A Redox Stimulation-Activated Amphiphile for Enhanced Photodynamic Therapy

Article abstract of DOI:10.1021/acs.biomac.9b00581

The development of more efficient photosensitizers with minimal damage to surrounding normal tissues has been a valuable and challenging subject during photodynamic therapy (PDT). Herein, a stimuli-activated porphyrinic photosensitizer (PEG-TPP-DNB; PEG =

Full text of DOI:10.1021/acs.biomac.9b00581

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Article
A Redox Stimulation-activated Amphiphile
for Enhanced Photodynamic Therapy
Yudong Xue, Jia Tian, Zhiyong Liu, Jianbo Chen, Mengsi Wu, Yongjia Shen, and Weian Zhang
Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.9b00581 • Publication Date (Web): 07 Jun 2019
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A Redox Stimulation-activated Amphiphile for
Enhanced Photodynamic Therapy
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†
†
Yudong Xue, Jia Tian, Zhiyong Liu, Jianbo Chen, Mengsi Wu, Yongjia Shen, Weian Zhang*
Shanghai Key Laboratory of Functional Materials Chemistry, East China University of
Science and Technology, 130 Meilong Road, Shanghai 200237, P. R. China
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ABSTRACT
The development of more efficient photosensitizers with minimal damage to surrounding
normal tissues has been a valuable and challengeable subject during photodynamic therapy
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(PDT). Herein, a stimuli-activated porphyrinic photosensitizer (PEG-TPP-DNB) with
capabilities of fluorescence and singlet oxygen remarkably quenching had been prepared
successfully for photodynamic therapy with high efficiency and biosecurity. The amphiphilic
PEG-TPP-DNB could be self-assembled into nanomicelles in aqueous media and dissociated
in response to reductive thiol such as glutathione (GSH). Meanwhile, the fluorescence and
singlet oxygen generation of porphyrinic photosensitizer would be activated to regenerate.
Moreover, the intracellular uptake and localization effectively confirmed the
redox-responsive and activated behavior of PEG-TPP-DNB micelles. The cytotoxicity in
vitro revealed that the micelles had low dark toxicity and great phototoxicity, and in vivo
bio-imaging and antitumor evaluation further indicated that the micelles possessed selective
tumor imaging and targeted PDT antitumor effect as well as low systemic toxicity. Overall,
this tumor microenvironment activated photosensitizer system may provide a useful strategy
for precise photodynamic therapy.
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INTRODUCTION
Remarkably, photodynamic therapy (PDT) has attracted more extensive attentions for cancer
treatments because of its unique advantages such as perceived controllability, less invasive,
site-specific selectivity and fewer side effects.1-3 Photosensitizers (PSs) play a decisive factor
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in PDT because of the fact that cytotoxic reactive oxygen species (ROS) such as singlet
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oxygen ( O ) produced by the PSs upon light irradiation with the suitable wavelength could
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induce irreversible damage to cancer cells.4-5 To date, porphyrin and phthalocyanine
derivatives as important photosensitizers for clinical applications have been extensively
studied and demonstrated excellent therapeutic effect in PDT, and applied topically or
systemically in clinical cancer treatments.6-7 However, most of clinical PSs due to their
hydrophobic conjugated large macrocycle structure also remained several fatal disadvantages
including poor solubility and biocompatibility, low singlet oxygen generation, unexpected
dark toxicity, unfavourable pharmacokinetics and inappropriate selectivity. In order to solve
these shortcomings, multitudinous nanocarrier systems have been widely developed and
applied in the transport of hydrophobic PSs including biological membrane and protein
carrieries,8-11 polymeric nanoparticles,12-17 liposomes,18-20 silica nanocarriers,21-23 amphiphilic
supramolecular compound.24-28 Those nanocarrier platforms can effectively improve the
solubility and biocompatibility of PSs through physical loading directly in the hydrophobic
core or chemical conjugation and significantly attain better tumor penetration via enhanced
permeability and retention (EPR) effects compared with free drugs, which give rein to the
multifunctional diagnostic and therapeutic characteristics of PSs.29-34 Despite these
developments, the untoward reproducibility and quantification of conjugates, the insecure
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transportation of PSs under pathophysiological conditions and the serious non-specific
photosensitive reaction and damage of PSs during cancer treatment are other obstacles in the
future clinical application of PDT.
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What's more, if accidentally exposed to light, PSs in the bloodstream and metabolism can
destroy heathy tissues, which can’t be conquered entirely by the utilization of active or
passive tumor targeting nanosystems due to the uncontrollable ROS generation of PSs. Thus,
it is urgent to build switchable systems with controllable release and photoactivity which can
be selectively activated at specific condition, particularly in the tumor microenvironment by
responding to the tumor-associated biological stimulus, while remaining inert throughout
these body's systemic circulation.35-39 With the aim of the switchable and controllable PSs
potently and specifically destructing cancer cells under a specific environment, various
stimuli-responsive approaches have been studies including internal and external stimuli, such
as pH,40-43 temperature,44-47 redox,48-53 light,54-57 enzymes58-60 and so on. Among current
diverse stimuli-responsive nanoparticles, the redox-responsive stimulation is one of the
highlights of nanocarriers due to the redox alterations including the overexpression of
reduced thiols in tumor intracellular microenvironment. And glutathione (GSH) as a major
over-expressed reduced agent is critically implicated in cell survival against oxidative stress
for the undesirable therapeutic selectivity and the development of drug resistance.49, 61-62
In general, a broad range of nanocarrier systems have been designed to conjugate
functional PSs by means of physical loading or covalent connection. These strategies can
improve the biostability of PSs, control the photoactivity and bring forth self-quenching at the
appropriate conjugation density, either of which can be reactivated in response to various
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stimuli.63-68 For instance, Yoon and co-workers designed and synthesized a versatile zinc (II)
phthalocyanine (Pc) derivative for biophotonic imaging and activatable PDT, which could
form assemblies with the switchable photoactivity in the presence of targeted proteins.69
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However, these architecting decorations always encounter many issues such as complex
molecular design and synthesis, unsatisfactory efficacy and unstable quenching and
activation properties. Moreover, the strategy of conjugating a quencher molecules or energy
acceptors to PSs via a switchable or cleavable linker has been investigated, which can be
inactivated at normal environment but rapidly regained the photoactivity at a specific site
with certain stimulation.70-72 There are some molecules which can greatly conquer the
photoactivity of PSs such as carbon nanostructure,73-74 2, 4-dinitrobenzene,75-76 black hole
quencher (BHQ-3).77-78 Defined and controlled molecular systems in these strategies insure
the uniformity of photosensitizer preparation and the high selective efficiency of the
activation of fluorescence and photodynamic properties.
In this work,
a
biocompatible stimuli-activated photosensitive nanosystem
(PEG-TPP-DNB) based on porphyrin molecules had been proposed, which exhibits
nonphototoxic in the absence of activation factor and possesses sensitive release and PDT
efficacy in site of the tumor under the overexpressed GSH (Scheme 1). 5, 15-bis
(
4-aminophenyl)-10, 20-diphenylporphyrin (NH -TPP-NH ) as the center of molecular
2 2
structure was individually modified at both ends with poly (ethylene glycol) (PEG) chain and
the functional group 2, 4-dinitrobenzene (DNB), respectively, while DNB had a strong ability
of withdrawing electrons on the porphyrin macrocycles to remarkably quench the
fluorescence and singlet oxygen generation of porphyrin via pH-deionized and thiol-labile
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sulfonamide linkages. Thus, the amphiphilic PEG-TPP-DNB could endow attractive features:
(i) the formation of applicable and stable micelles with high biocompatibility and rigorous
non-photoactivity under physiological conditions for the high-efficient and EPR effect, (ii)
the superior capacity of both selective release and efficient activation of hydrophilic
porphyrin for a maximized tumor enrichment through the responsive trigger of GSH, which
is severely overexpressed in cancer cells, (iii) the maximization of the cytosolic dose of
active PSs to achieve higher cellular internalization and cytotoxicity, (iv) both the reliable
prevention of photosensitive side effects during blood circulation and the sensitive recovery
of fluorescence and singlet oxygen generation in tumor tissues. On the whole, these features
could enable PEG-TPP-DNB micelles to accumulate into tumor tissues via efficiently EPR
effect and be specifically activated at tumor tissues, accompanying with the recovery of their
fluorescence and photoactivity, which could enhance the treatment efficacy and reduce side
effects of accidental photoactivity to healthy normal tissue during internal circulation.
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Scheme 1. Schematic illustration of GSH-responsive intracellular activation of
PEG-TPP-DNB micelles and the tumor-selective and targeted PDT performed in vivo.
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EXPERIMENTAL SECTION
Materials
Methoxy poly-(ethylene glycol) (PEG, M = 2 000 g/mol) was purchased from Aladdin
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Reagents of China and was purified by azeotropic distillation in toluene to remove the water
before using. Dimethylformamide (DMF) and dichloromethane (DCM) were dried over CaH2
and distilled just before using. Tetrahydrofuran (THF) was refluxed over sodium to remove
water. Sodium nitrite (NaNO ), tin (II) chloride dehydrate (SnCl ), trifluoroacetic acid (TFA),
2
2
4
-dimethylaminopyridine (DMAP), 2, 4-dinitrobenzenesulfonyl chloride, 5, 10, 15,
20-tetrakis
(4-sulfophenyl)
porphyrin
(TPPS),
1,
1
-(3-dimethylaminopropyl)-3-ethylcarbodiimide
hydrochloride
(EDC),
3-diphenylisobenzofuran (DPBF) and N-hydroxysuccinimide (NHS) were purchased from
Aladdin Reagents. Glutathione (GSH), glutathione monoester (GSH·OEt), 2,
7
-dichlorofluorescein
diacetate
(DCFH-DA),
3-(4,
5-dimethylthiazol-2-yl)-2,
5-diphenyltetrazolium bromide (MTT) and Hoechst 33342 were purchased from Sigma. All
other chemicals are of analytical grade and used without further purification.
Characterizations
1H NMR and 13C NMR spectra were acquired by a Bruker AV400 MHz NMR
spectrometer using CDCl with tetramethylsilane (TMS) as an internal reference. Mass
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spectrum (MS) was recorded on a Waters LCT Premier XE spectrometer with DCM as the
solvent. The number average weight (M ) and polydispersity index (PDI) of the samples were
n
determined using a Waters 1515 gel permeation chromatograph (GPC) equipped with a
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refractive index detector and ultrastyragel columns of 100-10000 Å porosities. The GPC
system was calibrated with polystyrene as the standards and tetrahydrofuran (THF) as the
eluent at a flow rate of 1 mL/min. A BECKMAN COULTER Delasa Nano C particle
analyzer was used to acquire the hydrodynamic diameter at a fixed angle of 165°, and all the
measurements were carried out at room temperature. Transmission electron microscopy
(TEM) analysis was performed using a JEOL JEM1400 electron microscope operated at 100
kv. Samples for TEM were prepared by dropping the micelle solution onto a carbon-coated
copper grid and then dried at room temperature. The UV-Vis spectra of the samples were
measured by a UV-2450 UV-Visible spectrophotometer. The fluorescence spectra were
measured on a Lumina Fluorescence Spectrometer (Thermo Scientific) at room temperature
with both excitation and emission slit widths of 20 nm. Flow cytometry was recorded by BD
FACS Calibur flow cytometer. Confocal laser scanning microscopy (CLSM) was performed
on a Nikon AIR. Optical imaging in vivo was imaged via a Kodak FX multispectral animal
imaging system.
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Synthesis of TPP
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, 10, 15, 20-tetraphenylporphyrin (TPP) was prepared successfully according to our
previous work.79-80 1H NMR (400 MHz, CDCl ), δ ppm: 8.88 (s, 8H, β-H), 8.25 (m, 8H, 2,
3
6-Ar-H), 7.79 (m, 12H, 3, 4, 5-Ar-H)), -2.76 (s, 2H, -NH-).
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Synthesis of NH -TPP-NH
2
2
5
, 15-bis (4-aminophenyl)-10, 20-diphenylporphyrin (NH -TPP-NH ) was synthesized
2 2
from TPP. Firstly, TPP (0.4 g, 0.65 mmol) and NaNO (0.37 g, 5.30 mmol) were mixed in
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TFA (30 mL), the mixture was stirred at room temperature for 5 min and quenched with ice
water (100 mL), and washed with water three times. The products were extracted with DCM
(3 × 50 mL) until the aqueous solution was colorless. Then the extract was washed with
saturated sodium bicarbonate solution and the color of the solution was changed from green
to purple. Then the solution was extracted with DCM for three times and dried with
anhydrous MgSO . After the solvent was removed by vacuum evaporation, the crude product
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was purified on a silica gel column with DCM/PE (2:1, v/v) as the eluent to obtain the
mixture
of
5,
15-bis(4-nitrophenyl)-10,
20-diphenylporphyrin
and
5,
1
0-bis(4-nitrophenyl)-15, 20- diphenylporphyrin (TPP(NO ) ). Yield: 0.24 g (60%).
2
2
Secondly, a stirred solution of the mixture (TPP (NO ) ) (0.71 g, 1 mmol) in 38% HCl (30
2
2
mL) was added SnCl (1.28 g, 6 mmol) under N protection conditions. The mixture was
2
2
heated to 65 °C and stirred for 2 h. After cooled to room temperature, the mixture was poured
into ice water (100 mL) and NH ·H O was added to adjust pH to 8 until the color of the
3
2
solution was purple. The aqueous phase was extracted successively with DCM (3×100 mL),
the organic phase was combined and dried with MgSO . After the solvent was removed by
4
vacumn evaporation, the crude product was purified on a silica gel column with
DCM/acetone (50:1, v/v) as the eluent to obtain 5, 15-bis (4-aminophenyl)-10,
1
20-diphenylporphyrin (NH -TPP-NH ). Yield: 0.15 g (21.1%). H NMR (400 MHz, CDCl ):
2
2
3
δ ppm: 8.91 (s, 4H, pyrrole C-H), 8.80(s, 4H, pyrrole C-H), 8.41 (d, 4H, Ar-H), 8.01 (d, 4H,
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Ar-H), 7.06 (d, 4H, Ar-H), 7.71-7.81 (m, 6H, Ar-H), 4.05(s, 4H, amino-H), -2.78(s, 2H,
1
3
pyrrole N-H). CNMR (400 MHz, CDCl ), δ ppm: 146.01, 142.41, 135.72, 134.61, 132.43,
3
1
31.09, 129.92, 127.65, 126.86, 120.51, 119.79, 113.48. Mass spectrometry (ESI-MS, m/z):
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[M + H] calcd for C H N O S, 645.2688; found, 645.2857.
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Synthesis of NH -TPP-DNB
2
Compound NH -TPP-DNB was synthesized via substitution reaction by coupling
2
NH -TPP-NH with 2, 4-dinitrobenzenesulfonyl chloride. Typically, NH -TPP-NH (129 mg,
2
2
2
2
0.2 mmol), and pyridine (0.1 mL) were dissolved in anhydrous DMF (20 mL) under N2
atmosphere. After the solution was cooled to 0 °C in an ice-water bath, 2,
4-dinitrobenzenesulfonyl chloride (54 mg, 0.2 mmol) in anhydrous DMF (5 mL) was added
dropwise, and the mixture was stirred at room temperature overnight. Then the mixture was
washed with DCM and 1% hydrochloric acid solution, and dried with anhydrous MgSO4.
After removing DCM and excess of pyridine by evaporation, the crude product was purified
1
on a silica gel column with PE/EA (1: 2, v/v) as the eluent. Yield: 0.15 g (86.7%). H NMR
(400 MHz, CDCl ): δ ppm: 8.87 (d, 2H, pyrrole C-H), 8.75(s, 4H, pyrrole C-H), 8.70 (d, 1H,
3
Ar-H), 8.57 (d, 2H, pyrrole C-H), 8.50 (d, 1H, Ar-H), 8.29 (d, 1H, Ar-H), 8.12 (m, 4H,
Ar-H), 8.06 (d, 2H, Ar-H), 7.89 (d, 2H, Ar-H), 7.68 (m, 6H, Ar-H), 7.50 (d, 2H, Ar-H), 6.95
1
3
(d, 2H, Ar-H), 4.01(s, 2H, amino-H), -2.76(s, 2H, pyrrole N-H). CNMR (400 MHz, CDCl ),
3
δ ppm: 149.86, 147.54, 141.43, 140.92, 140.88, 134.83, 133.51, 132.73, 130.12, 126.80,
125.72, 125.68, 125.33, 119.67, 119.42, 119.25, 119.21, 116.28. Mass spectrometry
+
(
ESI-MS, m/z): [M + H] calcd for C H N O S, 875.2322; found, 875.2614.
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Synthesis of PEG-TPP-DNB
Firstly, carboxylic PEG2000 (PEG2000-COOH) was synthesized via esterifcation by coupling
methoxy poly-(ethylene glycol) (PEG, M = 2000 g/mol) with succinic anhydride. Secondly,
n
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NH -TPP-DNB (88 mg, 0.1 mmol), PEG2000-COOH (188 mg, 0.09 mmol), NHS (115 mg, 1
2
mmol) and dry THF (20 mL) were added into a dry bottle under N , and EDC (192 mg, 1
2
mmol) in THF (5 mL) was added drop-wise into the bottle in an ice-water bath. After the
mixture was stirred at room temperature overnight, the mixture was washed with water and
DCM, and dired with anhydrous MgSO and filtered. After evaporation of the solvent, the
4
crude product was precipitated in cold diethyl ether for 3 times, centrifuged for 5 min at 12
0
00 rpm and further purified with a silica gel column with DCM and MeOH (20:1 v/v) as the
eluent. Then the product was dried in vacuum oven at room temperature. Yield: 235 mg
1
(
85.1%). H NMR (400 MHz, CDCl ): δ ppm: 8.90-8.67 (s, 9H, pyrrole C-H and Ar-H), 8.50
3
(d, 1H, Ar-H), 8.22 (d, 1H, Ar-H), 8.16-8.02 (m, 6H, Ar-H), 7.94 (d, 2H, Ar-H), 7.75-7.58
(m, 8H, Ar-H), 7.51 (d, 2H, Ar-H), 4.21 (s, 2H, -CH -), 3.84-3.37 (m, 178H, -CH -CH -),
2
2
2
3
.32 (s, 3H, -CH ), 2.84 (m, 4H, -CH -CH -), -2.76(s, 2H, pyrrole N-H).
3
2
2
Self-assembly of PEG-TPP-DNB in aqueous media
The micellar solution was prepared by the following process: PEG-TPP-DNB (2 mg) was
adequately dissolved in 1 mL of THF at 25 °C. Subsequently, the solution (1 mL) was slowly
injected drop-wise into 4 mL of ultrapure water under magnetic stirring and then dialyzed
against ultrapure water by using a dialysis membrane (MWCO = 1 000) for 8 h to remove the
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organic solvents THF at least 3 times. Finally, the PEG-TPP-DNB micelles were obtained
with a concentration of 0.40 mg/mL.
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Critical micelle concentration (CMC) of micelles
Typically, the critical micelle concentration (CMC) of micelles in aqueous solution was
determined by fluorescence spectroscopy using pyrene as a hydrophobic probe, which can
enter the hydrophobic core of micelles from the aqueous phase by hydrophilic and
hydrophobic interaction with significant spectroscopic variations. In detail, a fixed
concentration of pyrene solution (5 μL, 1.2 mmol/L) in acetone was added into a series of 10
mL volumetric flasks, and then the acetone was allowed to evaporate at room temperature.
Various concentrations of PEG-TPP-DNB micelles were prepared in volumetric flasks with
-4
-2
the concentration ranging from 2.5×10 to 2×10 mg/mL by using ultrapure water and then
sonicated for 1 h at room temperature. Fluorescence spectra of different micellar
concentrations were recorded using an F-4500 fluorescence spectrometer with an excitation
wavelength at 335 nm. Fluorescence emissions at 372 nm and 383 nm were monitored and
recorded. The I /I ratio remained almost constant at lower micelle concentration but
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increased sharply when the concentration reached a certain value. It meant that pyrene
molecules were trapped into the micelle core with a hydrophobic environment, which
actually reflected the formation of micelles. The CMC value was obtained from the
intersection of peak intensity ratios of fluorescence intensity at 383 nm to that at 372 nm
(I /I ).
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Stimulus responsive behavior of PEG-TPP-DNB micelles
PEG-TPP-DNB micelle aqueous solution (0.4 mg/mL) was mixed with GSH (10 mM) in
PBS solution (pH = 7.4), and then the mixed solvent was placed under stirring conditions for
24 h at 37 °C. The size of the micelles was checked by dynamic light scattering (DLS) and
transmission electron microscope (TEM).
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The fluorescence intensity and responsiveness of the compounds were determined by
Lumina Fluorescence Spectrometer (Thermo Scientific) with the emission slit widths of 20
nm. PEG-TPP-DNB micelles with the concentration of 5 μM were examined their changes of
the fluorescence over time under different concentrations of GSH (0 mM, 1 mM, 5 mM and
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0 mM) at 37 °C.
Singlet oxygen generation
1
The generation of singlet oxygen ( O ) for PEG-TPP-DNB micelles was determined using
2
DPBF as the detection agent by a UV-Visible spectrophotometer. The mixture solution of
DPBF and micelles with different concentrations of GSH was irradiated under a 650 nm light
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source at a powder density of 200 mW/cm in PBS with 1% DMF, and the absorbance of
DPBF decay derived by deducting the constant absorption of micelles at 410 nm was
monitored every 20 s under light irradiation. The singlet oxygen quantum Ф was calculated
∆
by comparing with H TPP as a standard which has a quantum yield of 0.64 in DMF
2
according to eqn (1):81
Std
RI
abs
Std
Ф = Ф
∆
(1)
∆
Std
R I
abs
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Where Ф S∆ td is the singlet oxygen quantum yield for the standard H TPP (Ф S∆ td = 0.64 in
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2
Std
DMF); R and R
are the DPBF photobleaching rates in the presence of the analyte and
the standard, respectively; Iabs and IStd are the rates of light absorption by the analyte and
abs
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the standard, respectively.
Cell culture
4
T1 cells (murine breast cancer cell line) were cultivated in DMEM (Dulbecco's modified
Eagle's medium) containing 10% FBS (fetal bovine serum) and 1% antibiotics (50 units per
mL penicillin and 50 units per mL streptopycin) at 37 °C in a humidified atmosphere
containing 5% CO2.
Intracellular uptake
CLSM and flow cytometry were used for studying the cellular uptake by 4T1 cells. Briefly,
4
4T1 cells were cultivated on sterile glass-bottomed dish at a density of 2×10 cells/well and
incubated in 2 mL culture medium for 24 h at 37 °C. Then the culture medium was replaced
by the culture medium containing either free porphyrin (TPP) or PEG-TPP-DNB micelles
(
porphyrin concentration: 20 μg/mL) to incubate at 37 °C for 4 h and 24 h, respectively. For
the CLSM measurement, the cells were washed with PBS for three times firstly. Furthermore,
the cells were fixed with 4% paraformaldehyde in PBS for 30 min, washed with PBS three
times, then stained by Hoechst for 3 min to mark the nuclei and washed three times with
PBS. Finally, the images of cellular uptake were carried out by using CLSM. The excitation
wavelength of Hoechst and porphyrin was performed with 405nm, while the emission
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spectrum was collected using a wavelength range of 425-475nm for DAPI and a wavelength
range of 660-750 nm for porphyrin fluorescence, respectively.
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plates and cultured for 24 h. Then, the culture medium was replaced with fresh culture
medium containing either free porphyrin (TPP) or PEG-TPP-DNB micelles (porphyrin
concentration: 20 μg/mL) to incubate for 4 h and 24 h, respectively. Then the cells were
washed thrice with PBS (pH = 7.4), digested by trypsin and harvested by centrifugation.
Then, the collected cells were resuspended in PBS (1 mL) for measuring the fluorescence
intensity via flow cytometer (BD FACS Calibur, Channel: Qdot 605-A (λ = 405 nm and λem
ex
=
550-650 nm)).
Intracellular ROS detection
The ROS generation within the cells was identified using DCFH-DA as the ROS indicator
by means of fluorescence as well as CLSM. DCFH-DA itself has no fluorescence and can
freely cross the cell membrane for entering the cell, which can be hydrolyzed by intracellular
esterase to generate DCFH. DCFH as the probe cannot penetrate the cell membrane and it can
label the cell inside swimmingly. As soon as the presence of ROS, DCFH can be oxidized to
form a green fluorescent substance (DCF). Generally, 4T1 cells were incubated with free
porphyrin and PEG-TPP-DNB for 24 h. And the cells without any treatment were used as
control. Then, the cells were washed for three times and the culture medium of DCFH-DA
(10 μM) was further incubated with the cells for 30 min. Subsequently, the cells were
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illuminated with a laser (200 mW/cm , 650 nm) for 8 min and immediately imaged with
CLSM (λ = 488 nm and λ = 500–550 nm).
ex
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In vitro cytotoxicity of PEG-TPP-DNB micelles
In vitro relative cytotoxicity of PEG-TPP-DNB micelles and free porphyrin against 4T1
cells was determined through a standard MTT assay. 4T1 cells were seeded in 0.2 mL culture
medium at 4 000 cells/well on 96-well plates for 24 h. After overnight of incubation, the cells
were cultured with the medium containing different concentrations of PEG-TPP-DNB
micelles and free porphyrin (porphyrin concentration: 0-20 μg/mL) in dark for 24 h at 37 °C.
Then, 200 μL of 0.5 mg/mL MTT solution in DMEM was added to replace the medium of
each well for a further incubation of 4 h. Finally, 150 μL DMSO was added to each well to
dissolve the precipitate, and then the absorbance was measured using a THERMO Multiskan
MK3 spectrometer at a wavelength of 492 nm. The cell viability was calculated as follows:
cell viability (%) = (ODtest - ODbackground) / (ODcontrol - ODbackground) × 100, where ODbackground
was the absorbance of blank well plate; ODtest and ODcontrol were the absorbance in the
presence of sample solutions and that without treatment, respectively. The cancer cells treated
without PEG-TPP-DNB micelles and free porphyrin in the dark were prescribed to the
viability of 100 % as a reference.
The phototoxicity of PEG-TPP-DNB micelles was measured using a similar process with
the dark cytotoxicity. After the cells were incubated with the DMEM containing different
concentrations of PEG-TPP-DNB micelles and free porphyrin (0-20 μg/mL of the porphyrin
content) for 24 h at 37 °C, the cells were irradiated by using a laser light source (200
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mW/cm , 650 nm) for different time (10 min, 20 min, 30 min) and then incubated by using
fresh DMEM for 24 h at 37 °C. MTT assay for the cell viability was described as above.
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Animal model
All animal studies were conducted on male Balb/c nude mice (4-5 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 East China University of Science &
Technology. To build the xenograft tumour-bearing mouse model, the suspension of 4T1
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cells (1 × 10 ) were subcutaneously inoculated into the flank of the right front of each mouse,
and the experiments were operated approximately after post-inoculation for 12 days when the
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tumor volumes reached about 100–150 mm . Then, mice with tumors were used for in vivo
fluorescence imaging and treatment experiment.
In vivo fluorescence imaging and PDT of PEG-TPP-DNB micelles
To observe the accumulation process in tumor site, TPPS (as a small molecule control) and
PEG-TPP-DNB micelles solution (porphyrin concentration: 1 mg/mL) were intravenously
injected into the 4T1 tumor-bearing male Balb/c nude mice, respectively. At 2 h, 6 h, 12 h
and 24 h after the injection, the mice were anesthetized and imaged via a Kodak FX
multispectral animal imaging system.
For antitumor treatment of PDT, the tumor-bearing mice were randomly divided into 6
groups (4 mice per group) which included (1) Saline, (2) Saline + 650 nm, (3) TPPS (4)
TPPS + 650 nm, (5) PEG-TPP-DNB, and (6) PEG-TPP-DNB + 650 nm. 4T1 tumor-bearing
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mice were intravenously injected into 200 μL of saline, TPPS (1 mg/mL), and
PEG-TPP-DNB micelles with porphyrin concentration of 1 mg/mL, respectively. For the
2
groups of light irradiation, the mice were only irradiated with 650 nm laser (0.8 W/cm ) for
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10 min after 12 h of the injection, respectively. The mice weight and tumor size were
measured every two or three days for 21 days. Tumor size was measured by a vernier caliper
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and tumor volume was calculated using the following formula: V = L × W /2. L and W were
the longest and shortest diameters of tumors, respectively. Relative tumor size was calculated
as V/V (V was the initial tumor volume). On day 21, all mice were sacrificed and the
0
0
tumors were excised and weighed.
Histological staining and analysis
In vivo histological analysis, after treatment for 21 days, the mice were sacrificed and the
tumour tissues and main organs (heart, liver, spleen, lung, and kidney) of the mice in
different groups were collected and fixed in 4 % paraformaldehyde, embedded in paraffin
sectioning at a thickness of 5 mm and then conducted for histological hematoxylin and eosin
(H&E) staining. The evaluation of histological alterations was examined by a digital
microscope.
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RESULTS AND DISCUSSION
Synthesis of PEG-TPP-DNB
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Scheme 2. Synthetic route of PEG-TPP-DNB.
As shown in Scheme 2, for the synthesis process of PEG-TPP-DNB, TPP was firstly
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prepared according to previous literature. After nitration and redox reaction from TPP, the
1
H NMR spectrum of NH -TPP-NH is shown in Figure S1. The proton signals at δ =
2
2
8
.89-7.08 and -2.78 ppm are separately assigned to porphyrin macrocyclic protons, and the
1
3
proton signal at δ = 4.06 ppm is ascribed to the two amino groups. Furthermore, the C NMR
and MS spectra also indicated that NH -TPP-NH was successfully synthesized (Figure
2
2
S2-3). Then, NH -TPP-DNB was synthesized by a substitution reaction between
2
1
13
NH -TPP-NH and 2, 4-dinitrobenzenesulfonyl chloride. The H NMR, C NMR and MS
2
2
spectra of NH -TPP-DNB are shown in Figure S4-6, indicating the purity and identity of
2
NH -TPP-DNB. In addition to the protons signals of the porphyrin, the proton signals at δ =
2
8
.70, 8.50 and 8.29 ppm are assigned to the protons of 2, 4-dinitrobenzene. Finally,
PEG-TPP-DNB was prepared by the esterification reaction between NH -TPP-DNB and
2
1
PEG2000-COOH (Figure S7). The H NMR and MALDI-TOF-MS of PEG-TPP-DNB are
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shown in Figure S8-9. The proton signals at δ = 8.94-7.01 and -2.78 ppm are attributed to
aromatic and pyrrole ring protons, and the proton signals at δ = 4.21, 3.79 - 3.40, 3.32 and
2
.72 ppm belong to methylene and methyl protons of PEG chain. Moreover, the proton signal
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at δ = 4.06 ppm disappears in comparison to that of NH -TPP-DNB after the reaction, which
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suggested that PEG-TPP-DNB was successfully synthesized. Simultaneously, the results of
Gel Permeation Chromatography (GPC) (Figure 1A) have clearly illustrated that
PEG-TPP-DNB shifted to the higher molecular weight than PEG. The number-averaged
molecular weights of PEG and PEG-TPP-DNB are 1893 g/mol with the PDI value of 1.07
and 2811 g/mol with the PDI value of 1.04, respectively. In addition, the similar UV-Vis
spectra of NH -TPP-NH and PEG-TPP-DNB (Figure S10 A) showed that DNB units had no
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significant effect on the basal properties of porphyrin nuclei. However, the fluorescence
spectrum (Figure S10 B) indicated that the fluorescence emission of PEG-TPP-DNB was
greatly quenched so that it was much weaker than that of NH -TPP-NH . The DNB unit is an
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electrophilic group which can vastly extinguish the fluorescence of porphyrins through
photo-induced electron transfer. The results showed that the porphyrin core was efficiently
inactivated by the DNB group with the significant fluorescence quenching.
Characterization of PEG-TPP-DNB micelles
The CMC value was calculated from the intersection of the ratios of fluorescence peak
intensity (I /I ) of pyrene with different concentrations of PEG-TPP-DNB micelles which
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was about 2.93 ×10 mg/mL (Figure S11). The low CMC value for PEG-TPP-DNB
suggested that it has high thermodynamic stability, which represents an advantage upon the
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excellent stabilization of micelles in aqueous solution. Synchronously, the UV–Vis
absorption of PEG-TPP-DNB micelles were broadened and red-shifted of about 8 nm
comparing to that of PEG-TPP-DNB solution (Figure S12), which displays that the
self-assembly process by hydrophobic π-π stacking interactions. Additionally, as shown in
Figure 1B, the hydrodynamic diameter of the PEG-TPP-DNB micelles characterized by
dynamic light scattering (DLS) was about 129 nm with polydispersity index (PDI) of 0.072.
And it also can be evidently observed that PEG-TPP-DNB could form spherical micelles
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(Figure 1C). For validating the colloidal stability of PEG-TPP-DNB micelles, DLS was also
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employed to assess the stability of the PEG-TPP-DNB micelles at 37 C over time in
DMEM+10% FBS media which is diffusely utilized to simulate physiological conditions. As
shown in Figure S13, the average size remained little change at around 156 nm for a week
which suggested that PEG-TPP-DNB micelles overall exhibited quite a steady biostability.
Meanwhile, we also found that as the decrease of pH value of solution, the size distribution of
PEG-TPP-DNB micelles became broader and the zeta-potential also changed to a certain
extent (Figure S14). These results may be attributed to the facts that the hydrogen atom of
sulfonamide bond can be ionized to proton in the solution. The sulfonamide group will be
negatively charged at neutral pH and loses its charge to bring about the pH-sensitivity in
faintly acidic conditions.83-85 Moreover, the change of pH value basically didn’t affect the
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fluorescence emission and the O generation, which suggested the effectiveness of the
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quenching strategy (Figure S15).
In addition, GSH containing thiol groups as the trigger can be further used to study the
stimulus-responsive behaviour of PEG-TPP-DNB micelles. After GSH was added in the
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micellar solution for 1 h, the average micellar size obviously increased from 129 nm to 196
nm with an appropriate increase of PDI from 0.072 to 0.117 (Figure 1B). Moreover, when
treated by GSH for 24 h, the average micellar diameter reached 446 nm with PDI of 0.237
accompanied by the emergence of large scattering peak. These variations were attributed to
the fact that GSH could selectively attack to the 2, 4-dinitrophenylsulfonamide to produce a
collapsible protonated intermediate, give rise to the cleavage of sulfonamide bond and
ultimately induce the detachment of DNB moiety from the porphyrin ring and the destruction
of assemblies via nucleophilic aromatic substitution.86-88 On the contrary, PEG-TPP-DNB
micelles were extremely steady without GSH and the average size remained almost invariant.
Meanwhile, the TEM image of micelles after treated with GSH (Figure 1D) displayed that
the micelles were ruptured and this result was consistent to that of DLS.
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Figure 1. (A) The GPC curves of (a) PEG, (b) PEG-TPP-DNB and (c) PEG-TPP-DNB
treated with GSH; (B) The DLS curves of PEG-TPP-DNB micelles in PBS (pH = 7.4) treated
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with 10 mM of GSH for 1 h or 24 h and without GSH for 0 h or 72 h; TEM images of
PEG-TPP-DNB micelles treated without GSH (C) and with 10 mM GSH for 24 h (D).
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GSH-triggered fluorescence activation and singlet oxygen generation
Redox-activated fluorescent property of PEG-TPP-DNB was further investigated using
fluorescence spectroscopy. Subsequently, with the different concentrations of GSH (0 mM, 1
mM, 5 mM and 10 mM) added into PEG-TPP-DNB micelles (5 μM) respectively, the
fluorescence of PEG-TPP-DNB could be detected at different time intervals. As shown in
Figure 2A and S16-19, the fluorescence intensity of PEG-TPP-DNB micelles treated with
different concentrations of GSH gradually increases with time, and it illustrates that the faster
rate of fluorescence intensity recovery was accelerated with the increase amount of GSH
which could cleave the sulfonamide bond more quickly. More importantly, compared with
little fluorescence change of no GSH treatment, the fluorescence intensity of porphyrin units
treated with 10 mM GSH could completely restored within two hours. In order to further
verify the dominant quenching effect of quenching agent, GSH-triggered fluorescence
recovery of PEG-TPP-DNB was tested at low concentrations (0.6 μM). And we obtained the
similar results of the fluorescence recovery (Figure S20). Thus, these results indicated that
the structure of PEG-TPP-DNB is the primary factor of fluorescence quenching, and the
fluorescence performance of PEG-TPP-DNB is provided with a strong stimulated response.
PEG-TPP-DNB can be destroyed by thiols so that the fluorescence intensity can be restored
rather than the original quenching state.
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Afterwards, the singlet oxygen generation of PEG-TPP-DNB was also measured by
UV-Vis spectroscopy by using 1, 3-diphenylisobenzofuran (DPBF) as the detection agent,
which can capture singlet oxygen to reduce its own absorbance. The absorbance of the
mixture of DPBF and PEG-TPP-DNB micelles with different concentrations of GSH (0 mM,
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mM, 5 mM and 10 mM) was monitored by a UV-Vis spectrophotometer after the
irradiation with the wavelength of 650 nm every 20 s (Figure S21-24). According to eqn (1),
the yield of singlet oxygen was calculated as 0.08 without GSH, and the yields were clearly
increased to 0.21, 0.48 and 0.61 with GSH of 1 mM, 5 mM and 10 mM, respectively, which
indicated that the singlet oxygen generation ability of PEG-TPP-DNB scarcely existed
without GSH but regenerated by means of the addition of GSH. It can be observed from
Figure 2B that singlet oxygen yields of the PEG-TPP-DNB after the activation of GSH are
higher than that without GSH, and the singlet oxygen yields are promoted with increasing
amounts of GSH. These results of fluorescence and singlet oxygen quantum yields
demonstrated that PEG-TPP-DNB micelles with a susceptive thiol-stimulus response could
be reactivated for the ability of singlet oxygen generation in the presence of GSH.
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Figure 2. (A) The changes of fluorescence intensity of PEG-TPP-DNB micelles (5 μM) in
PBS (pH = 7.4) at different time intervals with 0 mM GSH, 1 mM GSH, 5 mM GSH and 10
mM GSH, respectively. (B) The absorbance changes of DPBF versus time after irradiation at
different concentrations of GSH: 0 mM, 1 mM, 5 mM and 10 mM.
Cellular uptake of PEG-TPP-DNB micelles
For demonstrating the cellular uptake of PEG-TPP-DNB micelles by cancer cells, flow
cytometry and confocal laser scanning microscopy (CLSM) were employed to visualize the
efficient cellular internalization and activation of PEG-TPP-DNB micelles. As illustrated in
Figure 3A, B, the results of flow cytometric analysis quantitatively embodied that regardless
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of the duration of cell incubation, the fluorescence intensity of cells treated with
PEG-TPP-DNB micelles was always greatly stronger than free porphyrin. And the mean
fluorescence intensity value of cells incubated with PEG-TPP-DNB micelles for only 4 h was
also about 1.5-fold higher than that of free porphyrin cultured for 24 hours. All these results
indicated that PEG-TPP-DNB micelles could be effectively internalized and activated by
cells.
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Control
Free porphyrin 4 h
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Figure 3. (A) Flow cytometric analysis of free porphyrin and PEG-TPP-DNB micelles
incubated with 4T1 cells for 4 h and 24 h, respectively. (B) Quantitative results of flow
cytometry shown as mean fluorescence intensity.
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Subsequently, the cellular internalization of free porphyrin and PEG-TPP-DNB micelles in
T1 cells was evaluated by CLSM which can directly visualize the fluorescence of porphyrin.
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Apparently, the image of cells incubated with free porphyrin for 4 h could only be found
fairly weak fluorescence (Figure 4A), but the fluorescence intensity of cells treated by the
micelles was obviously stronger than that of the free porphyrin. Moreover, the similar result
was obtained after 24 h incubation which meant that more PEG-TPP-DNB micelles could be
endocytosed effectively with the passage of time and more readily activated the
internalization than free porphyrin by tumor cells. And it could also be perceived that when
glutathione monoester (GSH·OEt) as the additive of intracellular thiols pre-cultured with
cells, the porphyrinic fluorescence intensity delivered by PEG-TPP-DNB micelles in the
cytoplasm exhibited a further enhancement compared with that only treated with
PEG-TPP-DNB micelles. These results indicated that PEG-TPP-DNB micelles can be
effectively enriched in the cytoplasm and the intracellular GSH acted as a trigger for the
activation of PEG-TPP-DNB caused the intensive cytoplasmic release and strong
fluorescence emission.
Intracellular ROS production of PEG-TPP-DNB micelles
The efficiency of ROS generation as an essential prerequisite has great significance for
PDT. Thus, the capacity of intracellular ROS generation by PEG-TPP-DNB in cancer cells
was measured with 2, 7-dichlorofluorescein diacetate (DCFH-DA) as the intracellular ROS
detector by CLSM. As soon as the presence of ROS, the green fluorescence intensity from
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this oxidized ROS indicator (DCF) is directly proportional to the level of ROS in cells. As
shown in Figure 4B, the cells of blank control group under laser irradiation revealed almost
no green fluorescence and faint green fluorescence could be observed in free porphyrin
treated cells. In contrast, after the cells treated with PEG-TPP-DNB were exposed to laser
irradiation, awfully intense green fluorescence extended all over the cells. These results
suggested that there was little ROS detected in control cells and a small amount of ROS was
generated from free porphyrin with light irradiation. Furthermore, on account of the poor
biocompatibility of free porphyrin for extremely low levels of cell internalization,
PEG-TPP-DNB micelles in cancer cells could generate much more intracellular ROS than
free porphyrin upon laser irradiation due to the activation of the overexpressed GSH. It
reasonably showed that the activatable PEG-TPP-DNB micelles could retrieve its ability to
produce ROS accompanying with the fluorescence recovery in cancer cells.
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