CLSM images of cells were taken by using a Zeiss LSM
700 (Zurich, Switzerland). AO (Acridine Orange) staining method was used to
determine the changes in lysosomal membrane permeability. HeLa cells were plated
in a 6-well plate at a concentration of 300,000 cells per well. After incubation for 24 h,
the cells were incubated with Co-NPs for 4 h and irradiated 5 min with an 808 nm
laser, then AO containing PBS was added to each well (at a concentration of 2.5
µg/ml). After 30 min of incubation with AO, cells were washed three times with PBS
and examined with a fluorescence microscope (Nikon Eclipse TE2000-U). The

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excitation and emission wavelength of red fluorescence was 555 nm and 617 nm, and
that of green fluorescence was 490 nm and 528 nm. AO concentrated in lysosomes
emits a red fluorescence, whereas AO in the cytosol emits a green fluorescence. A
reduction in red fluorescence along with an increased intensity of green fluorescence
indicates a relocation of AO from the lysosomes to the cytosol, following by the
change of lysosome permeability.
In vitro cytotoxicities on HeLa and HepG2 cells
HeLa and HepG2 cells obtained at a logarithmic growth phase were seeded in
96-well plates at a density of 5 × 103 cells per well and incubated in culture medium
for 24 h. The media were then replaced by three different molar ratios of Co-NPs at
various concentrations. The incubation was continued for 6 h. Then, the three groups
were irradiated by a laser with a wavelength of 808 nm at 1 W/cm2 for 15 min. After
48 h, the above culture medium was replaced by PBS and then 20 µL of MTT
solution in PBS with the concentration of 5 mg/mL was added and the 96-well plates
were incubated for another 4 h at 37 °C, followed by removal of the PBS containing
MTT and addition of 150 µL of dimethyl sulfoxide (DMSO) to each well to dissolve
the formazan crystals formed. Finally, the plates were shaken for 3 min, and the
absorbance of formazan crystals dissolved was measured at 490 nm by a micro plate
reader.
Calcein-AM/PI test
To visually demonstrate the anticancer efficacy of Co-NPs in chemotherapy and
photodynamic therapy, the HeLa cells were stained with propidium iodide (PI) and
calcein-AM (AM) to distinguish dead (red) and live (green) cells, respectively. The
control and drug-treated cells were incubated in 96-well plates at 37 °C for 24 h in a
humidified atmosphere containing 5% CO2. Then, the cells were incubated with the
drugs for 6 h, and then 2PE-PS and Co-NPs dishes of cells were irradiated for 15 min
with an 808 nm laser at an intensity of 1 W/cm2. Then, the cells were further
o
incubated at 37 C for another 48 h. After 48 h, the cells were stained with
calcein-AM/PI for 35 min and washing with PBS, the samples of cells were imaged
with a fluorescence microscope.
In vivo studies
Male nude mice were obtained and maintained under appropriate conditions. All
the subsequent animal procedures have been approved and controlled by the local
ethics committee and carried out according to the guidelines of Chinese law
concerning the protection of animal life. To evaluate the antitumor activity,
subcutaneous human cervical carcinoma HeLa tumor xenografts were utilized as
animal modal. HeLa cells were subcutaneously inoculated into the rear backside of
mice (4×106 cells in 0.1 mL PBS). The tumor-bearing male mice were randomly

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divided into three groups (n=4): (1) blank control (PBS+Laser), (2) Co-NPs and (3)
Co-NPs+Laser groups. Before treatment, three groups of mice were marked, weighed
and measured, and then the tumor-bearing mice from Co-NPs and Co-NPs+Laser
groups were intratumorly injected with Co-NPs at the dose of 1.9 mg kg−1 (drug
weight/body weight), respectively. At designed time (every other day), body weight
and tumor volume of mice were measured. At day 20, all the mice were sacrificed,
and tumor was excised to intuitionally evaluate the tumor inhibition effect.
Results and discussion
Preparation and Characterization of Coassembly Nanoparticles (Co-NPs)
PTX-s-s-PTX conjugates (PTX-SS)26 and 2PE-PS32 were synthesized according to
a method described previously. Their chemical structures were well confirmed by 1H
and 13C NMR spectroscopy (Fig S1A-B and Fig S2A-B). The peaks at 1880 and 403.3
in the MALDI-TOF mass spectrum further validated the successful synthesis of the
targeting molecules (Fig S1C and Fig S2C). The elution time of PTX-SS at 6.72 min
obtained from high performance liquid chromatography (HPLC) method identified
the purity. (Fig S1D) PTX-SS and 2PE-PS can form the nanoparticles (PTX-SS and
2PE-PS NPs) individually, and coassemble into nanoparticles (Co-NPs) in aqueous
solution through a well-documented nano-precipitation method. Briefly, a solution of
PTX-SS and 2PE-PS in tetrahydrofuran was added dropwise into the deionized water
under regular stirring, and NPs were formed after evaporation of tetrahydrofuran and
dialysis against water. Different molar ratios of PTX-SS and 2PE-PS in feed could
form the corresponding PTX/2PE-PS0, PTX/2PE-PS2, PTX/2PE-PS5 and
PTX/2PE-PS10 NPs by the same precipitation method.
The morphology and size distribution of formed PTX-SS, 2PE-PS and Co-NPs NPs
were characterized by transmission electron microscopy (TEM) and dynamic light
scattering (DLS), respectively. As shown in Fig. 2A-C, the PTX-SS, 2PE-PS and
Co-NPs NPs all possess a spherical morphology with a size of about 80 nm, while the
corresponding DLS analysis shows that they have hydrodynamic diameters of 210 nm,
140 nm and 180 nm, respectively. The size, PDI and zeta potentials measured by DLS
were listed in Table S1. The size and size distribution of three as-obtained NPs almost
kept unchanged up to two weeks at room temperature, which indicated the resulting
NPs hold good stability in water and fetal bovine serum (FBS) (Fig S3). The zeta
potentials of PTX-SS, 2PE-PS, Co-NPs NPs are around -20, -17.3, -18.7 mV,
respectively (Fig 2D). This negative surface charge can not only stabilize the
as-prepared NPs by electrostatic repulsion but also reduce the serum protein
adsorption. All these favorable properties are beneficial for their future biomedical
applications.[42]

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Fig 2. (A-C) TEM images and size distribution (DLS) of PTX-SS NPs, 2PE-PS NPs,
Co-NPs. Scale bars: 200 nm. (D) Zeta potentials of PTX-SS NPs, 2PE-PS NPs,
Co-NPs. (E) UV-vis absorption of 2PE-PS NPs, PTX-SS NPs, Co-NPs in water and
2PE-PS in DMF. (F) Fluorescence spectra of Co-NPs in water and equal
concentration of 2PE-PS in DMF.
Photochemical Properties
We also recorded photophysical properties of 2PE-PS molecules in dimethyl
formamide (DMF) and the 2PE-PS, PTX-SS, Co-NPs NPs dispersed in water,
respectively. As shown in Fig 2E, the maximum absorption of 2PE-PS in DMF is 477
nm, which appeared red shifted to 484 nm when forming the aggregated nanoparticles.
The typical absorption of 2PE-PS could be observed in that of Co-NPs in aqueous
solution. The absorption band of PTX-SS is below 300 nm, imposing no obvious
effect on the absorption of Co-NPs. The 2PE-PS molecules in DMF display an
obvious orange florescence excited at 480 nm. By contrast, Co-NPs exhibit negligible
fluorescence due to aggregation-induced quenching (Fig 2F).
The photochemical ability of the 2PE-PS to generate reactive oxygen species upon
irradiation of 808 nm laser was studied by using 1,3-diphenylisobenzofuran (DPBF)
and 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) as the indicator,[43-45]
and the absorbance of DPBF was monitored by UV−vis absorption spectra. As a
control experiment, DPBF (15 µg/mL) was irradiated in DMF without 2PE-PS. As
shown in Fig 3B, only a negligible decrease in the absorbance of DPBF was found
upon light irradiation, which reveals that DPBF is stable upon 808 nm laser irradiation.
In sharp contrast, irradiation of a solution of DPBF (15 µg/mL) in DMF (3 mL) with
laser at an intensity of 0.1 W/cm2 in the presence of 2PE-PS (5 µg) causes a steady
generation of reactive oxygen species, as evidenced by the absorption peak of DPBF
at 414 nm decreasing upon oxidative degradation by 1O2 (Fig 3A). A time-dependent
plot of the DPBF absorbance at 414 nm (Fig 3B) reveals that the DPBF is consumed

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rapidly in the presence of 2PE-PS upon laser irradiation. As an important property of
photosensitizers, photostability of Co-NPs is investigated. As shown in Fig S4A, the
absorption of Co-NPs almost kept unchanged under irradiation of an 808 nm laser at 1
W/cm2 for 30 min, indicating the favorable optical stability. Above results identified
that the Co-NPs could efficiently generate ROS in vitro. Because the PTX-SS existed
in the Co-NPs, it was necessary to test the stability of PTX-SS molecule in the
presence of ROS. PTX-SS and 2PE-PS were dissolved in DMSO, and then irradiated
with an 808 nm laser at 1 W/cm2 for 20 min. As shown in Fig S4B, the NMR spectra
showed no changes before and after irradiation, validating that PTX-SS would not be
destroyed by ROS before it exerts the effect of subsequent anticancer therapy.
Fig 3. Singlet oxygen generation ability of as-prepared Co-NPs. (A) Time-dependent
UV absorption spectra of DPBF at 414 nm with 2PE-PS in DMF after irradiation with
an 808 nm laser from 0 to 12 min. (B) Comparison of the decay rate of DPBF with
and without irradiation, treated with 2PE-PS along with or without irradiation. (C)
The generation of intracellular ROS mediated by Co-NPs upon light irradiation of 0.1
W/cm2 for 5 min indicated by the fluorescence of DCF about the blank control with

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and without irradiation, pretreated with Co-NPs along with or without laser treatment.
Scale bars, 20 µm.
Besides, intracellular ROS generation by Co-NPs in cervical cancer cells (HeLa
cells) was further detected by using DCFH-DA as indicators under confocal laser
scanning microscopy (CLSM). As shown in Fig 3C, almost no green fluorescence is
observed in all control groups, because there is no adequate singlet oxygen in cells to
oxidize nonfluorescent DCFH-DA into fluorescent dichlorofluorescein (DCF). By
comparison, a bright green fluorescence appears in Co-NPs group under light
irradiation due to the formation of singlet oxygen. Hence, such Co-NPs can be
potentially applied as photosensitizers for photodynamic therapy.
Cell uptake properties
It is well known that effective cell internalization of drugs is vital to the efficacy
of both PDT and chemotherapy. The cellular endocytosis of Co-NPs in HeLa cells
was investigated by using CLSM and flow cytometry. The cell nuclei are stained with
the dye of DAPI. CLSM images (Fig S5) and flow cytometry (Fig 4A, A1) show an
enhanced intracellular distribution of Co-NPs in HeLa cells with the extension of
incubation time from 0.5 h to 6 h. The uptake of Co-NPs increased obviously when
the incubation temperature increased from 4 °C to 37 °C, indicating the
ATP-mediated endocytosis (Fig. S5).
Fig 4. (A) Flow analysis and (A1) mean fluorescence intensity of different time of
Co-NPs after uptaked by HeLa cells. (B–C) Flow cytometric analysis of HeLa cells
pretreated with Co-NPs and NPs (PTX-SS+Nile red) with (+L) or without (−L) 3 min
laser irradiation during internalization. Cells without any treatments were used as
control. (B1–C1) Mean fluorescence intensity of HeLa cells treated with the two NPs.
**p < 0.01, #p > 0.05.
Lysosome colocalization research was carried out by using the probe of
Lyso-tracker Red. HeLa cells were incubated with Co-NPs for 2 h and then stained

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o
with Lyso-tracker Red at 37 C for 30 min.[46-50] As shown in Fig 5, Co-NPs
nanoparticles mainly locate within the lysosome. Under the same condition, cells
were incubated for 2 h and then irradiated with an 808 nm laser for 5 min. Compared
to the non-irradiated control, the green fluorescence of Co-NPs and red fluorescence
of Lyso-tracker Red decreased remarkably, which indicated that the laser irradiation
facilitate the escape of Co-NPs from lysosome to cytoplasm (Fig 5).
Fig 5. (A-B) Confocal microscopy images of HeLa cells after incubation with Co-NPs
without (-L) with (+L) 5 min laser irradiation. (A1-B1) Quantitative analysis of the
fluorescence of Co-NPs and Lyso-tracker Red in cells from 2.5D image. For each
panel, the images from above to down show cell nuclei stained by DAPI (blue),
Co-NPs fluorescence in cells (green), lysosomes stained with Lyso-tracker Red (red)
and overlays of three images. Scale bars, 20 µm.
When light irradiation with just 3 min was employed at the initial time of cells
incubated with Co-NPs, enhanced cellular uptake was observed. Obviously, the
irradiated group emits a brighter fluorescence than the non-irradiated group (Fig S6).
The enhanced cell uptake was ascribed to the photochemical internalization (PCI)
effect.[51-53] PCI-induced internalization of the Co-NPs was furthered demonstrated
by flow cytometric analysis. In Fig 4B and 4B1, the group of Co-NPs in HeLa cells
with light irradiation displays the enhanced cellular internalization of the Co-NPs
compared with the control. In order to validate the PCI induced internalization result
from 2PE-PS, the flow cytometric analyses of NPs (PTX-SS + Nile Red) with or
without 3 min light irradiation were also carried out. The significant difference (**p <
0.01) in the cellular internalization after light illumination further demonstrate the
photochemical internalization process. The results (Fig 4C, C1, Fig S6B)
demonstrated that there was no noteworthy difference in the cellular endocytosis,
indicating that 2PE-PS play a vital role during the process of PCI-induced

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internalization. Moreover, the negligible significant difference result (#p > 0.05) also
corroborates that it is the photosensitizer-2PE-PS that induce the PCI process. In
addition, lysosomal permeability was investigated by (AO) staining.[54] When AO
concentrated in lysosomes, it emits a granular red fluorescence, whereas it would emit
a diffuse green fluorescence in cytosol. A reduction in red fluorescence along with an
increased green fluorescence in cytosol shows a relocation of AO from the lysosomes
to the cytosol, followed by the change of lysosome permeability because of the ROS
produced by 2PE-PS (Fig S7).
Fig 6. In vitro synergistic therapy efficacy of Co-NPs. (A) Relative cell viabilities of
HepG2 and HeLa cells incubated with different concentrations of 2PE-PS NPs. Cell
viabilities of HeLa cells (B) and HepG2 cells (C) incubated with different molar ratios
of Co-NPs upon an 808 nm laser irradiation at 1 W/cm2 for 15 min. (D) Fluorescence
microscope images of calcein AM (green, live cells) and Propidium Iodide (red, dead
cells) co-cultured HeLa cells pretreated with 2PE-PS NPs without or with irradiation,
Co-NPs in dark and Co-NPs upon irradiation for 15 min, Scale bars, 40 µm.
Cytotoxicity results
Standard thiazolyblue tetrazolium bromide (MTT) assay was used to evaluate the
cytotoxicity of Co-NPs against human hepatocellular carcinoma (HepG2) and HeLa
cells. 2PE-PS NPs exhibited a negligible cytotoxicity without irradiation at the
concentration of up to 5 µM after 48 h incubation, implying that 2PE-PS NPs have
favorable biocompatibility (Fig 6A). Although 2PE-PS shows better anticancer effect
under low power 450 nm light irradiation (one photon) contrast to 808 nm laser

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irradiation (two photon) (Fig S8), 808 nm light resource was employed because of the
depth of tissue penetration in subsequent experiments. Under irradiation with a laser
of 808 nm, a dose-dependent cytotoxicity of Co-NPs is observed towards both HepG2
and HeLa cells (Fig 6B-C). Co-NPs with three different molar ratios of PTX to
2PE-PS were designed to explore the synergetic effect of chemotherapy and
photodynamic therapy. Compared to Co-NPs in dark, the corresponding half-maximal
inhibitory concentration (IC50) against HepG2 and HeLa become lower and lower
along with increasing of proportion of 2PE-PS in Co-NPs. The corresponding IC50
values are listed in Table S2. The IC50 value of Co-NPs in dark against HeLa cells is
0.03 µM, while the values of Co-NPs after irradiation under an 808 nm laser are 0.011，
0.003，0.0027 µM for PTX/2PE-PS2, PTX/2PE-PS5 and PTX/2PE-PS10, respectively.
Moreover, the anticancer efficiency of Co-NPs was examined by the live/dead
staining (Fig 6D). Almost all of the HeLa cells were dead after being incubated with
the Co-NPs under irradiation, which was the synergistic therapeutic effect of
chemotherapy of PTX and photodynamic therapy of 2PE-PS. Obviously, the
combination therapy offers some advantages over single treatment.
Antitumor activity
To verify the in vivo anticancer efficacy, intratumor injection was employed to
deliver Co-NPs into the mice bearing human cervical cancer tumors at the doses of
1.9 mg/kg of PTX/2PE-PS5 with an 808 nm laser irradiation at 1.5 W/cm2 for 15 min,
followed by the tumor volume and body weight measurements. As shown in Fig 7A-F,
PBS injection and laser irradiation as a control displayed a sharp tumor growth. In the
absence of irradiation, Co-NPs had a relatively satisfied tumor inhibition effect as
compared to that of control group due to the favorable therapeutic effect of paclitaxel
(Fig 7B). Upon irradiation, tumors were almost completely clear away (Fig 7C). After
20 days of observation, the tumors were harvested from three groups. The weights
and sizes of tumors treated with Co-NPs were significantly smaller than for the
control group (Fig 7D, 7F). The obvious significant difference (***P < 0.001) in the
tumor volume and tumor weight between the control and drug-treated groups well
affirmed the tumor inhibition effect of as-prepared Co-NPs. Moreover, the body
weights of mice were not significant influenced by various treatments, suggesting that
Co-NPs treatment have no obvious system toxicity (Fig 7G).

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Fig 7. In vivo anticancer efficacy. Various groups treated with (A) PBS+Laser as
control, (B) Co-NPs, (C) Co-NPs+Laser. (D) Photo of excised tumor. From top to
down: PBS+Laser, Co-NPs and Co-NPs+Laser group. (E) Change of tumor volumes
of three groups. (F) Tumor weight of three groups. (G) Body weight of mice after
different treatments. Statistical significance: ***P < 0.001.
Conclusion
In conclusion, an organic nanoparticle was constructed to enhance anticancer
efficiency by combining chemotherapy and PDT. Due to the similar symmetric
structure, PTX dimers and two-photo dyes could assemble into spherical and stable
nanoscale Co-NPs. The Co-NPs indicate the enhanced cellular uptake and endosomal
escape upon irradiation due to the production of ROS, which result in the synergistic
enhancement of cytotoxicity towards cancer cells. These Co-NPs possess effective
ability of inhibiting the growth of human cervical cancer tumors upon NIR laser
irradiation. This work emphasizes the great potential of using molecular self-assembly
to develop state-of-art nanomedicines for cancer therapy.
Acknowledgements
This work was supported by the National Natural Science Foundation of China
(Project nos. 81602715) and Postdoctoral Science Foundation of China (Project nos.
2018T110256).
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Graphical abstract

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Research highlights
► The first study to apply nanoparticles coassembled by paclitaxel dimer and
two-photo photosensitizer.
► The nanoparticles indicates the enhanced cellular uptake and endosomal escape
upon irradiation due to the production of ROS
► The nanoparticles possesses synergistic enhancement of cytotoxicity towards
cancer cells and growth inhibition of human cervical cancer tumors