A [5]Rotaxane-Based Photosensitizer for Photodynamic Therapy

A Journal of

Accepted Article

Title: 5]Rotaxane-Based Photosensitizer for Photodynamic Therapy

Authors: Melis Ozkan, Yagmur Keser, Seyed Ehsan Hadi, and Donus

Tuncel

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To be cited as: Eur. J. Org. Chem. 10.1002/ejoc.201900278

Link to VoR: http://dx.doi.org/10.1002/ejoc.201900278

Supported by

European Journal of Organic Chemistry

10.1002/ejoc.201900278

[

5]Rotaxane-Based Photosensitizer for Photodynamic Therapy

[

a]

[a]

[a, b]

Melis Özkan, Yağmur Keser, Seyed Ehsan Hadi, Dönüs Tuncel*

[

a]

M. Özkan, Y. Keser, S. E. Hadi, Prof. D. Tuncel

Institute of Materials Science and Nanotechnology, National Nanotechnology Research

Center (UNAM), Bilkent University, Ankara, 06800, Turkey.

[

b]

Prof. D. Tuncel, Department of Chemistry, Bilkent University, 06800 Ankara, Turkey.

*

dtuncel@fen.bilkent.edu.tr

http://www.fen.bilkent.edu.tr/~dtuncel/index.html

Abstract

Here, a [5]rotaxane was synthesized through a catalytically self-threading reaction in which

CB6 serves as a macrocycle and acts as a catalyst to catalyse the 1,3-dipolar cycloaddition

reaction between the alkyne substituted porphyrin core and azide functionalized stopper groups

by forming triazole. Application of this rotaxane as a photosensitizer in photodynamic therapies

against cancer cells and bacteria inactivation have also been demonstrated. This photosensitizer

has an excellent water solubility and remains stable in biological media at physiological pH

(

7.4) for prolonged time. It has ability to generate singlet oxygen efficiently; while it shows no

dark cytotoxicity up to 300 µM to the MCF7 cancer cell line, it is photo cytotoxic even at 2

µM and reduces the cell viability to around 70% when exposed to white light. It also displays

light-triggered biocidal activity both against gram-negative bacteria (Escherichia coli, E. coli)

and gram-positive bacteria (Bacillus subtilis). Upon white light irradiation for 1 min with flux

2

of 22 mW/cm of E. coli suspension incubated with [5]rotaxane (3.5 µM), the killing efficiency

of 96% is achieved, whereas in the dark the effect is recorded as only around 9%.

European Journal of Organic Chemistry

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Introduction

Rotaxanes are fascinating class of compounds with many interesting properties and potential

applications.¹^,²They are composed of an axle-like molecule which is threaded by a macrocycle

and terminated with bulky stopper groups that prevent a macrocycle to slip off from the axle.

These mechanically interlocked supramolecules can be synthesized using a number of different

macrocycles including crown ethers, cyclophanes, calixarenes, cyclodextrins and cucurbiturils

(

CBs).³^-⁵The degree of threading and the nature of the macrocycle can significantly affect the

physical and chemical properties of axle molecule.

Among those macrocycles, CB is highly attractive as it offers a very rich host-guest

chemistry due to its two identical hydrophilic portals and a hydrophobic cavity. It binds with

guest through ion-dipole interactions as well as hydrophobic effect.⁵^,⁶Acid catalyzed

condensation of glycoluril with formaldehyde forms CB-homologues in various sizes. The

number of glycol units are denoted by n and for most common CB homologues, n can be 5, 6,

6

7

, 8. Among them CB6 has very important feature that is its ability to catalyze 1,3-dipolar

cycloaddition between properly substituted alkyne and azide substrates by forming 1,4-

disubstituted triazoles. This feature was extensively explored in the synthesis of rotaxanes and

polyrotaxanes.⁵^-⁷

In this paper, we have revisited our earlier work which was on the synthesis of novel

7

CB6-based rotaxane via 1,3-dipolar cycloaddition catalysed by CB6. Resulting rotaxane is

composed of a tetraphenyl porphyrin core which is surrounded by four CB6s and it is called

8

[

5]Rotaxane. Tetraphenyl porphyrin has been selected because porphyrin derivatives are very

appealing as a photosensitizer due to their absorption in the visible range of the electromagnetic

spectrum, long-lived triplet excited state, high molar extinction coefficient and their ability to

generate singlet oxygen when they are irradiated with visible light.⁹

European Journal of Organic Chemistry

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The light, photosensitizer, and molecular oxygen are main components to generate reactive

oxygen species (ROS) including singlet oxygen that are mainly responsible for the

photodynamic therapies against cancer cells or bacteria inactivation.¹⁰^aPhotodynamic

antimicrobial chemotherapy (PACT) can be considered as a very efficient approach in killing

pathogens with resistance to antibiotics¹⁰as the antibiotic resistance of microorganisms has

1

become a serious global challenge. An ideal photosensitizer to be used in the photodynamic

therapy should have good water solubility, be stable in the biological milieu and should not

exhibit dark cytoxicity.¹⁰^,¹¹Although attaching hydrophilic and ionic groups to the meso-

positions of porphyrin core improve the water solubility, aggregate formation caused by π-π

interactions and hydrophobic effect is still an issue that should be tackled.¹⁰^,¹²However, ionic

groups attached to porphyrin derivatives as solubilizers can also cause dark toxicity. Recently,

supramolecules have been employed as a photosensitizer in PACT,¹³^-¹⁶especially, the inclusion

complexes of cationic porphyrin derivatives with CB homologues seem to be promising as they

show limited dark cytotoxicity.¹⁶For example, Zang et al. reported a highly efficient

supramolecular photosensitizer based on the inclusion complex of CB7 with cationic porphyrin

decorated by naphthalene guests.¹⁶^aThey have demonstrated that inclusion complex formation

significantly reduces the dark toxicity but the complex is highly toxic when irradiated with

light. However, to the best of our knowledge, rotaxane-based photosensitizers have not been

reported to date. Rotaxane-based photosensitizer is expected to be more stable in a variety

biological media than the pseudorotaxane-based photosensitizers. This is especially important

for in vivo applications because biological media contain a number of different competitive

guests for CBs that will cause a premature disassembly of pseudorotaxane before reaching and

accumulating in the target. This, in turn, can increase the dark cytotoxicity.

Our porphyrin-cored [5]rotaxane confers the requirements of an ideal photosensitizer

as it has an excellent solubility in water and highly stable in biological media (PBS, at pH 7.4,

European Journal of Organic Chemistry

10.1002/ejoc.201900278

DMEM). Also, due to the presence of bulky CB units which limits the interaction of porphyrin,

no aggregate formation was observed. Moreover, it does not show any dark toxicity although

it contains cationic ammonium ions which are disguised by CB units by forming complexes

with carbonyl groups.

Results and discussion

[

5]Rotaxane was synthesized as shown in Scheme 1 by similar to our previously reported

8

procedure. The precursor Porphyrin 1 (1 equiv.) was dissolved in water, CB6 (4.4 equiv.)

and tert-butyl azidoethylammonium chloride (4.4 equiv.) was added to it. The reaction mixture

was stirred at room temperature for 24 hours. A clear greenish solution was obtained which

was poured into dialysis tube and dialyzed overnight against water to remove excess CB6 and

unreacted monomers. The resulting solution was freeze dried to obtain burgundy coloured

1

13

powders in 87% yield. [5]Rotaxane was characterized by spectroscopic techniques ( H, C

NMR and FT-IR) to elucidate its structure and its molecular weight was determined by ESI-

mass spectrometry (Supporting Information, Figure S1-S4). [5]Rotaxane dissolves well in

water and PBS buffer (pH 7.4), remains stable over long time period.

O

H

N

2

H

N

2

N

2

N

H

N

H

N

2

N

2

4

H

N

6

0

N

H

N

2

H

2

O, 24 h, 40 C

N

O

NH

HN

_N₃+ cucurbit[6]uril

+

4

NH

8

7%

HN

(

CB6)

N

H

N

2

H

N

2

H

N

2

N N

H

N

2

Porphyrin 1

N

H

N

2

N

[5]Rotaxane

N

H

2

Scheme 1. Synthetic scheme for [5]rotaxane.

Once we authenticated the structure of the desired [5]rotaxane, we set out to investigate

its photophysical properties and its ability as a photosensitizer. Figure 1 shows the UV-vis

absorption and fluorescence spectra of [5]rotaxane at various concentration in water. A sharp

European Journal of Organic Chemistry

10.1002/ejoc.201900278

Soret band (λmax at 415 nm) and four weak Q-bands (λabs at 517, 553, 582, and 636 nm) in the

spectrum indicating the typical absorption peaks of free base porphyrins. It is clearly seen that

the presence of bulky CB6 units prevents the interactions between porphyrin units and as a

result, an aggregate formation is prevented and the photophysical properties of porphyrin core

is preserved in aqueous media. The excitation of [5]rotaxane in water at 415 nm resulted

fluorescence emission above 600 nm as characteristic of porphyrin with two vibrational bands

at 646 and 707 nm. (Figure 1). While the concentration of [5]rotaxane increases, its emission

intensity decreases as can be seen from Figure 1, probably due to self-quenching caused by

short-range interactions between the fluorophores. Fluorescence quantum yield and life time

are measured as 10% and 7.5 ns, respectively in water (5 µM).

Figure 1. UV-vis absorption and fluorescence spectra of [5]rotaxane in water at various

concentrations.

Light triggered reactive oxygen species (ROS) generation ability of [5]rotaxane can be

investigated by using a probe, 2,7-dichlorofluorescein diacetate (DCFH-DA).¹⁶^,¹⁷2,7-

dichlorofluorescin (DCFH) which can be obtained by a hydrolysis of DCFH-DA in an alkaline

medium is very sensitive to ROS and could quickly turn into highly fluorescent 2,7-

European Journal of Organic Chemistry

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dichlorofluorescein (DCF) (Scheme 2) leading a significant increase in the fluorescence

intensity around 524 nm.

O

HO

Cl

O

OH

Cl

HO

Cl

O

C

Hydrolysis

ROS

Cl

0

.01 N NaOH

COOH

in EtOH

DCFH-DA

DCF

DCFH

Scheme 2. Molecular structures of 2-7 Dichlorofluorescin diacetate (DCFH-DA), 2,7-

dichlorofluorescin (DCFH) and the reaction of DCFH with ROS to produce highly fluorescent

2

,7-dichlorofluorescein (DCF).

As shown in Figure 2a, ROS generation ability of [5]rotaxane was evaluated upon exposure to

2

white light with a light intensity of 1 mW/cm . When DCFH was irradiated in the absence of

[

5]rotaxane, very weak emission band at 524 nm was observed due to autooxidation of DCFH

to DCF. Upon addition of [5]rotaxane and before light irradiation, the intensity of peak at 524

did not change much. However, with exposure to light and the peak intensity increased

significantly and continue to rise gradually over time as shown in Figure 2a. These results

indicate that [5]rotaxane has a remarkable light-triggered ROS generation ability and even

under a relatively low light intensity of white light with broad excitation wavelengths, ROS

can be generated efficiently.

European Journal of Organic Chemistry

10.1002/ejoc.201900278

(

a)

(b)

Figure 2. (a) Fluorescence intensity of DCF at 524 nm as blank and in the presence of

5]rotaxane (5 µM) under continuous white light illumination. I , I , I , I , I correspond to

blank and white light irradiated duration for 1, 2, 3, 4 minutes measurements, respectively.

[

0

1

2

3

4

(

[

b) Time response curve of DCFH oxidation in the presence of [5]rotaxane and without

5]rotaxane (R = 0.986 for blank, R =0.9976 in the presence of [5]rotaxane).

2

Investigation of cytotoxicity and phototoxicity on gram-negative bacteria, Escherichia

coli (E. coli) and gram-positive bacteria, Bacillus subtilis

After finding out that [5]rotaxane has ability to generate singlet oxygen efficiently, we

investigated its light-triggered biocidal activity. For this purpose E. coli, a Gram-negative

bacteria, which is responsible for half of the infections was selected as a model and their

interactions with [5]rotaxane were investigated. Bacterial survival experiments were carried

out using various concentrations of [5]rotaxane in the dark and upon exposure to white light

2

18

for 1 min. with flux of 22 mW/cm by a surface plating method (Figure 3 c-f). Colony

counting showed that the killing efficiency upon irradiation of E. coli suspension incubated

with [5]rotaxane (3.5 µM) was 96%, whereas the killing efficiencies in the dark is only around

9

% (Figure 3b). Studies of the effect of [5]rotaxane concentrations on killing efficiency toward

E. coli under irradiation showed that the killing efficiency enhanced with increasing

concentration of [5]rotaxane and reaches a plateau after 3.5 µM (Figure S6-S8).

European Journal of Organic Chemistry

10.1002/ejoc.201900278

Figure 3. (a), (b) Biocidal activities of [5]rotaxane toward E. coli in the dark and under photo-

irradiation. The values represent the mean ± standard deviation (SD) of six separate

experiments. Error bars represent SD of data from six separate measurements. Plate

photographs for E. coli on YTD agar plate treated (c) without photosensitizer in the dark, (d)

with photosensitizer in the dark, (e) without photosensitizer under photo-irradiation (22

2

mW/cm white light, 1 min.), (f) with photosensitizer under photo-irradiation (22 mW/cm

white light, 1 min).

We have also investigated the antibacterial activity of Porphyrin 1, which does not

contain CB6, toward E. coli in the dark and under light keeping similar conditions

(

concentration, light intensity, and exposure time) used for [5]rotaxane to find out the effect of

CB6. Porphyrin 1 showed biocidal activities around 60% and 70%, in the dark and under light,

respectively (Figure S12 and S13). High dark toxicity of Porphyrin 1 compared to [5]rotaxane

can be attributed to strong electrostatic interactions between the ammonium groups with

bacteria cell walls and insertion of the hydrophobic part of the porphyrin monomer through

hydrophobic interactions. In [5]rotaxane, ammonium groups co-ordinate with the carbonyl

European Journal of Organic Chemistry

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portals of CB6 and interact with bacteria cell walls less strongly. Additionally another

drawback of Porphyrin 1 is that it forms aggregates in PBS over time.

The light-triggered antibacterial effect of [5]rotaxane against the Gram-positive

bacteria (B. subtilis) was also investigated under the same conditions (concentration, light

intensity, and exposure time) used for E.coli. While only around 7% of bacteria survived in the

dark, almost 100% of the bacteria were inactivated under light (Fig. S14, S15). These findings

suggest that these nanoparticles can be used as a broad-spectrum antibacterial agent.

Photosensitizer and bacteria interactions investigated by scanning electron microscopy

(

SEM)

SEM is a powerful method to investigate the morphology of bacteria interaction with a

photosensitizer. We took the SEM images of bacteria (E. coli) which are treated with

[

5]rotaxane at a MIC (3.5 µM) in dark and under light. Figure 4b shows the SEM image of E.

coli treated with [5]rotaxane in the dark along with its control group (Figure 4a). As can be

seen from the images, in the dark most of the bacteria are in good shape and not affected by

the photosensitizer by preserving their smooth surfaces. However, in the case of bacteria treated

with light exposure, SEM images showed that the surface of E. coli bacteria treated with

[

5]rotaxane (Figure 4d) were collapsed, fused and their membranes were ruptured in

comparison with that of control group in the absence of [5]rotaxane (Figure 4c).

European Journal of Organic Chemistry

10.1002/ejoc.201900278

Figure 4. SEM images of E. coli (a) without photosensitizer in the dark, (b) with

photosensitizer (3.5 µM) in the dark, (c) without photosensitizer under photo-irradiation (22

2

mW/cm white light, 1 min.), (d) with photosensitizer (3.5 µM) under photo-irradiation (22

2

mW/cm white light, 1 min).

The result confirmed that [5]rotaxane could destroy the bacterial outer membrane upon light

irradiation but not in dark. These observations also are in line with antibacterial experiments.

Although the axle molecule is cationic with many ammonium ions that are known to cause

dark toxicity because of the strong electrostatic interaction between positive charges and

bacteria, CB6s disguise them by forming ion-dipole complexes and this, in turn, decreases the

cytotoxicity. However, [5]rotaxane can still interact with bacteria albeit with weaker binding

and accumulates on the surfaces, upon light exposure ROS are generated and destroy the

membrane of the bacteria as can also be seen in the SEM images.

European Journal of Organic Chemistry

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Binding of [5]rotaxane with E.coli was also supported by ζ-potential measurements.

When E.coli were mixed with [5]rotaxane, positive ζ-potential shifts were observed from −45.7

to −33.3 mV and from -50.3 to -22.9 mV, in the dark and under light, respectively (Figure S16).

Investigation of cytotoxicity and photocytotoxicity on the MCF7 cell

MTT assay was employed to determine the cytotoxicity of [5]rotaxane on mammalian cells in-

vitro. For this purpose, MCF7 was chosen as the representative breast cancer cell line and

treated with different concentrations of [5]rotaxane in dark and under white light exposure. On

the basis of dark cytotoxicity test, various concentrations of [5]rotaxane (10-300 µM) together

with DMEM control group were used to observe cytotoxicity of [5]rotaxane. [5]rotaxane did

not show any significant cytotoxic effect on MCF7 cells between control DMEM group and

any of the concentrations used (P=0.0653) and had a high cell survival rates. This finding

supported the safety of this supramolecular photosensitizer on mammalian cells in dark even

at a concentration (300 µM) higher than that used in antibacterial experiments (Figure 5).

We also determined the photodynamic activity of [5]rotaxane on cell viability upon light

2

irradiation, MCF7 cells were treated with white light (20 mW/cm ) with different exposure

times (5, 10 and 15 min) and different concentrations of [5]rotaxane (2-100 µM). In contrast,

MCF7 cells exhibited signiﬁcant reductions in relative cell viability after light irradiation. As

shown in Figure 5, significant reduction in cell viability was observed at the minimum

concentration of [5]rotaxane (2 µM) even after 5 min of light exposure when compared with

the DMEM control group (P<0.0001) (Supporting Information, Figure S9). Moreover, while

the light exposure time and concentration of [5]rotaxane was increasing, the rate of decrease

on cell viability did not change and remained around 70% of the DMEM control group at all

concentrations including the maximum (100 µM) (Figure 5, Figure S9). These results

demonstrated that white light efficiently activated [5]rotaxane, hence, reduced the cell viability

even at low concentrations with the same rate of reduction observed at high concentrations.

European Journal of Organic Chemistry

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Remarkable cell viability inhibition in treatment groups indicated that the potential superiority

of anti-tumorigenic activity under light. Thus, the use of [5]rotaxane under light provides a

potentially safe strategy for solving the problem of high cytotoxicity which may cause

unnecessary side eﬀects. We concluded that [5]rotaxane photosensitizer showed a relatively

less toxicity in the dark, whereas it exhibited a high cytotoxic eﬃciency under light irradiation

even at low concentrations.

Figure 5. Relative cell viability (%) measurements obtained from MTT analysis at the given

concentrations (µM) of [5]rotaxane treatments upon normalization with DMEM control group

2

in MCF7 cells (a) in dark (P=0.0653) and (b) upon white light irradiation (10 min, 20mW/cm )

(

P<0,0001). Diﬀerences in MTT relative cell viability (normalized to DMEM control group)

were analysed for each dose in each cell line separately using one-way ANOVAs followed by

multiple comparisons (Tukey’s at α = 0.05; Graphpad). ^*P<0.0001; ns: non-significant.

***

Conclusions

[

5]Rotaxane having a photoactive axle was synthesized and its use as an efficient

photosensitizer has been demonstrated in the photodynamic therapy. This rotaxane has an

excellent water solubility with an ability to generate ROS under light. It exhibits no dark

cytotoxicity towards the pathogens (both gram negative bacteria, E.coli and gram positive

bacteria, B. subtilis) and the mammalian cells (breast cancer cell line, MCF7) even at high

concentration (up to 0.3 mM). However, it is highly cytotoxic even very low concentration (2

µM) under white light with relatively low fluence and short exposure time towards both E.coli

European Journal of Organic Chemistry

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and B. subtilis as well as MCF7 cell line. The results show that [5]rotaxane is a remarkable

photosensitizer and its use under light provides a potentially safe strategy for solving the

problem of high cytotoxicity which may cause unnecessary side eﬀects. Moreover, owing to

its negligible dark cytotoxicity it can be used as a therapeutic agent in the light-triggered

antibacterial and anticancer therapies.

Experimental section

Materials and Measurements

All chemicals used in the syntheses were of analytical grade and were obtained from Sigma-

Aldrich and were used as received. Milli-Q water (18.2 MΩ·cm at 25 °C) was used when

needed. Solvents were dried and distilled before used and all reactions were performed under

air unless otherwise stated. Thin layer chromatography was performed on SiO

and flash column chromatography was carried out using SiO 60 (particle size 0.040–0.055

mm, 230–400 mesh). NMR spectra ( H, at 400 MHz and C at 100 MHz) were recorded on a

Bruker DPX-400 spectrometer in D O solvent and TMS (δ = 0.00 ppm) as an internal standard.

2

60 F-254 plates

2

1

13

2

Chemical shifts were reported as δ values in ppm as referenced to TMS. FT-IR spectra were

recorded by Bruker Alpha-II Platinum ATR FT-IR spectrometer. The mass spectra were

obtained with Agilent 6224 High Resolution Mass Time-of-Flight (TOF) LC/MS with

Electrospray Ionization method. UV-VIS absorption spectra were recorded on a UV–vis

spectrophotometer (Cary UV–vis) with 1 cm path length quartz cuvettes in the spectral range

of 300-800 nm. Emission spectra were recorded on a fluorescence spectrophotometer (Cary

Eclipse Fluorescent spectrophotometer). The quantum yields of fluorescence of the compounds

were determined using integrated sphere method. Optical density at 600 nm (OD600) of bacteria

and the absorbance for MTT analysis (at 570 nm) were measured by SpectraMax M5 multi-

detection microplate reader system. Samples for Scanning Electron Microscope (SEM) were

European Journal of Organic Chemistry

10.1002/ejoc.201900278

dried with Autosamdri-934 Critical Point Dryer (Cleanroom), Tousimis. SEM characterization

was conducted on FEI Quanta 200F, Thermo Fisher Scientific.

Synthesis of [5]rotaxane⁸

To the solution of porphyrin 1 (50 mg, 0.049 mmol) in DI water (5 mL), CB6 (214 mg, 0.215

mmol) was added. The resulting mixture was stirred for 1 hour to dissolve CB and a green

colour solution was obtained. After adding tert-butylazidoethylamine (38.3 mg, 0.215 mmol),

the mixture was stirred at room temperature for 24 hours. After the completion of reaction, the

reaction mixture was cooled to room temperature and poured into a dialysis membrane

(

regenerated cellulose, MW cut-off 12 kDa) and dialysed against water for 24 hours to remove

excess CB and monomers. Dialysate was freeze-dried to obtain a fluffy burgundy-colored

4

3

powders. Yield: 245 mg (87%). UV–Vis (H O): lmax: 415 nm (35 x10 ), 517 (14 x10 ), 553

2

3

(

8.5 x10 ), 582 (6.2 x10 ), 636 (4.2x10 ).

¹H-NMR (400 MHz, D

O): d 1.61 (s, 36H, o), 3.81 (t, 8H, ³JHH = 7.95 Hz, m), 4.13 (t, 8H, ³JHH

2

=

8.15 Hz, l), 4.25 (48 H, CB), 4.39 (s, 8H, i), 4.86 (s, 8H, h), 5.52 (48H, CB), 5.75 (48H, CB),

6

.62 (s, 4H, k), 8.52 (d, 8H, ³JHH = 9.9 Hz, e), 8.79 (d, 8H, ³JHH = 10.1 Hz, f), 9.09 (s, 8H, a).

¹³C-NMR (100 MHz, D

30.8, 132.7, 138.8, 138.9, 140.1, 145.1, 156.3, 156.7

ROS Measurement¹⁶^,¹⁷

O): d 25.2, 40.0, 42.5, 47.5, 51.3, 51.5, 52.5, 70.2, 120.9, 122.5, 129.9,

2

1

First a 40 µM solution of 2,7-dichlorofluorescin (DCFH) was prepared by hydrolyzing

dichlorofluorescin diacetate (DCFH-DA) in alkaline media and the resulting solution in sodium

phosphate buffer (pH 7.4) was kept in cold (2 °C) in dark place until further use. Highly

fluorescent DCF (excitation 488 nm, emission at 524 nm, quantum yield: 90%) was obtained

in the presence of ROS. To investigate the ROS generation ability of [5]rotaxane, first blank

measurements were performed as follows: 0.5 mL of 40 µM DCFH was diluted with 1 mL of

European Journal of Organic Chemistry

10.1002/ejoc.201900278

water and further excited at 488 nm. The emission intensity at 524 nm was measured (0 min)

2

then the solution was irradiated under white light (1 mW/cm ) for 4 minutes, and the emission

intensity of blank solution was recorded after every minute.

After blank measurements, 100 µL of 5 µM [5]rotaxane solution was added to the 0.5 mL of

4

0 µM DCFH, which was diluted with 1 mL of water. The same procedure as blank

measurements was followed for [5]rotaxane.

1

8

Determination of Minimum Inhibitory Concentration (MIC Assay): The respective MIC

of [5]rotaxane was determined by Broth microdilution method. Hereby, a single colony of E.

coli on a solid Luria-Bertani (LB) agar plate was transferred to 5.0 mL of liquid LB culture

medium and grown (37 °C, 200 rpm, 14 hours). Bacterial mixture was diluted 2-fold with pure

LB and initial OD600 value was adjusted to 1.0 and 10 different concentrations were prepared

as follows; 0.5, 1.0, 1.5, 2.0, 2.42, 2.90, 3.40, 3.89, 4.29 and 4.74 µM. 1.5 mL-eppendorf tubes

were inoculated with 100 µL of bacterial mixture and then 10 µL from each concentration was

added. For performing the control experiment, equal volume of 10mM PBS was used. Both

blank and [5]rotaxane containing tubes were exposed to white light for 1 minute with flux of

2

2 mW/cm . The same experiment was also carried out in the dark. After that, eppendorf tubes

were placed into incubator (at 37°C, 200 rpm, 14 hours). At the end of the incubation period,

bacterial mixtures were transferred from eppendorf tubes to 96-well plate. In microplate reader,

OD600 was measured. The results were repeated in triplicate.

Preparation of the Bacterial Solution: A single colony of E. coli on a solid LB agar plate

o

was transferred to 5.0 mL of liquid LB culture medium and grown (37 C, 200 rpm, 14 hours).

o

Bacteria were harvested by centrifuging (4 C, 7000 rpm, 2 minutes) and washing by phosphate-

buffered saline (PBS; 10 mM, pH 7.4) three times. The supernatant was discarded and

remaining E. coli were resuspended in PBS and then diluted so that the OD600 of the bacterial

suspension was adjusted to 1.0.

European Journal of Organic Chemistry

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Investigation of Antibacterial Activity of [5]rotaxane toward E. coli:

Suspension of E. coli (2.0 mL, OD600=1.0) was diluted 5-fold with PBS. According to the

results of MIC assay, 3.5 µM [5]Rotaxane was added to 2.0 mL of diluted E. coli suspension

o

and the mixture was incubated (37 C, 200 rpm, 15 min) and then irradiated upon white light

2

4

with a flux of 22 mW/cm for 1 min. Then the bacterial suspension was serially diluted (10

fold) in PBS. A 50 µL portion of the diluted bacterial mixture was spread on the solid LB agar

o

plate. The colonies formed after 14 hours incubation at 37 C were quantified. The same

procedure was repeated for the [5]rotaxane incubated with E. coli in the dark without exposure

to light. Control experiments were carried out with exposure to the light and in the dark by

using equal volume of PBS as blank.

Antibacterial activities of nanoparticles toward Gram-positive bacteria (Bacillus subtilis, B.

subtilis) were also studied fol- lowing the similar procedure described for E. coli.

z

-Potential Measurements

Suspensions of E. coli (100 µL, OD600=0.95) in 500 µL of PBS were incubated with 3.5 µM

of [5]rotaxane (37 °C, 200 rpm, 1 min). After that, they were kept in the dark and exposed to

2

white light for 1 min with flux of 22 mW/cm . The bacteria were harvested by centrifugation

at 7000 rpm for 2 min, followed by removal of the supernatant; they were then washed once

with water and suspended in 1 mL of H

2

O. The suspensions were kept on ice for ζ-potential

o

measurements, while the measurement itself was performed at 25 C.

Imaging of Antibacterial Activity by SEM: Prior to the experiment, silica wafers were cut 1

cm to 1 cm and incubated for 30 min with isopropanol and ethanol respectively in order to

remove organic contamination. They were completely dried in air and then placed to 6 well-

plate. The bacterial samples for SEM imaging experiments were prepared as follows:

Suspensions of E. coli (100 µL, OD600=1.0) in 1 mL PBS were incubated with [5]rotaxane and

(

3.5 µM, 37 °C, 200 rpm, 15 min) and then irradiated upon white light with a flux of 22

European Journal of Organic Chemistry

10.1002/ejoc.201900278

2

mW/cm for 1 min, equal volume of PBS was used for control experiment. The same protocol

was performed in the dark. 10 µL of each sample was taken and fixed onto silica wafers with

o

2

.5% glutaraldehyde solution in PBS for overnight at 4 C. Next day, wafers with cells were

washed one time with PBS, one time with pure water, one time with 25% (v/v) ethanol, one

time with 50% (v/v) ethanol, one time with 75% (v/v) ethanol and one time with pure ethanol

for 2 min each. Following the washing steps, the wafers were dried by critical point dryer

(

CPD) and then coated 5 nm with Au/Pd alloy. The Samples were examined at 15 kV.

In Vitro Cell Viability Assay

Cell Culture. Breast cancer cell line (MCF7) were cultured in Dulbecco's Modified Eagle

Medium (DMEM) supplemented with 10% fatal bovine serum, 1% penicillin/streptomycin, 0.1

mM non-essential amino acids and 1mM sodium pyruvate at 37°C and 5% CO2-humidiﬁed

atmosphere in the dark.

Cytotoxicity. In vitro cytotoxicity of [5]rotaxane was investigated with the MTT cell viability

3

assay. To determine cell viability under dark, 4×10 cells were seeded in 96-well tissue culture

plates for 24 h, and the next day cells were treated with diﬀerent concentrations of [5]rotaxane

(

0-300 µM) dissolved in 1X PBS. All dilution series and negative control (without [5]rotaxane)

included same volume of PBS and DMEM (Supporting Information Table X), each with three

replicates, and incubated for 48h at 37 °C. Thiazolyl blue tetrazolium bromide (MTT),

membrane-permeable dye (ab146345) was used according to the manufacturer’s protocol.

Briefly, the medium was removed and replaced with 110 µL of DMEM containing [3-(4,5-

dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] (MTT, 1.2 mM). The mixture was

further incubated for 4h at 37 °C. The purple formazan product was solubilized in 110 µL of

SDS-HCl solution (1g SDS in 10mL of 10 mM HCl). The plates were incubated overnight at

3

7°C, and the absorbance of each microwell was measured at a wavelength of 570 nm using a

microplate reader. To determine the photo-cytotoxicity of [5]rotaxane, the MCF7 cells were

European Journal of Organic Chemistry

10.1002/ejoc.201900278

seeded and incubated in DMEM as described above. After 24 h, cells were treated with diﬀerent

concentrations of [5]rotaxane (0–100 µM). All dilution series and negative control (without

[

5]rotaxane) had the same volume of PBS and DMEM (Supporting Information Table X), each

with three replicates and incubated for 24h at 37°C. Irradiation treatment was performed with

2

white light (20 mW/cm ) from a ZEISS - Cold light source CL 4500 for 5, 10 and 15 mins, and

then placed in the dark for another 24 h at 37°C. The above MTT assay was performed and the

absorbance of each well was read at 570 nm.

Statistical Analysis. Diﬀerences in MTT relative cell viability (normalized to DMEM control

group) were analysed for each dose in each condition separately using one-way ANOVAs

followed by multiple comparisons (Tukey’s at α = 0.05; Graphpad).

Acknowledgments

We gratefully acknowledge the financial support of the Scientific and Technological

Research Council of Turkey (TUBITAK) grant number 215Z035. We would like to thank

Prof. Özlen Konu and Dr. Şahika Cıngır Köker for their valuable advices on the biological

assays.

Keywords: rotaxane • cucurbituril • supramolecular photosensitizer • porphyrin •

Photodynamic antimicrobial chemotherapy (PACT)

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Entry for the Table of Contents

Key Topic: Rotaxane-based photosensitizer

[

5]rotaxane synthesized through CB6-catalyzed 1,3-dipolar cycloaddition reaction between

alkyne substituted porphyrin and azide containing stopper groups serves as a remarkable

photosensitizer in photodynamic therapies against cancer cells and bacteria inactivation.

Article Doi

A [5]Rotaxane-Based Photosensitizer for Photodynamic Therapy

DOI: 10.1002/ejoc.201900278

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Article abstract of DOI:10.1002/ejoc.201900278

Full text of DOI:10.1002/ejoc.201900278

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