Sensitization of Hypoxic Tumor to Photodynamic Therapy via Oxygen Self-Supply of Fluorinated Photosensitizers

Article abstract of DOI:10.1021/acs.biomac.9b01368

Photodynamic therapy (PDT) utilizes photosensitizers to convert innoxious oxygen to cytotoxic reactive oxygen species under an appropriate light, thus inducing cancer cells necrosis. However, PDT performs in an oxygen-dependent method to destroy cells whi

Full text of DOI:10.1021/acs.biomac.9b01368

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Article
Sensitization of hypoxic tumor to photodynamic therapy
via oxygen self-supply of fluorinated photosensitizers
Zhiyong Liu, Yudong Xue, Mengsi Wu, Guoliang Yang, Minbo Lan, and Weian Zhang
Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.9b01368 • Publication Date (Web): 11 Nov 2019
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Sensitization of hypoxic tumor to photodynamic
therapy via oxygen self-supply of fluorinated
photosensitizers
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Zhiyong Liu, Yudong Xue, Mengsi Wu, Guoliang Yang, Minbo Lan, Weian Zhang*
Shanghai Key Laboratory of Functional Materials Chemistry, East China University of Science
and Technology.130 Meilong Road, Shanghai 200237, China
KEYWORDS: photodynamic therapy; fluorinated photosensitizers; hypoxic; cancer
ABSTRACT: Photodynamic therapy (PDT) utilizes photosensitizers to convert innoxious oxygen
to cytotoxic reactive oxygen species under an appropriate light thus induce cancer cells necrosis.
However, PDT performs in an oxygen-dependent method to destroy cells while hypoxia is a
feature for most solid tumors. To effectively improve the PDT effect against solid tumors, an
oxygen self-supplying and pH-sensitive therapeutic nanoparticle (PTFC) has been developed by
the self-assembly of a tetrakis(pentafluorophenyl) chlorin (TFPC)-conjugated block copolymer
(POEGMA-b-P(DEAEMA-co-GMA)). PTFC nanoparticles can transport oxygen to tumor site
with their accumulation in tumor on account of the good oxygen solubility, therefore relieving the
oxygen deficiency of solid tumor and enhancing the PDT efficacy. It's worth noting that the oxygen
loading was realized by fluorinated photosensitizer itself. In addition, the phototoxicity of PTFC
nanoparticles is greatly improved in an acidic aqueous environment due to the DEAEMA unit
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protonation, which not only enhanced the cellular uptake of nanoparticles but also weakened the
aggregation of photosensitizers. Taking the hypoxia and acidic microenvironment of solid tumors,
PTFC nanoparticles could be efficiently taken up and disassembled to release oxygen upon
accumulated at tumor sites, thus significantly improving the PDT efficacy against solid tumors.
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INTRODUCTION
Tumor hypoxia, which resulted from overwhelming oxygen consumption, contributes to the
growth and metastasis of tumors and impacts their responses to oxygen-consuming therapies, such
as photodynamic therapy (PDT), radiotherapy, sonodynamic therapy and chemotherapy (CT).1-2
Among them, PDT is one of most attractive therapeutic approaches, where oxygen is converted to
cytotoxic reactive oxygen species by photoexcited photosensitizers for eliminating tumor cells
under a light with a specific wavelength.3-4 For enhancing the therapeutic effect, a great number
of contributions have been recently dedicated to defeating the hypoxia of tumor,5 including in situ
oxygen production at tumor sites via catalytic reactions,6-12 oxygen delivery into tumors,13-19
oxygen-independent photosensitizers for PDT20-25 as well as hypoxia-reactivated prodrugs for
combination therapy of PDT and CT.26-29 More recently, perfluorocarbons (PFC)-based oxygen
delivery systems have attracted much attention for their inherent ability to solubilize oxygen.30
PFCs are highly hydrophobic and lowly reactive due to the relatively nonpolarity and inertness of
the carbon-fluorine bond, endowing them with good biocompatibility and oxygen-dissolving
capability.31 For example, Hu and her co-workers developed an oxygen self-enriching PDT system
to enhancing the PDT effect against hypoxia tumor by loading photosensitizers into PFC
nanodroplets.32 Shuai and his co-workers fabricated an ultrasound /fluorescent bimodal imaging-
guided and oxygen-potentiated PDT system by construction of perfluorohexane-cored
nanodroplets.16 Besides, we also reported an oxygen self-sufficient nanosystem through
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conjugating PFCs to cyanine 7 for improving combination therapy efficacy of PDT and CT in prior
study.33 Although the antitumor efficiency has been significantly enhanced, the use of PFCs may
complicate the preparation process. Hence, it could be an attractive approach to supply oxygen by
fluorinated photosensitizer itself.
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Another limitation of PDT is that the therapeutic effect is greatly dependent on the effective
concentration of photosensitizers. As one of the three elements of photodynamics, most
photosensitizers tend to aggregate in aqueous solution due to their poor water solubility, resulting
in low tumor selectivity and severely quenched photosensitivity.34 To overcome this limitation,
photosensitizer-based nano-organic frameworks (metal organic frameworks, MOF35-36 and
covalent organic frameworks, COF37-38), stimuli-responsive polymeric nanoparticles39-47 and
organic/inorganic hybrid nanoparticles48-50 have been developed to improve the therapeutic effect
of photosensitizers. In consideration of the specific tumor microenvironment (TME), such as low
pH,51-54 over-expressed glutathione,55-58 enzyme59-61 or reactive oxygen species,62-65 significant
efforts have been focused on construction of TME-associated stimuli-responsive systems for
biologically activatable PDT. Among them, the pH-responsive nanoparticles are of particular
interest as a result of the spatiotemporal diversity of pH values in different tissues (e.g. the
extracellular environment of tumor (pH 6.5-6.8) is more acidic than that of normal tissues (pH
7.4), and the pH values of intracellular endocytic vesicles are even lower than 6.0.).66-68 The cell
internalization could be effectively improved by activating surface positive charge of nanoparticles
at tumor acidic extracellular microenvironment.69-72 Moreover, photosensitizers can also be
selectively activated for enlarged imaging or treatment of tumors through incorporating them into
pH-responsive nanoparticles.73-75 Therefore, it would be of great significance to exploit smart
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photosensitizer delivery systems that combine oxygen transport and acid responsiveness for
hypoxic tumors.
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Herein, we developed an oxygen self-supplying system by the construction of a fluorinated
photosensitizer-based POEGMA-b-P(DEAEMA-co-TFPCMA) (PTFC) block copolymer for
overcoming the hypoxia of solid tumors, thereby enhancing the photodynamic effect (Scheme 1).
After amphiphilic PTFC block copolymers self-assembled into nanoparticles through hydrophilic
and hydrophobic interactions, the hydrophilic shell formed by POEGMA segments endowed
PTFC nanoparticles with good biocompatibility, which could partially reduce nonspecific cell
uptake or protein adsorption during the accumulation of nanoparticles to tumor sites. Due to the
high affinity of fluorine to oxygen, oxygen can be effectively adsorbed into the TFPC-containing
hydrophobic core and simultaneously delivered to tumor sites with the accumulation of PTFC
nanoparticles for relieving the hypoxic environment of solid tumors. Moreover, nanoparticles can
be more effectively taken up by cancer cells since DEAEMA are pH-responsive resulting in the
negative to positive surface charge transition under weak acidic tumor microenvironment. After
successful uptake by cells, the nanoparticles can be broken by the lower pH state inside the cells,
reducing the aggregation caused quenching (ACQ) effect of photosensitizers. Consequently, the
photodynamic effect could be significantly improved by the integration of oxygen transport
capacity and pH-responsiveness, where oxygen can be carried by aggregated photosensitizers and
then liberated at hypoxia tumor sites, but also, the cellular uptake and photo-sensitiveness of
photosensitizers can be improved via pH-induced transitions in surface charge and aggregation
state of nanoparticles.
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Scheme 1. Schematic illustration of the PTFC nanoparticles formation and the enhancing PDT
efficiency resulted from the acidic pH-responsiveness and oxygen transport of PTFC
nanoparticles.
EXPERIMENTAL SECTION
Materials. Pentafluoro benzaldehyde and pyrrole were purchased from Macklin Reagents
(Shanghai). p-Toluenesulfonyl hydrazide was acquired from J&K Scientific Ltd (Shanghai,
China). Potassium carbonate (K2CO3), sodium sulfate (Na2SO4), 1-(3-Dimethylaminopropyl)-3-
ethylcarbodiimide hydrochloride (EDC) and 4-dimethylaminopyridine (DMAP) were obtained
from Aladdin Reagents of China. Dichloromethane (DCM) and N, N-dimethylformamide (DMF)
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were dried over with calcium hydride (CaH2) and distilled before use. All other chemicals were
directly utilized after they are received without further purification.
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Synthesis of TFPP. Pyrrole (268 mg, 4 mmol) and pentafluoro benzaldehyde (784.3 mg, 4
mmol) were dissolved with 300 mL dry dichloromethane in dry and clean flask and then degassed
with nitrogen for 30 min. Subsequently, activated molecular sieve (10 mL) and boron trifluoride
etherate (47% solution in diethyl ether, 0.3 mL, 1.12 mmol) were added into the DCM solution.
The obtained mixture was shielded from ambient light. After stirring for 4 h at room temperature,
p-tetrachlorobenzoquinone (1 g, 4.06 mmol) was added into the reaction flask and then the mixture
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was heated to 50 C and refluxed for 1 h. Subsequently, the solution containing products were
obtained by filtration under reduced pressure and concentrated by vacuum rotary evaporation. The
pure product 5, 10, 15, 20-tetrakis-(2, 3, 4, 5, 6-pentafluorophenyl)-porphyrin (TFPP) was obtained
by column chromatography (dichloromethane: petroleum ether = 1:4) (400 mg, yield 41%).
Synthesis of TFPP-C6-OH. 100 mg (0.1 mmol) of TFPP was dissolved in mixed solution of
tetrahydrofuran (2 mL) and dimethyl sulfoxide (2 mL), 11.8 mg (0.1 mmol) of 6-amino-1-hexanol
and 138 mg of K2CO3 were added into the above solution. The reaction was performed at room
temperature for 4 h and then diluted by dichloromethane and washed with deionized water. The
obtained organic layer was dried by anhydrous Na2SO4 and then evaporated to obtain dry powder.
The powder was further purified by column chromatography elution with dichloromethane to
obtain TFPP-C6-OH (30 mg, yield 26 %).
Synthesis of TFPC-C6-OH. 200 mg of TFPP-C6-OH and 1 g of p-toluenesulfonyl hydrazide
was carefully mixed and then kept under vacuum for 1 h. Subsequently, the reaction was proceeded
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at 158 C for 15 min. After cooling to room temperature, the mixture was dissolved in
dichloromethane and then tetrachloro-o-benzoquinone was added into the solution. After the
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absorption of at 750 nm disappeared, the solution was washed with a saturated solution of sodium
thiosulfate (2×), with distilled water (2×), and then dried over anhydrous Na2SO4.
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Synthesis of POEGMA Homopolymer and POEGMA-b-P(DEAEMA-co-GMA) Block
Copolymer. OEGMA (1 mL, 2 mmol), CDB (10.8 mg, 0.04 mmol), AIBN (1.3 mg, 0.008 mmol)
and 1 mL anhydrous THF were charged in a reaction vial equipped with a magnetic stir bar. The
mixture was degassed by several freeze-thaw cycles and sealed in vacuum. Then the reaction was
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carried out in a preheated oil bath at 70 C. After 135 min, the flask was plunged into liquid
nitrogen. The reaction solution was dialyzed against pure water and lyophilized under vacuum.
Mn, NMR = 11, 880 g/mol, Mn, GPC = 6970 g/mol, PDI = 1.05. POEGMA-b-P(DEAEMA-co-GMA)
block Copolymer were synthesized in a similar way by charging POEGMA (100 mg, 10 µmol),
DEAEMA (0.15 mL, 0.7 mmol), GMA (0.038mL, 0.3mmol), AIBN (0.1 mL, 3 µmol) and 1 mL
anhydrous THF into a reaction vial. After 3.75 h, the flask was plunged into liquid nitrogen. The
solution was transferred into a dialysis bag (MWCO, 8000-14000) and dialyzed for 72 h against a
mixture of water and THF. Finally, the product-containing solution was frozen and lyophilized
under vacuum to afford the white powder. Mn, NMR = 24, 250 g/mol, Mn, GPC = 24, 400 g/mol, PDI
= 1.15.
Synthesis of PTPP, PTFP and PTFC Block Copolymers. 20 mg of POEGMA-b-
P(DEAEMA-co-GMA) and 35 mg of TFPC-C6-OH were dissolved in 2 mL anhydrous DMF, and
then 0.5 mL anhydrous TEA was added into the mixture. The reaction was carried out at room
temperature for 14 days under nitrogen atmosphere. Finally, the product PTFC copolymer was
purified by dialyzing against water and subsequently centrifuging to remove the sediment. PTFC
copolymer was obtained after lyophilized under vacuum. PTPP and PTFP copolymers were
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synthesized in a similar way by replacing TFPC-C6-OH with TPP-C6-OH and TFPP-C6-OH,
respectively.
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Self-Assembly PTFP, PTFC and PTPP Copolymers. 5 mg of PTFC, PTFP and PTPP
copolymers were separately dissolved in 5 mL of dimethyl formamide. Under vigorous stirring, 1
mL of the solution was added dropwise to deionized water (9 mL) at room temperature. After
stirring for 1 h, the organic solvent was removed by dialysis against deionized water for 24 h using
a dialysis membrane (MWCO = 3500).
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Singlet Oxygen production. As a singlet oxygen scavenger, 1, 3-diphenylisobenzofuran
(DPBF) was employed to verify the singlet oxygen production of nanoparticles. A solution
containing a fixed concentration nanoparticle and DPBF was added into a quartz cuvette and
irradiated at 655 nm for 100 s. The 1O2 generation of nanoparticles can be directly correlated with
the decrease of the DPBF absorbance in the UV-vis spectrum, thus the absorbance of DPBF at 415
nm was measured every 20 s.
Cell Culture. 4T1 murine breast cancer cells were cultivated in Dulbecco’s modified Eagle’s
medium (DMEM) supplemented with 1% antibiotics (penicillin and streptomycin) and 10% fetal
bovine serum (FBS) in a humidified standard atmosphere of 5% CO2 at 37 oC.
Intracellular ROS Generation Assay. Dichlorofluorescein diacetate (DCFH-DA) was
employed as a probe to measure the intracellular ROS production. The ROS-induced oxidation of
DCFH-DA could result in the generation of highly fluorescent DCF. Typically, 4T1 cells with a
density of 1 × 105 containing complete DMEM media were incubated on glass bottom cell culture
dish for 24 h. Then, the 4T1 cells were chosen to incubate with PTFP, PTFC or PTPP
nanoparticles for 24 h with or without light irradiation (655 nm, 10 min, 100 mW/cm2). The group
without any treatment was acted as the control. The cells medium was replaced with DMEM
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containing DCFH-DA solution at the concentration of 10 × 10-6 M at 37 ºC in dark. After treatment
for 30 min the cells were observed via confocal laser scanning microscopy (CLSM) to evaluate
the ROS generation. The excitation wavelength was 488 nm and the emission wavelength was 525
nm for the DCFH-DA detection.
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Cellular Uptake Evaluation. The cellular uptake was determined by the utilization of CLSM.
4T1cells (2 × 104 cells/well) were seed on glass bottom cell culture dish for 24 h, and then the cells
were cultured with fresh medium containing PTFP, PTFC or PTPP nanoparticles at the same
concentration 50 µmol/mL of porphyrin for 24 h. Next, the cells were washed carefully with PBS
and then treated with 4% paraformaldehyde. After paraformaldehyde was removed, hoechst were
utilized to stain the cells nuclei for 3 min and the staining were terminated by washing three times
with PBS. Finally, intracellular fluorescence of porphyrin was observed by CLSM with excitation
at 404 nm and emission at 660 nm.
Cytotoxicity Assay. The cytotoxicity of PTFC nanoparticles was evaluated by a standard MTT
assay. 200 µL of 4T1 cell suspension (1 × 104 cells/mL) was seeded in a 96-well plate and then
incubated for 24 h at 37 oC. Different concentrations of PTFP, PTFC and PTPP nanoparticles in
fresh DMEM media were added into the wells and co-cultured for another 24 h. The cells were
washed and irradiated with 660 nm laser (200 mW/cm2) for 10 min. Before the media was replaced
with 200 µL of MTT solution (0.5 mg/mL in DMEM) and cultured for 4 h, the cells were incubated
for further 24 h. Finally, 150 µL of DMSO per well was added to replace the MTT solution and
dissolve the formazan, and the absorbance value was recorded with a SpectraMax spectrometer at
the wavelength of 492 nm. The in vitro dark cytotoxicity of nanoparticles was checked using the
same procedure described above but without illumination.
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Animals and Tumor Xenograft Model. BALB/c mice bearing xenograft tumor were
established and employed for in vivo imaging and PDT performance: 200 µL of 4T1 cells in PBS
(5 ×107 cells/mL) were subcutaneously injected into the mice, respectively. The tumor volumes
(V) were measured by the length and width of tumors and calculated as V = 0.5 × (tumor length)
× (tumor width)2. Relative tumor volume was defined as V/V0 (V0 was the tumor volume when
the treatment was initiated). All animal studies were conducted on male Balb/c nude mice (four to
five weeks) in compliance with the National Institutes of Health Guide for the Care and Use of
Laboratory Animals approved by the Animal Ethics Committee of the East China University of
Science & Technology.
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In vivo Fluorescence Imaging. For fluorescence imaging, mice with 4T1 tumor were
administrated with 200 µL of nanoparticles labeled by Cy7 (1 mg/mL of porphyrin) through the
tail vein. All the image acquisitions were performed with in vivo multispectral imaging system
(Kodak FX) equipped with excitation bandpass filter at 750 nm and emission 830 nm when the
mice were anesthetized at 0.5 h, 2 h, 4 h, 6 h, 12 h, 24 h, and 36 h post-injection.
In vivo Therapeutical Evaluation of PTFC Nanoparticles. The 4T1 tumor-bearing mice were
chosen for therapeutical evaluation of the PTFC nanoparticles. After the tumor volumes of the 4T1
tumor-bearing mice reached about 100 mm3, the mice were divided into five group (n=4 for each
group) and administrated with different agents: (1) Control, (2) PTPP, (3) PTFC, (4) PTPP +
laser, (5) PTFC + laser (porphyrin concentration at 1 mg/kg). The power density of 655 nm laser
was 200 mW/cm2. The tumor volumes were measured by vernier caliper every two days. The body
weights of mice were recorded during the whole experiments. The mice were sacrificed at day 18
and the tumors were dissected and weighed. The dissected tumors of the groups (1) Control, (2)
PTPP, (3) PTFC, (4) PTPP + laser, (5) PTFC + laser were embedded in paraffin and made as
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slices by cryotomy. Furthermore, the frozen slices were stained with H&E and imaged under an
inverted fluorescence microscope to further characterize the therapeutic effects.
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RESULTS AND DISCUSSION
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Synthesis and Self-assembly of PTFC. To fabricate the pH-responsive oxygen-carrying PTFC
nanoparticles, the fluorinated photosensitizer, 5-((2,3,5,6-tetrafluoro-4-((6-hydroxyhexyl) amino)
phenyl)-10, 15, 20-tri(perfluorophenyl) chlorin (abbreviated as TFPC-C6-OH) and pH-responsive
block copolymer (POEGMA-b-P(DEAEMA-co-GMA)) were prepared, respectively. First, we
synthesized TFPC-C6-OH by the diamine reduction of 5-((2, 3, 5, 6-tetrafluoro-4-((6-
hydroxyhexyl) amino) phenyl)-10, 15, 20-tri(perfluorophenyl) porphyrin (TFPP-C6-OH), which
was obtained by the nucleophilic substitution of tetrafluorophenyl porphyrin (TFPP) with 6-
amino-1-hexanol (Scheme S1).76 The successful syntheses of above compounds were verified by
proton and fluorine nuclear magnetic resonance spectrometry measurements as shown in Figure
S1-S5. Next, pH-responsive block copolymer (POEGMA-b-P(DEAEMA-co-GMA)) was
prepared through RAFT polymerization (Scheme S2). Oligo ethylene glycol methacrylate
(OEGMA) monomers were polymerized with cumyldithiobenzoate to form the hydrophilic
POEGMA homopolymer, and then POEGMA as a macro-RAFT agent was used for
copolymerization of glycidyl methacrylate and 2-(diethylamino)ethyl methacrylate to produce the
POEGMA-b-P(DEAEMA-co-GMA) block copolymer. Gel permeation chromatography (GPC)
1
and H NMR were employed to characterize the structures and molecular weights of these
polymers, respectively (Figure S6-S8). We found that the GPC traces of POEGMA and
POEGMA-b-P(DEAEMA-co-GMA) were symmetrical, suggesting the polymerizations
proceeded in a living manner. The average molecular weight calculated from 1H NMR was in line
with that from GPC. Finally, PTFC was obtained by conjugating TFPC-C6-OH to POEGMA-b-
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P(DEAEMA-co-GMA) through ring-opening reaction of hydroxyl and epoxy group. Meanwhile,
POEGMA-b-P(DEAEMA-co-PTFPMA) (PTFP) and POEGMA-b-P(DEAEMA-co-TPPMA)
(PTPP) block copolymers were also prepared in a similar way by replacing TFPC-C6-OH with
TFPP-C6-OH or TPP-C6-OH, respectively. Detailed synthesis process and characterization were
given in supporting information (Scheme S3-S5 and Figure S9-S11).
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Figure 1. TEM images of PTFC nanoparticles in different condition (a) pH = 7.4, (b) pH = 6.0.
(c) Size distribution of nanoparticles determined by DLS. (d) Zeta potential of nanoparticles under
different conditions. (e) Absorbance spectra and (f) emission spectra of PTFC nanoparticles.
The nanoparticles were prepared by a nanoprecipitation method. Briefly, the POEGMA-b-
P(DEAEMA-co-TFPCMA), POEGMA-b-P(DEAEMA-co-TFPPMA) and POEGMA-b-
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P(DEAEMA-co-TPPMA) block copolymers were separately dissolved in tetrahydrofuran (THF),
and then these THF solutions were added drop by drop into deionized water under rapid stirring.
Finally, after removal of THF through dialysis against ultrapure water, the corresponding
nanoparticles were obtained and abbreviated as PTFC, PTFP and PTPP, respectively.
Transmission electron microscopy (TEM) was utilized to observe the morphology of
nanoparticles, as depicted in Figure 1a, S12 and S13. We found PTFC, PTFP and PTPP
nanoparticles dispersed in PBS at pH 7.4 were all sphere-like and had an average diameter of 160
nm, 165 nm and 170 nm, respectively. However, when the nanoparticles were stored in PBS at pH
6.0 (acidic microenvironment), the assembled aggregates became irregular as shown in Figure 1b,
which might be resulted from the hydrophobic to hydrophilic transformation of copolymers
through the protonation of DEAEMA units. The diameter distribution of nanoparticles was also
characterized by dynamic light scattering (DLS) as displayed in Figure 1c. In PBS at pH 7.4, the
hydrodynamic diameters of PTFC, PTFP and PTPP nanoparticles were 171 nm (PDI = 0.21),
173 nm (PDI = 0.23) and 174 nm (PDI = 0.24). In addition, the size of nanoparticles increased
gradually with the pH decrease, due to the responsive ability of nanoparticles to acid
microenvironment. These consequences matched well with those of TEM. Zeta potential was
further utilized to determine responsiveness of the nanoparticles to acid pH as shown in Figure
1d. The potentials of all nanoparticles were negative and lower than -40 mV at pH = 7.4, while
they were positive under acidic condition and even higher than 30 mV at pH = 6.0. This outcome
suggested that the cell uptake of pH-responsive nanoparticles may be facilitated at acid tumor site
due to the charge reverse. Absorption (Figure 1e) and fluorescence emission spectra (Figure 1f)
of nanoparticles were measured as well. It can be seen that PTFC nanoparticles have a strong
absorption peak in red light region thus PTFC is beneficial for PDT of deep-seated tumor.
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Meanwhile, the fluorescence intensity of PTFC under acid condition is significantly higher than
that under neutral condition, indicating that the aggregation-induced quenching effect of
photosensitizers in PTFC nanoparticles could be greatly weakened. As a consequence, PTFC
nanoparticles should be efficient for performing PDT against solid tumors.
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Figure 2. Cell internalization and ROS generation. (a) CLSM images of 4T1 cells incubated with
PTPP, PTFP and PTFC nanoparticles under physiological conditions (pH = 7.4) or acidic
condition (pH = 6.5). (b) Extracellular ROS generation detected by DPBF (n = 3, mean ± s.d.) and
(c) fluorescence images of ROS generation in 4T1 cells treated with nanoparticles, as detected
with DCFH-DA.
Extracellular and Intracellular ROS Production and Cellular Internalization. To achieve
effective PDT, photosensitizers need to be effectually taken up by cells. Therefore, the cell uptake
ability of obtained nanoparticles was first explored using 4T1 cells as a model. Confocal laser
scanning microscope (CLSM) was employed to evaluate the uptake of nanoparticles by cells via
detecting the fluorescence of porphyrin as shown in Figure 2a. The intracellular red fluorescence
of porphyrin indicated nanoparticles could be successfully internalized by 4T1 cells. Moreover,
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the amount of endocytosed nanoparticles at acid condition was significantly higher than that at
normal condition, which was confirmed by the stronger intracellular fluorescence of nanoparticles.
These results showed the nanoparticles could be used as a potential carrier of photosensitizers for
PDT.
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Encouraged by the excellent internalization capacity of the nanoparticles, we next investigated
the photoinduced extracellular and intracellular ROS production of the nanoparticles under 655
nm laser. For extracellular ROS production, 1, 3-diphenylisobenzofuran (DPBF) was utilized as a
ROS indicator.77 The characteristic absorption of DPBF at around 415 nm was detected by an
ultraviolet detector to indicate the generated ROS by the nanoparticles under light illumination.
As shown in Figure 2b, both PTFC and PTPP nanoparticles could result in obvious decrease of
absorbance of DPBF at 415 nm, however, PTFP nanoparticles showed almost no variation. These
results could be attributed to the absorbance differences between PTFP with PTFC and PTPP
nanoparticles, indicating that the diamine reduction of TFPP could significantly enhance the PDT
ability of TFPP in red light region. In addition, the decrease rate of DPBF of PTFC nanoparticles
was faster than that of PTPP nanoparticles, which may be induced by the oxygen-carrying capacity
of PTFC nanoparticles. To confirm our hypothesis, we also investigated the ROS generation of
PTFC and PTPP nanoparticles under hypoxia. It can be seen that the ROS production of PTPP
nanoparticles was strongly inhibited while that of PTFC nanoparticles was still remarkable,
demonstrating that PTFC nanoparticles have the good oxygen-carrying capacity. The oxygen-
carrying capacity was further confirmed by testing the dissolved oxygen through using a dissolved
oxygen detector. As shown in Figure S14, PTFC and PTFP nanoparticles both showed higher
dissolved oxygen value in comparison with the control without fluorine (PTPP). Finally, we
further explored the ROS production of PTFC nanoparticles under acid condition, the most rapid
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decay of the absorbance of DPBF was observed, indicating that the aggregation of porphyrin in
nanoparticles could be effectively attenuated by the protonation of DEAEMA units.
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As for the intracellular ROS production, it was observed via CLSM using DCFH-DA as a ROS
indicator as shown in Figure 2c and Figure S15. In contrast to the control, strong green
fluorescence intensity was observed in both PTFC and PTPP nanoparticles upon irradiation with
655 nm laser while that of PTFP nanoparticles was no obvious change, which was in line with the
extracellular result of ROS production. When the cells were incubated with the nanoparticles under
hypoxia, the generation of ROS of PTFC were slightly restrained compared to that under normoxia
while fluorescence intensity of PTPP was almost same as the control, indicating that PTFC
nanoparticles could carry oxygen thus enhancing the PDT effect under hypoxia. More importantly,
for the cells cultured with PTFC nanoparticles in acidic medium, we found the green fluorescence
intensity was significantly increased after irradiated with 655 nm laser, demonstrating prominent
production of ROS. In consideration of all the above results, PTFC nanoparticles could be a
potential system for PDT against solid tumors.
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Figure 3. Photo-cytotoxicity studies by MTT assay for 4T1 cells after incubation with various
concentrations of different nanoparticles. (a) Normoxia conditions (pH = 7.4), (b) normoxia and
acid (pH = 6.5) condition, (c) hypoxia condition (pH = 7.4) and (d) hypoxia and acid (pH = 6.5)
condition. (n = 3, mean ± s.d.).
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In vitro Cytotoxicity Assay. To investigate the antitumor ability of PTFC nanoparticles, the
cytotoxicity of nanoparticles was performed via MTT assay under different conditions (normoxia,
hypoxia or acid condition). We first investigated the cytotoxicity of nanoparticles without laser
irradiation as shown in Figure S16, and it can be seen that all the nanoparticles did not showcase
obvious toxicity after cultured with 4T1 cells for 24 h even under hypoxia and acid condition.
These results indicated that the obtained nanoparticles have a good biocompatibility. Next, the
photodynamic antitumor effect was verified by laser illumination at 655 nm for 10 min after the
4T1 cells were incubated with the nanoparticles for 24 h. Under normoxia, the cell viabilities
decreased with increased concentration of both PTFC and PTPP nanoparticles at pH = 7.4 (Figure
3a), and a similar result was observed at pH = 6.5 (Figure 3b) with a more significant inhibiting
effect. These consequences demonstrated the nanoparticles have a higher phototoxicity in acid
environment. Moreover, the lowest viabilities were observed in cells treated with PTFC
nanoparticles at both pH = 7.4 and pH = 6.5, which may be resulted from the oxygen self-supply
of PTFC nanoparticles. Subsequently, the phototoxicities of the nanoparticles were performed
under hypoxia at pH = 7.4 or pH = 6.5 as well. As shown in Figure 3c, the cell viabilities were
higher compared to that under normoxia. PTPP nanoparticles showed extremely weak toxicity
against tumor cells while the cell viability treated by PTFC nanoparticles even reduced to around
40%, indicating the antitumor effect of PTFC nanoparticles was evidently stronger than that of
PTPP nanoparticles. All these results revealed that the PTFC nanoparticles could also efficiently
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destroy tumor cells under hypoxia. The similar consequences were also found under hypoxia at
pH = 6.5 (Figure 3d), and the antitumor effect of PDT was slightly enhanced as compared with
that at pH = 7.4, suggesting that the phototoxicity of the nanoparticles could be amplified under
acidic condition. As a control, PTFP nanoparticles showed almost no toxic in all conditions after
exposing to 655 nm laser. As a consequence, the amplified PDT effect may be well realized via
PTFC nanoparticles in hypoxia and acidic tumors.
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Figure 4. In vivo fluorescence imaging and PDT performance of intravenously injected PTFC
nanoparticles in mice bearing 4T1 subcutaneous tumor model. (a) Fluorescence imaging for
different times. (b) Relative tumor volume and (c) body weight change during the treatment. (d)
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Average weight of representative tumors. (e) Photographs of the tumor dissection. (f) Pictures of
H&E stained tumors. (n = 4, mean ± s.d. ***p < 0.005).
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In vivo Fluorescence Imaging and Anticancer Efficacy of PTFC. To further demonstrate the
excellent PDT effect of PTFC nanoparticles, the BALB/c mice bearing xenografted tumor were
fabricated by injecting with 4T1 tumor cells subcutaneously for performing in vivo PDT. The
nanoparticles were injected into 4T1 tumor-bearing mice via the tail vein when tumor volume
reached to about 100 mm3 for investigating the accumulation and antitumor effect in vivo. The
accumulation of nanoparticles was real-time recorded by in vivo fluorescence imaging after the
cyanine (Cy7)-labelled nanoparticles were given through tail vein injection as shown in Figure
4a. The increased fluorescence intensity was observed with the extension of time, which could be
ascribed to the enhanced permeability and retention (EPR) effect of tumors. This result indicated
that the size of nanoparticles was suitable as a delivery system for achieving successfully cargo
transport to tumor sites. The highest fluorescence intensity was observed at 24 h post-injection and
could be kept even at 36 h post-injection. Therefore, it would be an optimal method to perform
antitumor test after the nanoparticles were given for 1 d.
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Tumor-bearing mice were randomly divided into five groups (the control, PTPP, PTFC, PTPP
+ Laser and PTFC + Laser) and then administrated with saline, PTPP or PTFC nanoparticles,
respectively. Mice in PTPP + Laser and PTFC + Laser groups were illuminated with 655 nm
laser for 10 min after 24 h of the administration to determine the antitumor PDT effect, while
PTPP and PTFC groups were used as contrast groups to verify the biocompatibility of the
nanoparticles. Figure 4b revealed PTFC nanoparticles mediated PDT exhibited the most
significant inhibition effect to tumors at day 18 after light illumination. In contrast, we found PTPP
nanoparticles showed a modest therapeutic effect to tumors. The remarkable antitumor effect of
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PTFC nanoparticles may benefit from its oxygen-carrying ability. Moreover, PTPP and PTFC
nanoparticles both demonstrated that there was almost no impact on the tumor growth without
laser irradiation, which could be ascribed to the well biocompatibility of these nanoparticles. In
addition, the body weights of all mice were recorded in Figure 4c and there was also no significant
change during PDT treatments, indicating the nanoparticles had no obvious systemic toxicity. The
tumors were excised from tumor-bearing mice at day 18 after the treatment to further investigate
the tumor inhibition effect. As shown in Figure 4d and 4e, the mean tumor weight and size of all
groups indicated that the greatest inhibition effect was well carried out by PTFC nanoparticles,
which was in agreement with the former results. The antitumor efficacy and biocompatibility were
also investigated via hematoxylin and eosin (H&E) staining of excised tumors and main organs,
respectively. The pictures of H&E stained tumors (Figure 4f) demonstrated that PTFC
nanoparticles resulted in prominent necrosis to tumor under laser illumination, which further
confirmed the significant PDT effect of PTFC nanoparticles. As proved by H&E staining of major
organs (Figure S17), there was no obvious tissue destruction to the major organs of mice injected
with the nanoparticles, which suggested that these nanoparticles were high biocompatible.
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CONCLUSIONS
In summary, we fabricated an oxygen self-suppling system by conjugating a far-red-absorbing
fluorinated photosensitizers (TFPC) to a pH-responsive block copolymer (POEGMA-b-
P(DEAEMA-co-GMA) for sensitization of hypoxic tumor to PDT. The block copolymers can form
PTFC nanoparticles with an appropriate size to accumulate at tumor site by EPR effect. The
hypoxia tumor environment can be improved through the release of oxygen carried by
nanoparticles, the oxygen carry was well realized by photosensitizers itself without the additional
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reagent such as perfluorocarbons. Moreover, due to the pH-responsive ability, the uptake of
nanoparticles can be effectively improved and the aggregation of photosensitizers can be
attenuated as well, leading to enhanced PDT effect. Thus, the efficient PDT can be well realized
by PTFC nanoparticles, which incorporated the oxygen-carrying ability and acidic
responsiveness. This work offers a smart nano-photosensitizer with effective regulation of tumor
microenvironment and activation of photosensitizers to realize a safer and more reliable PDT
efficacy.
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ASSOCIATED CONTENT
Supporting Information.
The Supporting Information is available free of charge on the ACS Publications website at DOI:
10.1021/acs.biomac
Figures S1−S17 of the following data: additional TEM, 1H NMR and 19F NMR spectrum, GPC,
and CLSM images, and cell viability (PDF).
AUTHOR INFORMATION
Corresponding Author
*E-mail: wazhang@ecust.edu.cn
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENT
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This work was financially supported by the National Natural Science Foundation of China (No.
21574039 and 21875063).
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