NIR aza-pentamethine dyes as photosensitizers for photodynamic therapy

Journal Pre-proof NIR aza-pentamethine dyes as photosensitizers for photodynamic therapy Haiqiao Huang, Daipeng Huang, Mingle Li, Qichao Yao, Ruisong Tian, Saran Long, Jiangli Fan, Xiaojun Peng PII:

S0143-7208(19)32807-4

DOI:

https://doi.org/10.1016/j.dyepig.2020.108284

Reference:

DYPI 108284

To appear in:

Dyes and Pigments

Received Date: 3 December 2019 Revised Date:

21 January 2020

Accepted Date: 14 February 2020

Please cite this article as: Huang H, Huang D, Li M, Yao Q, Tian R, Long S, Fan J, Peng X, NIR azapentamethine dyes as photosensitizers for photodynamic therapy, Dyes and Pigments (2020), doi: https://doi.org/10.1016/j.dyepig.2020.108284. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier Ltd.

This work was finished through contributions of all authors.

Haiqiao Huang: The design and synthesis of aza-pentamethine dyes used in this work,

most of tests in vitro, Data curation, Writing- Original draft preparation, Writing - Review & Editing. Daipeng Huang: Data curation, Some tests in vitro, Formal analysis. Mingle Li: Methodology, Formal analysis. Qichao Yao: Data Curation. Ruisong Tian: Visualization. Saran Long: Resources. Jiangli Fan: Visualization, Supervision. Xiaojun Peng: Project administration, Funding acquisition, Writing - Review & Editing.

All authors have given approval to the final version of this manuscript.

We firstly used aza-indole to increase the absorption wavelength of pentamethine dyes and used it for photodynamic therapy at 730 nm.

• ARTICLES •

NIR aza-pentamethine dyes as photosensitizers for photodynamic therapy Haiqiao Huang a, Daipeng Huang a, Mingle Li a, Qichao Yao a, Ruisong Tian a, Saran Long a, b, Jiangli Fan a, b and Xiaojun Peng a, b * a State Key Laboratory of Fine Chemicals, Dalian university of Technology, 2 Linggong Road, Dalian 116024, P. R. China. b Research Institute of Dalian University of Technology in Shenzhen, Shenzhen 518057, China.

Photodynamic therapy (PDT) as an appealing modality has been used to treat various malignant tumors. Compared with conventional PDT treatment activated by ultraviolet or visible light, near infrared (NIR) light triggered PDT possessing deeper penetration to lesion area and lower photodamage to normal tissue holds great potential for deep-seated tumor. To address these obstacles for PDT treatment, we firstly used aza-indole to modify pentamethine dyes to facilitate a large bathochromatic shift of the absorption maximum from 650 nm to 730 nm in the photo-therapeutic window. Interesting, BY-Br have higher molar extinction coefficient than BY-H, allowing BY-Br to absorb more light for enhance its therapeutic effect. Meanwhile, the introduction of bromine atoms has been enhanced reactive oxygen species generation (ROS) after laser irradiation. Furthermore, the new photosensitizer, BY-Br could specifically target to mitochondria, and substantial kill tumor cells through apoptotic pathway upon irradiation.

Keyword: aza-pentamethine, NIR, mitochondria, photodynamic therapy

1 Introduction

intersystem crossing (ISC)-mediated singlet-to-triplet

As a specific spatiotemporal selective and minimal non-invasive

therapeutic

method

for

tumours,

photodynamic therapy (PDT) induced by the external laser irradiations has attracted tremendous research interest

[1-5].

PDT

employs

a

light-excited

photosensitizer (PS) generate the triplet PS then via

transfer its excited triplet state energy to the surrounding molecular oxygen to form cytotoxic singlet oxygen (1O2) and other reactive oxygen species (ROS) [6,7]. The attractive feature of PDT is that photodynamic reactions just happen in the immediate locale of light-absorbing PS. However, most of the available PSs clinically approved for PDT were only active under UV-visible light (<700

*Corresponding authors. State Key Laboratory of Fine Chemicals, Dalian university of Technology, Dalian 116024, China. E-mail addresses: [email protected]

nm) with low tissue penetration. So far, a range of

heptamethine cyanines have been synthesized and used

Scheme 1 illustrates the structure of conventional

for PDT by NIR light [8-10]. Most of synthetic PSs are

pentamethine

hydrophobic in nature with poor solubility in water and

aza-pentamethine dyes BY-Br and BY-H. We firstly used

the structural instability due to the long of methine chain

aza-indole to modify pentamethine dyes. Having

[11].

introduced

nitrogen

atoms,

applications [12-15]. Herein, a near-infrared (NIR)

wavelength

of

modified

excited PS for deeper penetration against deep-seated

increased from 650 nm to 730nm compared of traditional

tumours is still urgently required.

pentamethine dyes. Bromine atom has been introduced at

This

disadvantage

impedes

their

further

dyes

the

CY-H

and

the

maximum

new

NIR

absorption

penthamethine

dyes

Mitochondria are essential cellular organelles and

5-position of the aza-indole to increase the ability of

play crucial roles in energy supply and cell apoptosis

intersystem crossing through the heavy atom effect to

[16,17]. Moreover, mitochondria are reported to be

produce more ROS, thereby improving the therapeutic

susceptible to excessive ROS. When mitochondria were

effect on the cancer [22]. More importantly, the

photo-damaged,

their

introduction of the bromine atom also increases its molar

mitochondrial membrane potential and initiate apoptosis

extinction coefficient, allowing PS to absorb more light

[18-20]. Recently, we reported a mitochondrial targeting

to enhance its therapeutic effect. In addition, the cationic

bromo-pentamethine dyes anticancer photosensitizers

of PS itself give it not only have better solubility but also

that induced cancer cells apoptosis under light [21].

locate in mitochondria. Furthermore, aza-pentamethine

However, this symmetric pentamethines were also active

were utilized for the evaluation of the photodynamic

under the UV-visible light (<700 nm), limiting tumour

therapeutic effect in vitro.

they

immediately

lose

deeper penetration and making biomedical applications more difficult. Therefore, a great deal of research work has focussed on developing new PSs with absorption maximum

in

the

phototherapeutic

window,

and

mitochondria targeted in an attempt to broaden the applicability of PDT. In this work, we have designed brominated aza-pentamethine with near-infrared absorption and combined mitochondria-targeting for enhanced PDT. 2

orded on a Bruker Avance II 400 MHz and 500 MHz spectrometer. Chemical shifts (δ) were reported as ppm in MeOD with TMS as the internal standard. Bovine Serum Albumin (BSA) was purchased from Shanghai Sangon Biotech Co., Ltd. Water used in all experiments was doubly purified by Milli-Q Academic A10 Ultrapure water system equipment. The preparation procedures of BY-Br and BY-H and related intermediates are given in the Supporting Information (Scheme S1). The solutions of BY-Br Scheme 1 Chemical structures of the conventional pentamethine dyes and the aza-pentamethine dyes

and BY-H were typically prepared from 5.0 mM stock solutions in DMSO.

2

Materials and methods

2.1 Material and sample preparation All solvents and reagents used were reagent grade. All reactions were performed in a nitrogen atmos-

2.2 Measurements of absorption and fluorescence Absorption spectra were measured on a Perkin Elmer Lambda 35 UV/VIS spectrophotometer (Perkin Elmer). Fluorescence spectra were obtained with

phere with dry, freshly distilled solvents under an-

a VAEIAN CARY Eclipse fluorescence spectropho-

hydrous conditions. Silica gel (100-200 mesh) which

tometer (Serial No. FL0812:M018). The fluores-

obtained from Qingdao Ocean Chemicals was used

cence quantum yield (Φf) was measured with A

for flash column chromatography for purifications.

Quantaurus-QY spectrometer (Hamamatsu, C11347)

Reversed-phase preparative HPLC was performed

[23].

using a CHEETAH medium pressure rapid purifica-

2.3 Fluorescence Lifetime Measurements

tion preparation system utilizing a Claricep Flash

Time resolved fluorescence measurements were

C18 column (100 Å, 20-35 μm, 40 g) obtained from

performed on freshly prepared samples using the

Tianjin Bonna-Agela Technology Co., Ltd. (Tianjin,

time-correlated single photon counting (TCSPC)

China). Mass spectrometric data were obtained using

method (PicoQuant PicoHarp 300) at room tempera-

HP1100LC/MSD MS and an LC/Q-TOF-MS in-

ture. Using deconvolution/fit program (PicoQuant

struments. 1H NMR and

FluFit), the time resolution was reached down to 10

3

13

C NMR spectra were rec-

carried

out

by

trapping

1

ps. The second harmonic of a titanium sapphire laser

was

O2

with

(Mai Tai DeepSee) at 400 nm (150 fs, 80 MHz) was

1,3-diphenylisobenzofuran (DPBF) [26]. In fact

selected as excitation source. Emission was moni-

DPBF reacts rapidly with 1O2 forming the colorless

tored at the wavelength of maximum fluorescence.

o-dibenzoylbenzene derivative, resulting from the

Data analysis was performed with FluoFit software

disappearance of DPBF’s characteristic absorption

(Picoquant) using an exponential decay model as

band at 415 nm. Thus, a solution of DPBF (45 μM)

described in the Supporting Information.

and Aza-pentamethine dyes (optical density was ad-

2.4 Determination of octanol-water partition coeffi-

justed to around 0.2-0.3 at the irradiation wave-

cient (log Po/w)

lengths) in 3 mL Dichloromethane (DCM) was irra-

The 1-octanol/water partition coefficient (log Po/w)

diated monochromatic light (700 nm or 730 nm) in-

was determined according to a reported procedure

tervals. The power density of the laser point is 10

[24,25]. Briefly, Solution A and Solution B were

mW·cm-2. The solutions were irradiated for 1.75 or 3

prepared by premixing 1-octanol and PBS buffer (10

minutes and their absorbance spectra were recorded

mM, pH 7.4) in a ratio of 1:4 and 4:1, respectively.

at 15 or 30 second intervals using a spectrometer.

Then, the solutions were shaking gently at 220 rpm,

Measurement singlet oxygen quantum yield (ΦΔ) [27]

at RT for 24 h. Thereafter, small stock solutions (5

The decrease in quencher absorption at 415 nm, as

μL) of a 5 mM probe solution in DMSO was added

a function of the pentamethine irradiation time, has

to Solution A and Solution B (each 5 mL), respec-

been compared to the values obtained from meth-

tively. The above solutions were then mixed and

ylene blue (MB), an efficient and well-known singlet

stirred for 30 min. The concentration of the probe in

oxygen generator. The quantum yields were calcu-

each layer was measured by UV-Vis spectroscopy

lated by using methylene blue in dichloromethane

using the molar coefficients of the probes. The log

with ΦΔ = 0.57 the standard reference [28]. The ab-

Po/w value was calculated using the following equa-

sorbance

tion:

3-diphenylisobenzofuran (DPBF) was adjusted to

log Po/w=log C[probe]oct – log C[probe]PBS

(1)

2.5 Detection of singlet oxygen in vitro

of

the

1

O2

scavenger,

around 1.0 in air-saturated DCM. Then the photosensitizer was added and its absorbance was adjusted

A qualitative and comparative study of the abili-

to around 0.2-0.3. Then the cuvette was exposed to

ties of aza-pentamethine to generate singlet oxygen

monochromatic light (700 nm or 730 nm) for 3.5

4

minutes and their absorbance spectra were recorded

before light irradiation. As control, DHR123 aqueous

at 30 seconds intervals. The slope of the absorbance

solution without photosensitizers was subjected to

maxima of DPBF at 415 nm versus time graph was

irradiation.

calculated for each photosensitizer. The singlet oxy-

Hydroxyl radical (OH•) detection:

gen quantum yield (ΦΔ) is calculated using the fol-

For OH• evaluation, both PS and HPF were prepared

lowing equation (2) [25]:

as 5 μM in aqueous solution, and others procedure

Φ  ΦMB   KPS  FMB  PFMB  /  KMB  FPS  PFPS 

(2)

Where K is the slope of the difference in the change

were consistent with that of O2•− measurement. 2.7 Intracellular PDT analysis by MTT

in the absorbance of DPBF (415 nm) with irradiation

The phototoxicity and dark toxicity of BY-Br and

time, and F is the absorption correction factor, which

BY-H were examined with or without light irradia-

is given by F=1-10-OD (OD at the irradiation wave-

tion at 700 nm and 730 nm respectively using MTT

length). PF is an absorbed photonic flux (μ Einstein

assay. MCF-7 cells were plated in 96-well plate with

dm-3 s-1). ΦMB is the singlet oxygen quantum yield of

5×104 cells/well, and incubated in 100 μL growth

MB.

medium under a humidified 5% CO 2 atmosphere at

2.6 Detection Production of Superoxide Radical (O2•−)

37°C for 24 h. Thereafter, cells were exposed to

and Hydroxyl radical (OH•) in Solution.

various concentrations of BY-H (0-5 μM), BY-Br

Superoxide Radical (O2•−) Detection:

(0-5 μM) separately, incubated for 2 h, then irradia-

For normoxia superoxide radical measurements, di-

tion with 700 nm (width of half wave 19 nm) or 730

hydrorhodamine123 (DHR123) was used as the spe-

nm (width of half wave 15 nm) NIR light at 20

cific indicator, which can be converted to Rhoda-

mW·cm-2 for 0, 5 min. After overnight incubation,

mine123 in the presence of O2•− and emit strong

the standard MTT assay was carried out to determine

green fluorescence at 526 nm. PS (5 μM) and

the cell viability. The cells were labelled with 0.5

DHR123 (5 μM) were prepared as in water. Then the

mg·mL-1 solution of MTT in full culture medium for

tube was exposed to 700 nm laser (20 mW cm-2) for

4 h, then the supernatant was removed and replaced

different time (0, 1, 2, 3, 4, 5 min), and the emission

with 200 μL of DMSO and the formazan absorbance

spectra were observed immediately after each irradi-

was analysed by a microplate reader at 490 nm.

ation (Ex: 500 nm). For O2•− quenching experiment, 50 μM Vc was added to the above aqueous solution 5

2.8 Cell imaging and localization [29]

ROS generation was then carried out. Briefly, the

The cellular localization of a photosensitizer

culture medium of cells that was exposed to the re-

within tumour cells is a critical determinant of treat-

spective BY-Br (1 μM) was incubated for 2 h, and

ment efficacy. Confocal laser scanning microscopy

then DCFH-DA (10 μM) was added incubated for 10

(CLSM) and organelle-specific probes make it con-

min ensure DCFH-DA that sufficiently entered the

venient and precise to study the cellular localization

cells. The cells were then subjected to photosensiti-

of PS [30]. MCF-7 Cells were cultured in Dulbecco's

zation by using 730 nm NIR lamp light irradiation

modified Eagle's medium (DMEM, Invitrogen) sup-

for 0 or 5 min. Fluorescent images of DCFH, stain-

plemented with 10% fetal bovine serum (Invitrogen).

ing on the cells were promptly captured by excitation

Cells were seeded in 24-well flat-bottomed plates

at

and then incubated for 24 h at 37°C under 5% CO 2.

(OLYMPUS, FV3000).

Before imaging, the cells were incubated with

2.10 Confocal imaging of photo-induced cell death

BY-Br or BY-H for 2 h and Mito-Tracker Green FM

and Flow cytometric analysis [3,31]

488

nm

using

a

fluorescence

microscope

for 30 min, then stained with Hoechst 33342 (for 10

Annexin V-FITC/propidium iodide (PI) Apoptosis

min), Mito-Tracker Green FM to stain mitochondria

Detection Kit (Beyotime, China) was used for detec-

and Hoechst 33342 to stain the cell nucleus. Then

tion of BY-Br mediated photo-induced cell death.

washed with phosphate-buffered saline (PBS) three

Briefly, MCF-7 cells were seeded onto 35 mm con-

times. Fluorescence imaging was performed using an

focal dishes for 24 h, then cells were treated with

OLYMPUSFV-3000 inverted fluorescence micro-

following different treatments: group 1, irradiated

scope with a 60×oil objective lens.

with 730 nm xenon lamp light for 5 min at a power

2.9 ROS generation during PDT treatment

density of 20 mW cm-2 (Light); group 2, incubated

The abilities of BY-Br to generate ROS within

with BY-Br (1 μM) at 37°C for 2 h (BY-Br +

MCF-7 cells were investigated by a singlet oxygen

nolight); group 3, incubated with BY-Br (1 μM) at

capture agent 2, 7- dichlorofluorescein diacetate

37°C for 2 h and irradiated with 730 nm xenon lamp

(DCFH-DA) using CLSM. MCF-7 cells were

light for 5 min at a power density of 20 mW cm -2

pre-treated with BY-Br and incubated with the cel-

(BY-Br + light); group 4, incubated with 10 mM

lular ROS indicator 2, 7-dichlorofluorescein diace-

Nacetyl-L-cysteine (NAC) at 37°C for 0.5 h, then

tate

incubated with BY-Br (1 μM) at 37°C for another 2

6

(DCFH-DA).

Irradiation-dependent

cellular

h and irradiated with 730 nm xenon lamp light for 5

λem = 671 nm in DCM) respectively (Fig. 1a b). Sig-

min at a power density of 20 mW cm-2 (NAC +

nificantly, after the conventional indole is replaced

BY-Br + light). After treatments, cells were stained

by aza-indole, the maximum absorption wavelength

with Annexin V-FITC/propidium iodide (PI) Apop-

of the dye molecules is red-shifted. BY-Br and

tosis Detection Kit according to the agent instruction

BY-H display an intense absorption profile that lo-

(KeyGEN, China). The cell death process was visu-

calizes in the therapeutic window, promising deeper

alized by fluorescence microscopy and flow cytome-

permeability against deep-seated tumors. Detail op-

try. (AnnexinV-FITC, λex = 488 nm, λem = 500–550

tical properties of two dyes in different solvents

nm; PI, λex = 488 nm, λem = 600–680 nm).

shown in Table S1, Fig. S15. Although their wave-

3

length peaks are similar to each other, the molar ex-

Results and discussion

3.1 Spectral Properties of BY-Br and BY-H

tinction coefficient of them are different. As shown

The two aza-pentamethine dyes (BY-Br and

in Table S1, the extinction coefficients of the two

BY-H) have different substitutents (H and Br re-

dyes BY-Br and BY-H at the maximum absorption

spectively) at the 5-position of the aza-indole. As

wavelength are 2.79×10 5 L mol-1 cm-1 and 1.38×105

shown in Fig. 1, two dyes (BY-Br and BY-H) dis-

L mol-1 cm-1, respectively. We know that BY-Br

play a large bathochromatic shift of absorption

have higher molar extinction coefficient than BY-H,

maximum: BY-Br (λabs = 736 nm and λem = 756 nm

meanwhile, the fluorescence quantum yields repre-

in DCM), BY-H (λabs = 716 nm and λem = 736 nm in

sent opposite properties (Φf =0.501 for BY-Br and

DCM), and conventional CY-H (λabs = 651nm and

Φf =0.571 for BY-H in DCM).

Fig. 1 UV−vis−NIR absorption (a) and emission (b) spectra of Cy-H, BY-H and BY-Br in DCM with 1 μM, c) Crystal structures of BY-Br. All H atoms are omitted for clarity atomic scheme, Br: dark yellow, C: gray, I: red, N: blue.

7

Additionally, the fluorescence lifetime of the two

730 nm Xe lamp leads to a comparable 1O2 generation.

compounds has been also measured, which is shown

As shown in Fig. 2, the absorbance of DPBF degraded

in Fig. S16. The fluorescence lifetime of BY-Br is

gradually under the irradiation. However, when they

also shorter than that of BY-H, further demonstrated

were exposed to light, the decrease of DPBF absorbance

that the introduction of bromine shortens the lifetime

at 415 nm caused by the same PS was similar. The sin-

of the first excited singlet (S1) of dyes. This is due to

glet oxygen quantum yield (ΦΔ in Table 1) of BY-Br and

the introduction of bromine atoms to enhance the

BY-H irradiation with 730 nm Xe lamp is higher than

Intersystem Crossing (ISC) process [32]. Further-

irradiation with 700 nm Xe lamp, indicated that BY-Br

more, the single-crystal X-ray analysis of BY-Br

and BY-H have better utilization of light at 730 nm. This

fully confirmed that the carbon skeleton of the entire

also has a consistent reflection on the absorption coeffi-

molecule is on a plane except for the N-substituted

cient [33]. It has been predicted that higher the absorp-

ethyl group (Fig. 1c). It is cleared that the introduc-

tion coefficient, such as BY-Br, possesses more effec-

tion of N atoms extends delocalized system of

tively photon utility due to more photon trap, accordingly

BY-Br. Moreover, in order to ensure that the con-

more 1O2 generated. As known that an important charac-

centration used dyes is not aggregated, we measured

teristic for a compound to be considered a potent PS is to

the solubility with different concentrations. The re-

have an intense absorption within the “phototherapeutic

sults show BY-Br and BY-H have excellent solubil-

window”. Inspired by this result, we have verified the

ity in aqueous solutions (5.0 × 10 −6 mol L-1 for

effect of its photodynamic properties at the cellular level.

BY-Br and 10 × 10−6 mol L-1 for BY-H, Fig. S17).

Table 1 The photophysical characters of BY-Br and BY-H in

3.2 Generation of singlet oxygen

DCM PS

λabs(nm)a

λem(nm)b

Φ fc

ΦΔ-700d

ΦΔ-730e

μ af

BY-Br

736

756

0.501

0.027

0.031

0.94

ating singlet oxygen upon NIR excitation, DPBF as a O2

BY-H

716

736

0.571

0.014

0.017

0.43

detector was utilized. The measurement mechanism of

The all data of the solvents were measured at 25°C. a The

DPBF for 1O2 was illustrated in Fig. S18. The degrada-

max absorption peaks of dyes (nm). b The max fluorescence

To determine the ability of BY-Br and BY-H for gener1

tion of DPBF the absorption signal at 415 nm is indica-

peaks of dyes (nm). c The fluorescence quantum yield. d The singlet oxygen quantum yield upon the presence of 700 nm

tive of the amount of singlet oxygen generated. As indiirradiation. e The singlet oxygen quantum yield upon the

cated by the DPBF decolorization curves, irradiation on solution of BY-Br and BY-H in DCM with 700 nm or 8

presence of 730 nm irradiation. f The absorption coefficient upon the presence of 730 nm irradiation.

Fig. 2 Change in the absorbance spectrum of the trap molecule DPBF in the presence of BY-Br and BY-H in DCM.

3.3 Generation of other ROS

DHR123 signal was indeed caused by generated O2•−, as

To distinguish different ROS productions by BY-Br

expected, an increase in DHR 123 fluorescence at 526

and BY-H, we used dihydrorhodamine123 (DHR123) for

nm was inhibited after the addition of Vc. These results

superoxide radical detection and hydroxyphenyl fluores-

fully validated that the halogen increase the Intersystem

cein (HPF) for hydroxyl radical detection. Firstly, we

Crossing (ISC) process. On the other hand, no •OH was

confirmed the O2•− production by O2•− probe DHR123,

observed when we employed hydroxyphenyl fluorescein

which is nonfluorescent but can react with O2•− to emit

(HPF) as the specific indicators for •OH (Fig. S20 b).

strong green fluorescence centered at 526 nm. BY-Br

This result indicated that BY-Br and BY-H could be

substantially increased the fluorescence intensity of

generating O2•−. Several research groups have suggested

DHR123 under normoxia upon 700 nm Xe lamp irradia

that the Type II reaction dominates, while Type I occurs

tion (Fig. S20 a). Remarkably, BY-Br also led to a faster

only when the PS is highly concentrated in the tumour or

O2•− generation rate in comparison with BY-H. Moreo-

if the tumour is hypoxic in nature [34]. Taken together,

ver, vitamin C (Vc), a radical scavenger, was added into

our studies of ROS generated by BY-Br and BY-H indi-

PS solutions to further validate that the enhanced

cate that BY-Br exhibits relatively high oxygen sensitiv-

9

ity, resulting in its relatively high ΦΔ and generation of

with experimental data of ROS generation in solu-

O2•−, with subsequent photodynamic cytotoxicity.

tion. Hence, light 730 nm Xe lamp was chosen as a

3.4 In vitro photodynamic activity

suitable illumination source for subsequent testing.

A successful PDT photosensitizer is one that exhibits low cytotoxicity in dark and potent cytotoxicity in the presence of photo-irradiation. The cytotoxicity of aza-cyanine PS was evaluated under both light and dark conditions. The dark and light cytotoxicity of BY-Br and BY-H in MCF-7 cells were examined by using the MTT assay. Both compounds displayed negligible toxicity in the absence of light, suggesting their acceptable biocompatibility. The

Fig. 3 Comparison of the cytotoxic effects of BY-Br (blue bar) and BY-H (rad bar) on MCF-7 cells in the absence and

light cytotoxicity was tested under the irradiation by

presence of light (λ =730 nm or 700 nm, 20 mW·cm −2).

two different wavelength light sources (700 nm or 730 nm) with the dose of 20 mW cm -2. The results showed that BY-Br exhibited more potent activities than BY-H under two different wavelength irradiations. Meanwhile, compared with 700 nm irradiation, upon the presence of 730 nm irradiation, cell viability was rapidly decreased with the increase of dosage of PS. As can be observed from Fig. 3, 56% of cell viability remained in the presence of 2.5 μM of BY-H, and 2.5 μM of BY-Br caused almost complete cell death with only 9% of cell viability remained. It is suggested that BY-Br is considerably powerful for cancer cell ablation through PDT pathway, and therapeutic output is better on 730 nm irradiation than on 700 nm, which is in good accordance 10

3.5 Cell imaging and intracellular localization The co-localization of BY-Br and BY-H in mitochondria of MCF-7 cells with commercially available Mito-Tracker Green FM (green) and Hoechst 33342 (blue) were investigated. As shown in Fig. 4, BY-Br exhibited high level of co-localization with that of Mito-Tracker Green FM, and the Pearson’s correlation coefficients was 0.918. In contrast, for BY-H comparatively a fewer overlap with Mito-Tracker Green FM, and the Pearson’s correlation coefficients was 0.856, which confirms less selectivity to mitochondria. On the other hand, both compound exhibited no overlap with the signals from

Hoechst 33342, indicate that aza-cyanine were not

diacetate (DCFH-DA) probe was selected to detect

located in the nucleus. The lipophilicity of BY-Br

the singlet oxygen generation under the irradiation of

and BY-H were determined by octanol/PBS partition

730 nm. DCFH-DA was non-fluorescence status

coefficient measurements using Poctanol/PBS = [C]octanol

could be transformed from into fluorescent 2,

layer / [C]PBS layer. BY-Br with two bromine was

7-dichlorofluorescein (DCF) in the presence of ROS.

found more lipophilic (P = 365, log P = +2.56) than

In the Fig. 5, DCFH-DA alone was almost non emis-

BY-H (P = 265, log P = +2.42). It is well known that

sive whether irradiated. In contrast, the cell incubat-

mitochondria targeting were numerically assigned in

ed with DCFH-DA and BY-Br in dark exhibits weak

accordance with the following criteria: electric

green fluorescence, while its emission intensity was

charge Z (cation number) > 0 and 0 < logP < +5.

triggered and rapidly raised after irradiation 5 min,

BY-Br and BY-H fully meet the criteria for mito-

illustrating that the high-efficiency ROS generation

chondrial accumulation. This result indicated that

of BY-Br.

such cationic aza-cyanine agents’ BY-Br are superior to target cell mitochondria.

Fig. 4 Co-localization of PS (1 μM) with a Mito-Tracker Green FM (200 nM) and Hoechst 33342 (2 μg/mL) in MCF-7

Fig. 5 Morphology changes and cellular ROS generation

cells imaged using a confocal microscope. Scale bars: 20 μm.

within MCF-7cells incubated with DCFH-DA (No BY-Br) or DCFH-DA+ BY-Br (BY-Br) with diffirent deal.(before light)

3.6 Intracellular singlet oxygen generation Encouraged by the attractive properties of BY-Br

control cell incubated indark, (light) with irradation 5 min; Scale bars = 20 μm.

in both superior photo-toxicity and mitochondria-specific targeting for MCF-7 cells. BY-Br was used as PSs for PDT application. In evaluating the intracellular ROS levels, 2, 7-dichlorofluorescein

3.7 photo-induced cell death by CLSM and flow cytometry observation For further demonstrating the PDT effect of BY-Br, the annexin V-FITC/PI kit has been applied

11

in this work for investigating the cell apoptosis and

could be generated the destructive 1O2 for killing

death pathway (Fig. 6 and Fig. S21). After MCF-7

MCF-7 cells through apoptosis.

cells were incubated with 1 μM BY-Br for 2 h fol-

4.

Conclusions

lowed by stained with Annexin V-FITC/PI, the sig-

In summary, we have developed a simple proto-

nal of annexin FTIC/PI cannot be observed, similar

col to prepare a novel type of pentamethine dye

to those treated with light alone. However, intense

BY-Br and BY-H possessing near-infrared absorp-

green and red fluorescence were detected in the PDT

tion characteristics and potential therapeutic agent

group, indicating that most cancer cells underwent

applied for PDT. To the best of our knowledge, this

late-stage apoptosis. Moreover, Nacetyl-L-cysteine

work is the first report using aza-indole to decorate

(NAC) as a ROS cleaner significantly prevented the

pentamethine dyes to facilitate a large bathochro-

cell damage, which further indicated that the ROS

matic shift of the absorption maximum from 650 to

induced by BY-Br were indeed responsible for the

730 nm in the photo-therapeutic window. The optical

cell destruction [35,36]. The flow cytometry has also

property measurements indicated that higher ab-

been used to evaluate BY-Br-induced cell death in

sorbance coefficient of BY-Br enabled it to be more

MCF-7 cells. As shown in Fig. 6, the group of PDT,

effectively photon utility can improve the generation

the population of apoptotic cells obviously increased

of 1O2. Meanwhile, bromine atom at the 5-position of

compare with the group without irradiation( Fig. 6c)

the aza-indole can enhance ROS generation. In vitro

and the control group (Fig. 6a and 6b), while the

studies show BY-Br can localize in mitochondria,

population of apoptotic cell had significantly de-

and efficiently kill cancer cell by generation ROS in

creased

cells and may be a potential single-molecule based

in the group of NAC+PDT. Overall, this

result clearly indicated that BY-Br under irradiation

12

PS candidate for PDT.

Fig. 6 Flow cytometer analysis of MCF-7 cells treated in different conditions a-e) Cells were cultured with PBS only, light irradiation only, BY-Br without irradiation, BY-Br under irradiation, and NAC & BY-Br under irradiation respectively. f) the apoptosis rate of different Group

Conflicts of interest There are no conflicts to declare.

Acknowledgements This work was supported by the National Natural Science Foundation of China (project 21421005, 21576037, and U1608222).

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Declaration of interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Highlight 1.

This paper firstly used aza-indole to modify pentamethine dyes to facilitate a large bathochromatic shift of the absorption maximum from 650 nm to 730 nm in the photo-therapeutic window.

2.

Bromine atom has been introduced at 5-position of the aza-indole to increase the ability of inter-system crossing of BY-Br to produce more ROS.

3.

The introduction of the bromine atom also increases its molar extinction coefficient, allowing PS to absorb more light to enhance its therapeutic effect.

4.

BY-Br can localize in mitochondria, and efficiently kill cancer cell by generation ROS in cells.