Photoswitchable Micelles for the Control of Singlet-Oxygen Generation

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Photoswitchable micelles for the control of singletoxygen generation in photodynamic therapies Yan Zhai, Henk J Busscher, Yong Liu, Zhenkun Zhang, Theo G. van Kooten, linzhu Su, Yumin Zhang, Jinjian Liu, Jianfeng Liu, Yingli An, and Linqi Shi Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.8b00085 • Publication Date (Web): 27 Mar 2018 Downloaded from http://pubs.acs.org on March 27, 2018

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Photoswitchable micelles for the control of singlet-oxygen generation in photodynamic therapies Yan Zhai,† Henk J. Busscher,§,* Yong Liu,§ Zhenkun Zhang,† Theo G. van Kooten,§ Linzhu Su,† Yumin Zhang,¶ Jinjian Liu,¶ Jianfeng Liu,¶ Yingli An,† Linqi Shi †,‡, * †

State Key Laboratory of Medicinal Chemical Biology, Key Laboratory of Functional Polymer

Materials of Ministry of Education, Institute of Polymer Chemistry, College of Chemistry, Nankai University, Tianjin 300071, China ‡

Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), Nankai

University, Tianjin 300071, China §

University of Groningen and University Medical Center Groningen, Department of Biomedical

Engineering, Antonius Deusinglaan 1, 9713 AV Groningen, The Netherlands ¶

Tianjin Key Laboratory of Radiation Medicine and Molecular Nuclear Medicine, Institute of

Radiation Medicine, Chinese Academy of Medical Science & Peking Union Medical College, Tianjin, 300192, P.R. China

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ABSTRACT

Inadvertent

photosensitizer-activation

and

singlet-oxygen

generation

hampers

clinical

application of photodynamic-therapies of superficial tumors or sub-cutaneous infections. Therefore, a reversible photoswitchable system was designed in micellar nanocarriers using ZnTPP as a photosensitizer and BDTE as a photoswitch. Singlet-oxygen generation upon irradiation didnot occur in closed-switch micelles with ZnTPP/BDTE feeding ratios >1:10. Deliberate switch closure/opening within 65-80 min was possible through thin layers of porcine tissue in vitro, increasing for thicker layers. Inadvertent opening of the switch by simulated daylight, took several tens of hours. Creating deliberate cell damage and prevention of inadvertent damage in vitro and in mice could be done at lower ZnTPP/BDTE feeding ratios (1:5) in open-switch micelles and at higher irradiation intensities than inferred from chemical clues to generate singlet-oxygen. The reduction of inadvertent photosensitizer activation in micellar nanocarriers, while maintaining the ability to kill tumor cells and infectious bacteria established here, brings photodynamic-therapies closer to clinical application.


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KEYWORDS: Photoswitchable micelles, Singlet-oxygen, Porphyrin, Diarylethene, FRET


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1. INTRODUCTION Photodynamic therapy dates back several thousands of years to ancient Egyptian, Indian and Chinese medicinal cultures in which combinations of natural products and light were used to treat superficial skin diseases.1 The start of photodynamic therapy can arguably be marked in the late 19th century, when Finsen treated lupus vulgaris with photodynamic therapy.2 Nowadays, photodynamic therapy with appropriate photosensitizers is considered more and more as a promising method for early treatment of superficial tumors, palliative stand-alone treatment of early diseases, palliative care or salvage treatment3 and eradication of sub-cutaneous, infectious biofilms.4 Photodynamic therapy is firstly based on the administration of a light-activatable photosensitizer to generate singlet-oxygen (1O2) upon light-activation of the photosensitizer5 to cause irreversible damage to tumor cells or infectious microorganisms.6 Roughly three different types of photosensitizers can be distinguished: (1) first generation photosensitizers of bloodderived hematoporphyrin, showing extremely low selectivity and causing excessive damage to healthy tissue,7 (2) second generation, synthetically-made porphyrins possessing improved selectivity towards target cells,8 and finally (3) porphyrins encapsulated in micellar nanocarriers or liposomes, equipped with antibody recognitions, all aimed to facilitate transport through the bloodstream, selective-targeting and avoidance of collateral tissue damage by singlet-oxygen production.9 Yet, clinical application is hampered by inadvertent photosensitizer activation and singlet-oxygen generation causing irritation of the skin, pain, inflammation, fever or nausea, that not only occur during the drug to light-activation interval between administration of the first photosensitizer injection and light-activation, but that can also extend till weeks after therapy.10 Currently, drug to light-activation intervals can range from 3 h to 96 h during which patients


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have to avoid sunlight and high artificial light intensities, which is often extremely bothersome considering the condition of most patients requiring photodynamic therapy.11 This drawback has led to the development of so-called “photoswitches” for the control of singlet-oxygen generation by photosensitizers. Photoswitches of porphyrin and diarylethene derivatives have been reported,12 but these mostly operate in organic solvents or mixed solvents due to poor water solubility and not in blood-borne systems.13 Photochromic metal-organicframeworks demonstrated good control of singlet-oxygen generation in aqueous systems,14 but could not be circulated in the blood-stream for prolonged periods of time, nor were they sufficiently stable, self-targeting and biodegradable.15, 16 Micellar nanocarriers have been successfully utilized for transport through the bloodcirculation of poorly water soluble drugs17 and offer advantages such as improved bioavailability, reduced toxicity, targetability and easy functionalization.18, 19 Poly(ethylene glycol)block-poly (ε-caprolactone) (PEG-b-PCL) copolymers, both approved polymers by the Food and Drug Administration, USA, provide for the synthesis of hydrophilic-shell micelles that resist protein adsorption and clearance by mononuclear phagocytes, thereby increasing their bloodcirculation time and chances to reach their target tumor or infection site in the body.20-22 This paper aims to reduce side effects and the necessity of patients to stay out of the light during the drug to light-activation interval and after photodynamic therapy through the fabrication of a reversible photoswitch system for the control of singlet-oxygen generation incorporated in PEG-b-PCL micellar nanocarriers. Zinc−tetraphenylporphyrin (ZnTPP) was used as a photosensitizer because of its high efficiency in light harvesting,23 while 1,2-bis(5-(4carbonxyphenyl)-2-methylthien-3-yl)cyclopent-1-ene (BDTE) was chosen as a photochromic switch due to its thermal stability, fatigue resistance and high efficiency in photoisomerization.24


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ZnTPP and BDTE were non-covalently coupled and encapsulated in PEG-b-PCL micelles in different ratios for on/off switching of singlet-oxygen generation upon irradiation with light of different wavelengths. Switch closure/opening by UV, respectively Vis, will be demonstrated using Electron Paramagnetic Resonance (EPR) and UV/Vis absorption and emission spectroscopy, including the possibility for on-off switching through different thicknesses of porcine tissue. Biocompatibility and efficacy of photoswitchable ZnTPP/BDTE micellar nanocarriers to cause tumor and bacterial cell death or prevent cell damage upon irradiation through singlet-oxygen generation, will be further demonstrated in vitro towards 3T3 fibroblast and HeLa epithelial tumor cells in an in vivo murine model.

2. EXPERIMENTAL SECTION 2.1. Chemicals. Monomethoxy poly(ethylene glycol) (CH3O−PEG− OH; Mw = 5000; Mw/Mn = 1.05; 99% purity) was purchased from Sigma-Aldrich (Massachusetts, USA) and dried under vacuum before use. ε-caprolactone (ε-CL; 95% purity) obtained from Alfa Aesar (Saint Louis, USA), was dried over calcium hydride and subsequently purified by vacuum distillation. ZnTPP and TPP were purchased from Hang Zhou B&P Biotech Co., Ltd. (Hangzhou, China). 4Bromopyridine hydrochloride (99% purity), Aluminium chloride (99% purity), Nitromethane and 2-chloro-5-methylthiophene (99% purity) were purchased from Energy Chemical (Beijing, China). n-butyllithium (1.6M solution in hexane), 2,2,6,6-tetramethyl piperidine (TEMP; 98% purity), Stannous octoate (Sn(Oct)2; 95% purity), TiCl4 (95% purity) was obtained from J&K (Beijing, China) and used as received. All other materials and solvents were used as received without further purification from commercial suppliers.


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2.2. Synthesis of photoswchitchable, porphyrin-loaded PEG-b-PCL micelles. 1,2-bis(5-(4carbonxyphenyl)-2-methylthien-3-yl)cyclopent-1-ene(BDTE) was synthesized according to literature (Supporting Information Scheme S1)14 and BDTE and intermediate products were characterized by NMR (Supporting Information Figure S1) and high resolution electrospray ionization mass spectroscopy (Supporting Information Figure S2). PEG-b-PCL was synthesized as previously reported (Supporting Information Scheme S2),25 through ring-opening polymerization of monomeric ε-caprolactone with PEG−OH as an initiator and Sn(Oct)2 as a catalyst in refluxed toluene. Afterward, the solvent was removed in vacuum and the crude product was dissolved in dichloromethane, followed by precipitation into an excess amount of diethyl ether and the precipitate was dried in vacuum. (1H NMR spectrum of PEG-b-PCL was shown in Supporting Information Figure S3). Based on the ratio between the areas underneath the peaks characteristic of PEG and PCL in the NMR spectrum, the degree of polymerization (Dp) of the PCL block in the block polymer was estimated as 62, corresponding to a number averaged molecular weight of 7 kDa. Photoswitchable micelles were prepared using a modified nanoprecipitation method26 in which ZnTPP and BDTE were separately dissolved in anhydrous, inhibitor-free THF (99.9%) to make stock solutions with a concentration of 1mg/ml, while preparing a stock solution of PEG5k-bPCL7k in THF with a concentration of 3.0mg/ml. Different amounts of ZnTPP, BDTE and PEG5k-b-PCL7k stock solutions were mixed by sonication to form a homogeneous solution and then added to MilliQ pure water and sonicated for 15 min. The resulting solutions possessing different feeding ratios of ZnTPP and BDTE, were dialyzed (cut-off number >3.5 k) against phosphate buffered saline (PBS; 10 mM, pH 7.4) or water for 2 days to remove THF. The different micelle suspensions in water (for physico-chemical experiments) or PBS (for biological


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and animal experiments) were filtrated over a 0.22 µm pore size polyether sulfone (PES) filter (STERIVEXTM, Millipore Corp., MA, USA) and the micelle concentration was adjusted immediately before experiments to the concentration optimal for the different experiments. 2.3. Light sources and irradiation conditions. Different light sources and irradiation conditions were applied for singlet-oxygen generation (Multimode fiber laser; Shanghai Xilong Optoelectronics Technology Co., Ltd, China), opening (Xe-light source CEL-S500 with 450 nm long-wavelength

pass-filter; Au-light Instruments, Beijing, China) and closing (308 nm

Spectroline; Xilong Optoelectronics Technology Co., Ltd, Shanghai, China) of the photoswitch and for the simulation of daylight (Xe-light source CEL-S500 with an AM1.5 filter; Au-light Instruments, Beijing, China), as summarized in Table 1. Light intensities were measured using optical power meter (NP190; Au-light Instruments, Beijing, China).

Table 1. Overview over the different light sources, their intensities, wavelengths and purposes with respect to singlet-oxygen generation, switch opening or closing and the simulation of daylight conditions*. Wavelength

Intensity

(nm)

(mW/cm2)

Deliberate singlet-oxygen generation

405

8 (in vitro), 50 (in vivo)

Multimode fiber laser

Deliberate switch opening

> 450

120

Xe-light source long-wavelength, pass-filtered

Deliberate switch closing

308

10

308 nm spectroline UV light source

Purpose

Source type


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Simulated daylight

300-1100

0.06 (40 lux)

AM1.5 filtered Xelight

*

40 lux represents family living room light, while 320-500 and 10,000-25,000 lux represent office light and full daylight (no direct sun) conditions, respectively.

2.4. Micelle characterization. Micelles in suspension (water; 0.1 mg/ml) were first characterized by their diameter, using a Zeta-sizer Nano-ZS (Malvern Instruments, Worcestershire, UK) at 25°C. Furthermore, TEM was carried out to visualize the morphology of the micelles on a JEM100CXII, (Tokyo, Japan), at a voltage of 7 kV and a magnification of 12000x. In order to calculate the actual ratio of ZnTPP and BDTE from the feeding ratios applied, a calibration series of ZnTPP and BDTE solutions were prepared and their UV/Vis absorption spectra (see below under section 2.6) analyzed and compared with the ones of the different micelles, allowing to calculate an actual ratio of ZnTPP/BDTE micelles (see Supporting Information Figure S4). Drug loading content (DLC) and drug loading efficiency (DLE) were calculated subsequently according to DLC (wt %) = (Wloaded drug/Wloaded drug and polymer) x 100%

and

DLE (wt %) = (Wloaded drug/Wdrug in feed) x 100%

in which W indicates weight.


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2.5. Evaluation of the singlet-oxygen generation ability of photoswitchable micelles by Electron Paramagnetic Resonance (EPR). For EPR (Bruker A-300 EPR spectrometer; Karlsruhe, Germany), singlet oxygen was detected as TEMP-1O2 adduct (TEMPO) using TEMP as a singlet oxygen trap, as described previously.27 Briefly, TEMP (7 mg/ml) was first dissolved in ethanol and mixed with ZnTPP/BDTEX micelles in suspensions (water; 0.1 mg/ml) in quartz cuvettes. Next, for singlet-oxygen generation the micellar suspension was irradiated for 30 min with a multimode fiber laser at the distance of 15 cm above the quartz cuvettes. During EPR, open switches were deliberately closed during 6 min and closed switches were deliberately reopened during 25 min of irradiation with the respective light sources (see Table 1). EPR was carried out at a microwave frequency of 9.8 GHz and power of 10 mW with a modulation amplitude and frequency of 1.0 G and 100 kHz, respectively, time constant 0.64 s, scan time 200 s, receiver gain 4.03105 and center field setting 3483 G. 2.6. UV/Vis absorption and emission spectroscopy. UV/Vis absorption spectra were recorded on Shimadzu UV-2550 spectrophotometer (Shimadzu Co. Ltd., Kyoto, Japan). Micelles in suspension (water; 0.1 mg/ml) were placed in 1ml quartz cuvettes a and measurements were performed at 25°C, taking three scans on each sample with a slit width of 2.0 nm. The photoswitches in their open form were deliberately closed by UV irradiation until absorption at 560 nm reached a maximum value (A0). Afterwards, the photoswitches were deliberately reopened by irradiation with visible long-wavelength, pass-filtered Xe-light. Light sources were placed 20 cm above the cuvettes. Fluorescence emission spectra were recorded on a Hitachi F-4600 spectrometer (Hitachi Co. Ltd., Tokyo, Japan). Micelles in suspension were placed in 1 ml quartz cuvettes and inserted in the spectrometer. All measurements were performed at 25°C, taking three scans on each sample


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with excitation and emission slits of 5.0 nm, scan speed of 1200 nm/min and photomultiplier voltage of 700V. The excitation wavelengths were 425 nm and 429 nm for ZnTPP and ZnTPP/BDTE micelles, respectively. During recording of the emission spectra, photoswitches were closed or re-opened as described above, taking the maximum fluorescence intensity at 609 nm as indicative of full closure of all switches upon UV irradiation. 2.7. Subcutaneous tissue model. In order to determine whether the photoswitch could be controlled through the skin, a subcutaneous tissue model was applied, placing different thicknesses of porcine tissue in between the quartz cuvette containing the micellar suspension in water (0.1 mg/ml) and the appropriate light sources applied to deliberately or inadvertently open and close the switch (see Table 1). To this end, the top of the cuvette was covered with a 1 mm thick quartz plate covered with a layer of porcine tissue. Porcine tissue was purchased commercially (“belly bacon”) and cut into slides with a thickness of 1.0, 1.5 and 3.0 mm. Porcine tissues were used immediately after purchasing and cutting. Spectroscopy was carried out as described above (section 2.6). 2.8. Biocompatibility and efficacy of ZnTPP/BDTE micellar nanocarriers in vitro. 3T3 murine fibroblasts were obtained from Shanghai Institutes for Biological Sciences, CAS, China and routinely grown in monolayer cultures in high-glucose Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% (v/v) Fetal Bovine Serum and 1% antibiotics (penicillin/streptomycin, 100 U/mL) at 37°C in a humidified atmosphere with 5% CO2 for 24 h. At 70-80% confluence, the fibroblasts cultured were passaged using a Trypsin-EDTA solution (Gibco, California, USA). Thus cells grown between passages 4-10 were used in all experiments. To determine the biocompatibility of the ZnTPP/BDTE micelles, cells were first seeded in 96well culture plates to an initial density of 1 × 104 cells per well in 100 µL DMEM. After 24 h


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incubation at 37ºC under 5% CO2 atmosphere, DMEM was removed and 100 µL fresh medium containing different concentrations of ZnTPP/BDTE micelles with open switches was added. Fresh DMEM without micelles was used as a control. Subsequently, cells and micelles were coincubated for 24 h at 37 ºC under 5% CO2 in the dark,. Finally, a standard Cell Counting kit-8 assay (CCK-8, Dojindo, Kumamoto, Japan) was used to quantitate the number of viable cells, according to the manufacturer’s instructions. 100 µL solution of CCK-8 and DMEM (1:9 v/v) was added to each well, followed by 2 h incubation and the absorbance at 450 nm (A450) due to interaction of CCK-8 with metabolically active, viable cells was measured using a SpectraMax i3x microplate reader (Molecular Devices, Sunnyvale, USA). The percentage cell viability was calculated after background subtraction of the absorbance at 650 nm (A650) with respect to the absorbance of the control according to

Cell Viability = [(A450-A650) micelle/ (A450-A650)control] × 100%.

Next the efficacy of open-switch ZnTPP/BDTE micelles in comparison with closed-switch micelles to inflict damage to tissue cells, including a HeLa-epithelial tumor cell line (grown in essence similarly as described above for fibroblasts) and a methicillin-resistant Staphylococcus aureus (SCC mec type II) (grown in TSB at 37°C in ambient air and suspended in PBS at a concentration of 106 colony forming units (CFU/ml) were compared. The above described cellular experiments were carried out essentially as described above, while adding 5, 10 or 15 µM of ZnTPP/BDTE micelles with open- or closed-switches for 12 h co-incubation at 37ºC under a 5% CO2 atmosphere. After 12 h, cells were irradiated for 40 min with 405 nm light at 50 mW/cm2 for singlet-oxygen generation, after which the percentage cell viability was determined,


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also as described above. In addition, bacterial killing was assessed by adding 5, 10 or 15 µM of ZnTPP/BDTE micelles with open- or closed-switches, essentially as described above but using TSB as a medium. Immediately after adding micelles (i.e. without a co-incubation phase), suspensions were irradiated for 40 min with 405 nm light at 50 mW/cm2 for singlet-oxygen generation, after which the numbers of CFUs were determined by plate counting using TSB plates. Note that a higher irradiation intensity was used than in the chemical detection of switch opening/closing and singlet-oxygen generation, as irradiation at lower intensity did not generate sufficient amount of singlet-oxygen for biological effects for reasons discussed later in this article. All experiments were performed in triplicate, with separately grown cells or bacteria and differently prepared micelles. 2.9. Efficacy of ZnTPP/BDTE micelles in a murine model. To evaluate the efficacy of closed and open switches to prevent or inflict tissue damage, respectively, ZnTPP/BDTE5 micelles were injected sub-cutaneously in mice, in agreement with all relevant guidelines and regulations set by Nankai University and with approved institutional protocols set by the China Association of Laboratory Animal Care. BALB/c nude female mice weighing 16-20 g each were obtained from Vital River Laboratory Animal Technology Co. (Beijing, China). The mice were anaesthetized using aqueous 4 wt% chloral hydrate (8.25 ml/kg) intramuscularly injected into the posterior thighs. PBS or PBS containing closed- or open-switch ZnTPP/BDTE5 micelles (0.3 mg mL-1, corresponding to 40 µM ZnTPP) was sub-cutaneously injected into the skin tissue of the mice (100 µL per mice per injection). Nine animals were randomly assigned into three groups consisting of three animals, each to receive sub-cutaneous injection of (i) PBS (as control), (ii) PBS containing open-switch ZnTPP/BDTE5 micelles and (iii) PBS containing closed-switch ZnTPP/BDTE5 micelles. 30 min after injection, each group was irradiated at the injection site


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with a laser (405 nm at 50 mW/cm2) for 1 h in order to generate singlet-oxygen. Injections and irradiation were repeated after 24 h. Animals were anaesthetized and sacrificed 24 h after the second injection irradiation, and skin tissue biopsies taken from the injection sites that were subsequently fixed, stained with hematoxylin/eosin and processed for histological evaluation using standard techniques.

3. RESULTS AND DISCUSSION 3.1. Micelle Characterization. PEG-b-PCL micelles with ZnTPP as a photosensitizer and BDTE incorporated as a photochromic switch, were prepared by mixing and precipitation. Photosensitizer and photoswitch were connected through non-covalent binding (Figure 1A). Since the photoswitching efficiency may depend on the ratio between ZnTPP and BDTE,28 photoswitchable micelles were prepared with different feeding ratios ( 1:1, 1:5, 1:10, 1:15, and 1:20 ), corresponding with actual ratios between ZnTPP and BDTE of ( 1:1, 1:4, 1:9, 1:12 and 1:16 ), respectively. This was concluded using UV/Vis absorption spectroscopy (see Supporting Information Figure S4) on calibration series of ZnTPP or BDTE solutions and comparison with absorption spectra of micelles prepared with different feeding ratios. Hence from here on, photoswitchable micelles will be indicated by their feeding ratio 1:X as ZnTPP/BDTEX. DLCs with only ZnTPP or BDTE, were similar and amounted 9.2% and 8.6%, respectively (Table 2), while DLEs were slightly higher for ZnTPP (56.1%) than for BDTE (45.6%). Depending on the feeding ratio X, DLC for ZnTPP varied between 0.8% and 6.4%, and for BDTE between 3.1% and 8.1%. Overall ZnTPP loading was more efficient than loading of BDTE (see also Table 2).


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Table 2. Drug-loading content (DLC, %) and drug-loading efficiency (DLE, %) of PEG-b-PCL micelles for different feeding ratios X. All data are means of triplicate experiments with separately prepared micelles and ± indicating standard deviations. ZnTPP/BDTE1 ZnTPP

ZnTPP/BDTE5

ZnTPP/BDTE10

ZnTPP/BDTE15

ZnTPP/BDTE20

BDTE ZnTPP

BDTE

ZnTPP

BDTE

ZnTPP

BDTE

ZnTPP

BDTE

ZnTPP

BDTE

D L C

9.2 ± 0.8

8.6 ± 1.2

6.4 ± 1.1

3.1± 0.7

2.7 ± 0.4

6.6 ± 0.8

1.4 ± 0.2

7.4 ± 0.9

1.1 ± 0.3

7.9 ± 0.6

0.8 ± 0.1

8.1 ± 1.1

D L E

56.1 ± 1.1

45.6 ± 2.4

52.3 ± 1.5

41.± 1.2

50.1 ± 3.6

40.2 ± 0.6

53.1 ± 1.6

45.9 ± 2.6

51.8 ± 4.6

40.6 ± 1.9

53.2± 1.7

41.8 ± 0.8

Micelles with only ZnTPP or BDTE had hydrodynamic diameters in water of 50 and 55 nm respectively (Figure 1b) that increased with the incorporation of ZnTPP/BDTEX. ZnTPP/BDTEX micelles had a typical diameter of around 55-75 nm regardless of the feeding ratio (see Figure 1c). DLS as a function of time showed that the hydrodynamic diameter of the micelles remained stable over at least 6 days, ruling out their aggregation (see Figure S5). TEM micrographs showed single micelles with a well-defined spherical morphology (Figure 1d) albeit with somewhat smaller diameters than obtained using DLS due to lyophilization shrinkage. Release of ZnTPP and BDTE from micelles was minor (see Table S1).


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Figure 1. Preparation and characterization of photoswitchable micelles. (a) Formation of ZnTPP/BDTEX micelles by precipitation. (b) Diameter distributions f(Dh) in water of micelles loaded with ZnTPP (1.2 µM ZnTPP) or BDTE (9 µM BDTE) only, as determined using dynamic light scattering (DLS). Corresponding polydispersity indices were 0.11 and 0.12, respectively. The micelle concentration used in DLS was 0.1 mg/ml. (c) Examples of the diameter distributions f(Dh) in ZnTPP/ BDTEX (X=1, 5, 10, 15, 20; 1.2 µM ZnTPP), with corresponding polydispersity indices of 0.14, 0.21, 0.18, 0.16 and 0.13, respectively. (d) TEM micrograph showing a spherical morphology of the ZnTPP/ BDTE20 micelles (1.2 µM ZnTPP). Scale bar equals 200 nm.

3.2. Photoswitchability of ZnTPP/BDTE Micelles. Photoswitchability of the ZnTPP/BDTEx micelles was first evaluated using Electron Paramagnetic Resonance (EPR) spectroscopy, using TEMP as a spin trap through the formation of nitroxyl radicals (TEMPO) by interaction of TEMP with singlet-oxygen.29


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Figure 2. EPR spectra of TEMP-1O2 adduct formation in different ZnTPP/BDTEX micelles (1.2 µM ZnTPP) in water during irradiation with 405 nm laser light at 8 mW/cm2 to initiate singletoxygen generation. (a) TEMPO formation in a cycle of closing (308 nm, UV, 10 mW/cm2) and re-opening (Vis > 450 nm, 120 mW/cm2) in ZnTPP/BDTE20 micelles, indicated by hyperfine splitting in the open forms. (b) Same as panel (a), now for ZnTPP/BDTE15 micelles. (c) Same as panel (a), now for ZnTPP/BDTE10 micelles. (d) Same as panel (a), now for ZnTPP/BDTE5 micelles.


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Upon irradiation of ZnTPP/BDTPE20 micelles with open photo-switches with 405 nm laserlight irradiation (8 mW/cm2) to initiate singlet-oxygen generation, hyperfine splitting was observed (Figure 2a) with hyperfine splitting constant AN and g-value of the radicals formed of 17.2 G and 2.0055 respectively. This confirms singlet-oxygen generation.30 After closure of the BDTE photo-switch by UV irradiation at 308 nm (10 mW/cm2), near complete inhibition of singlet-oxygen generation was observed, while subsequent irradiation with filtered Xe-light (> 450 nm at 120 mW/cm2) converted BDTE back to its open, singlet-oxygen generating form (see also Figure 2a). Note that a lower feeding ratio of 1:15 still yielded full quenching of singletoxygen formation (Figure 2b), but a lower feeding ratio of 1:10 or 1:5 did not completely impede singlet-oxygen generation (compare Figure 2b with Figures 2c and 2d, respectively), as detected TEMP-1O2 adduct formation in EPR. Note EPR is highly sensitive and, for instance does not indicate whether the singlet-oxygen generated is sufficiently long-lived to exit the micelles and become biologically active. Use of fluorescent single oxygen sensor green to monitor singlet oxygen generation confirmed effective closing and re-opening essentially over the entire range of feeding ratios applied (Figure S6a), with minor generation of singlet oxygen at feeding ratios below of 1: 1 and 1 : 5.


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Figure 3. UV/Vis absorption spectra of ZnTPP/BDTEX micelles in water and proposed1O2 control scheme. (a) UV/Vis absorption spectra of ZnTPP (2.7 µM ZnTPP) micelles and BDTE (9 µM BDTE) micelles with BDTE, in its open and closed form. (b) UV/vis absorption spectra of ZnTPP micelles (1.5 µM ZnTPP) and ZnTPP/BDTE20 (open form, 1.2 µM ZnTPP) micelles. (c) UV/Vis absorption spectra of ZnTPP/BDTE20 micelles (ZnTPP concentration, 0.8 µM) at different times during closing and re-opening of the photoswitch upon UV (308 nm at 10 mW/cm2) or Vis (> 450 nm at 120 mW/cm2) irradiation, respectively. (d) UV/Vis absorbance at 560 nm of ZnTPP/BDTE20 micelles as a function of time during closing and re-opening of the photoswitch upon UV (308 nm) or Vis (> 450 nm) irradiation, respectively. (e) Proposed FRET


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scheme of 1O2 control upon opening and closing the photoswitch, showing different energytransfer routes of excited TPP for BDTE in its open or closed state.

ZnTPP micelles can be further distinguished from micelles with BDTE both in its open and closed form using UV/Vis absorption spectroscopy (Figure 3a). ZnTPP micelles uniquely have a distinct absorption peak around 425 nm, while BDTE in closed form clearly distinguishes itself from its open form by the possession of absorption peaks at 381 and 560 nm. Note that the UV/Vis absorption peak of ZnTPP in micelles at 425 nm, undergoes a red shift towards 429 nm when connected with BDTE (open form) in ZnTPP/BDTEX micelles (Figure 3b), indicating their axial coordination in ZnTPP/BDTEX micelles.31 Switchability of BDTE micelles upon UV and filtered visible Xe-light irradiation was further evaluated by collecting UV/Vis absorption spectra upon switch-closing by irradiation with UV for increasing time periods, followed by reopening of the switch with long-wavelength, pass-filtered visible Xe-light irradiation (Figure 3c). Re-opening of the photoswitch by filtered Xe-light led to a decreased absorption bands around 381 nm and 560 nm. Closure of the switch using UV light (308 nm) led to a return of the absorption bands to their initial absorbance values. Note from the absorbance changes at 560 nm that closing of an open switch upon UV (308 nm) irradiation occurred faster (within 6 min) than re-opening of the switch by high intensity Vis (> 450 nm), requiring around 25 min (Figure 3d). The combination of hyperfine splitting in EPR (Figure 2) and the red shift observed in UV/Vis absorption spectra (Figure 3b) for BDTE incorporated in ZnTPP/BDTE20 micelles in their openswitch form (Figure 3b), suggests Förster resonance energy transfer (FRET) between the two light-sensitive donor (ZnTPP) to acceptor (BDTE) molecules (see Figure 3e) which implies that


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ZnTPP and BDTE are coupled within the micelles at a distance of less than 10 nm, as required for energy transfer.32

Figure 4. Reversibility of the photoswitchability of ZnTPP/BDTE20 micelles (1.2 µM ZnTPP) in water and fluorescence quenching. (a) Fluorescence emission spectra for ZnTPP and ZnTPP/BDTE20 micelles in their open form upon excitation at the Soret band of maximal absorption (429 nm). (b) Fluorescence emission spectra of ZnTPP/BDTE20 micelles in their open form and after closing by UV (308 nm at 10 mW/cm2) irradiation upon excitation at the Soret band of maximal absorption (429 nm). (c) Percentage fluorescence intensity at 609 nm, expressed relative to the initial fluorescence at 609 nm over multiple cycles of closing and re-


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opening for ZnTPP/BDTE20 micelles upon UV (308 nm) or Vis ( >450 nm at 120 mW/cm2) irradiation, respectively. (d) Percentage fluorescence intensity at 609 nm after closure of the photoswitch by UV irradiation (308 nm) for ZnTPP/BDTEX micelles prepared with different feeding ratios X at the same amount of ZnTPP. Percentage fluorescence was expressed relative to the initial fluorescence emission at 609 nm of open form micelles. Data are means over three independent experiments, with error bars indicating standard deviations.

Photoswitchability of the ZnTPP/BDTE micelles was further evaluated using fluorescence emission spectroscopy. Upon excitation at the Soret band of maximal absorption (429 nm) to avoid intervention with switching, ZnTPP and ZnTPP/BDTE20 micelles in their open-switch form showed two emission maxima at 606 and 657 nm (Figure 4a), that nearly completely disappeared upon closure of the switch by UV irradiation (308 nm) (compare Figure 4b), due to FRET energy transfer between ZnTPP and BDTE.33 Note that also fluorescence emission peaks with maxima at 606 nm and 657 nm in open-switch micelles, underwent a red shift from 606 to 609 nm and 657 to 662 nm upon switch closure (Figure 4a), confirming axial coordination as required for a FRET mechanism to control singlet-oxygen generation. Closing of the photoswitch using UV-light (308 nm) for 6 min reduced fluorescence emission at 609 nm to 4% of its initial emission (Figures 4b and 4c). Repetition of this cycle of closing and re-opening of the photoswitch yielded near complete reversibility (Figure 4c). However, fatigue resistance of the photoswitch developed slowly after more than two cycles, probably due to the formation of an annulated isomer as a byproduct of the photochromic reaction slightly decreasing the photoswitch reversibility.34


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Fluorescence quenching of ZnTPP/BDTEx micelles increased with feeding ratio (Figure 4d). However, fluorescence quenching of TPP in presence of BDTE in an unbound state amounted 82%, which is much lower than when bound in a ZnTPP/BDTEX photoswitch incorporated in micelles (compare Figures 4d and 5). This indicates the dependence of FRET energy transfer on donor−acceptor distance and accompanying fluorescence quenching in our photoswitchable micelles.35

Figure 5. Fluorescence emission spectra for TPP/BDTE20 (no binding; 1.2 µM TPP) micelles in their open-switch form and after switch closure by UV (308 nm at 10 mW/cm2) irradiation in water, showing a minor emission peak at 654 nm after closing.


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Figure 6. Deliberate closing and re-opening of photoswitches in ZnTPP/BDTE20 (1.2 µM ZnTPP) micelles in water and inadvertent opening under simulated daylight conditions through different thicknesses of subcutaneous porcine tissue in vitro. (a) UV/Vis absorbance ratio A/A0 at 560 nm of ZnTPP/BDTE20 micelles as a function of time during deliberate closing and reopening of the photoswitch upon UV (308 nm at 10 mW/cm2; left of the dotted line) and Vis (>


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450 nm at 120 mW/cm2; right of the dotted line) irradiation respectively, through different thicknesses of porcine subcutaneous tissue. The absorbance ratio A/A0 is expressed as a percentage relative to the maximal absorbance at 560 nm of ZnTPP/BDTE20 micelles in their closed form (see Figure 3d). (b) The percentage fluorescence emission I/I0 at 609 nm upon inadvertent opening of closed switches under simulated daylight conditions (0.06 mW/cm2), expressed relative to the initial fluorescence at 609 nm of ZnTPP/BDTE20 micelles (open form: see Figure 4a). Note a drop in fluorescence emission after 25–40 h due to photolysis of the photosensitizer. (c) The change of percentage fluorescence intensity of ZnTPP micelles without the photoswitch feature at 606 nm under simulated daylight conditions, expressed relative to the initial fluorescence at 606 nm of ZnTPP micelles.

3.3. Opening and closing of the switch in an in vitro, subcutaneous tissue model. Next, deliberate opening and closing and inadvertent opening of the photoswitch in ZnTPP/BDTE20 was evaluated in an in vitro subcutaneous model through different thicknesses of porcine tissue. Interestingly, a 1 mm thick tissue layers did not inhibit deliberate opening or closing of the photoswitch within several tens of minutes, although it did require longer irradiation times than in absence of tissue layers, both for opening and closing (Figure 6a). Irradiation times to open or close the photoswitches may be shortened by applying higher irradiation intensities. Inadvertent opening of the switch occurred after several hours up to days of low intensity simulated daylight, but slowed down in the presence of layers of subcutaneous tissue. After 24-36 h, UV/Vis emission at 609 nm decreased in absence of a layer of subcutaneous tissue and in presence of the thinnest layer (Figure 6b), probably to photolysis of the ZnTPP and not by fatigue of the photochromic switch,36 as also fluorescence emission by micelles containing only ZnTPP


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decreased after prolonged exposure by simulated daylight (Figure 6c). Also here, an increasing thickness of intermediate porcine tissue reduced photolysis of ZnTPP. Note, that the demonstration of switchability of BDTE inside micelles by means of UV/Vis absorption and fluorescence emission spectroscopy, does not necessarily imply singlet-oxygen generation in biological effective amounts upon irradiation by 405 nm laser light, nor does it imply absence of inadvertent opening of switches in micelles with closed photoswitches accompanied by negative side effects under daylight conditions. 3.4. Biocompatibility and biological efficacy of ZnTPP/BDTE micelles in vitro


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Figure 7. Biocompatibility and possibility to inflict deliberate damage of ZnTPP/BDTE5 micelles in vitro. (a) Percentage cell viability of 3T3 fibroblasts with respect to a medium control without micelles during 24 h co-incubation with different concentrations of ZnTPP/BDTE5 micelles (open-switch) in the dark expressed with respect to the viability in absence of micelles. (b) Percentage cell viability of 3T3 fibroblasts in absence and presence open and closed form ZnTPP/BDTE5 micelles in various concentrations upon irradiation with 405 nm light (50 mW/cm2), expressed with respect to cell viability after irradiation in absence of micelles (irradiation control). (c) Deliberate killing of HeLa-epithelial cells in absence and presence open and closed form ZnTPP/BDTE5 micelles in various concentrations upon irradiation with 405 nm light (50 mW/cm2), expressed with respect to cell viability after irradiation in absence of micelles (irradiation control). (d) Deliberate killing of infectious methicillin-resistant S. aureus (SCC mec type II) in absence and presence open and closed form ZnTPP/BDTE5 micelles in various concentrations upon irradiation with 405 nm light (50 mW/cm2), expressed with respect to the number of CFUs after irradiation in absence of micelles (irradiation control). Data represent averages over 3 experiments with separately cultured cells with error bars representing standard deviations and asterisks indicating statistical significances according to ***p < 0.001 and ****p < 0.0001 (ANOVA).

ZnTPP/BDTE5 micelles in their open form were fully biocompatible when co-incubated with 3T3 fibroblasts in the dark (Figure 7a), with a minor decrease in percentage cell viability upon increasing the micellar concentration to above 20 µM. Micelles with a higher feeding ratio X were less biocompatible (data not shown), but such high feeding ratios were not needed to


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prevent inadvertent biological effects. Reduced viability of 3T3 fibroblasts due to singlet-oxygen generation upon irradiation with 405 nm laser light (8 mW/cm2) of ZnTPP/BDTE5 micelles with open switches could not be observed (data not shown). Increasing the intensity to 50 mW/cm2, as also common in clinical applications,37 was required to observe significant reductions in fibroblasts viability, that in addition were only demonstrable for open-switch form ZnTPP/BDTE5 micelles with a relatively low amount of BDTE (Figure 7b). Importantly, not only benign fibroblasts but also malignant HeLa epithelial cells could be killed according to a similar pattern as observed for fibroblasts (Figure 7c). Considering the focus of our work on demonstrating the possibility of incorporating an effective photoswitchable system inside micellar nanocarriers, we did not study whether tumor cell killing occurred through necrotic or apoptotic pathways, despite its potential importance in complete tumor cell eradication.

38-39

Moreover, Figure 7d shows that also methicillin-resistant staphylococci are killed according to the same pattern as mammalian cells, demonstrating potential applicability of our photoswitchable micelles with controlled singlet-oxygen generation in the treatment of superficial

tumors

or

sub-cutaneous

infectious

biofilms.

EPR experiments evaluating singlet-oxygen generation based on TEMP-1O2 adduct formation (Figure 2) indicated that the BDTE feeding ratio should be above 10 to prevent singlet-oxygen generation, while biological clues indicated a feeding ratio of 5 to be sufficient. This can be explained by a variety of reasons. First of all, in EPR, TEMP not only measures singlet-oxygen outside the micelles, but due to its hydrophobic nature TEMP has the ability to enter the micelles40-41 and also measure singlet-oxygen inside micelles. Obviously, singlet-oxygen inside the micelles does not contribute to any biological activity. Moreover, once outside a micelle, singlet-oxygen is short-lived traveling around 125 nm in water before recombining and only


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traveling 20-25 nm intra-cellularly.42-43 Therefore, higher concentrations of singlet-oxygen than the threshold for its chemical detection may be required for biological efficacy. Furthermore the necessity to lower the ZnTPP/BDTE feeding ratio to 1:5, i.e. lower than indicated by EPR, may suggest that higher amounts of BDTE may have left part of the switches in their closed form. SOSG experiments with micelles suspended in PBS as done in biological experiments, yielded similar data as obtained with micelles suspended in water (compare Figures 6a and 6b), ruling out an impact of the ionic environment on singlet-oxygen generation. Biocompatibility and potential to inflict damage of ZnTPP/BDTEX micelles in vitro therefore indicated that the high feeding ratios of ZnTPP/BDTEX micelles (feeding ratios X > 15) inferred from highly sensitive chemical clues, were neither desirable nor required for biocompatibility and biological efficacy. 3.5. In vivo tissue reactions to activation of open- and closed-switch ZnTPP/ BDTE5 micelles in a murine model. Accordingly, biological efficacy of open-switch and its absence for closed-switch ZnTPP/BDTE5 micelles was demonstrated in a murine in vivo model, also applying 50 mW/cm2 for singlet-oxygen generation (note that fat tissue has an extremely high absorption coefficient for wavelengths of 405-410 nm compared to other wavelengths,44 also explaining the clinical use of high light-activation intensities).


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Figure 8: In vivo tissue reactions to activation of open- and closed-switch ZnTPP/BDTE5 micelles upon high intensity (50 mW/cm2) irradiation with 405 nm laser light. All micrographs are taken of mouse skin tissues at the injection sites after staining with haematoxylin/eosin. a-c represent overviews, while, d-f more detailed images. Solid black arrows indicate dermal fat tissue, single-headed arrows point at individual inflammatory cells (→), while double-headed arrows point at infiltrated dermal connective tissue layers (↔). (a, d) Injection with PBS only (irradiation control). (b, e) Injection with open-switch micelles. (c. f) Injection with closedswitch micelles. Scale bars represent 100 µm.

No tissue damage was observed upon irradiation with high intensity (50 mW/cm2), 405 nm laser light at sites injected with PBS only (Figures 8a and d), indicating that the light irradiation did not contribute to the cell damage seen with the open-switch ZnTPP/BDTE5 micelles. Sites injected with closed form ZnTPP/BDTE5 micelles neither showed significant skin damage upon


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irradiation (Figs. 8c and f), indicating insufficient singlet-oxygen generation for biological effects. Tissue damage upon irradiation of sites with open-switch ZnTPP/BDTE5 micelles manifested itself as the presence of dermal edema and massive infiltration of inflammatory cells, with neutrophilic granulocytes being the most pronounced cell type (Figures 8b and e). Both sites injected with the PBS control (Figures 8a and d) as well as sites injected with closed form ZnTPP/BDTE5 micelles (Figures 8c and f) displayed similarly low numbers of inflammatory cells which is probably due to the presence of mild tissue damage associated with the injection itself. Comparison of Figs. 8d with 2f subsequently leads to the conclusion that the micelles themselves do not contribute to an inflammatory tissue response that can be related to the presence of foreign bodies. When present in their open-switch form and being activated to produce singlet oxygen, ZnTPP/BDTE5 micelles clearly provoked an inflammatory response (compare Figs. 8d with 2f with Figure 8e). Thus the tissue damage observed must be due to generation of singlet-oxygen and not by the presence of the micelles themselves. Tissue responses were similar in all mice comprising one group. Herewith we have demonstrated the ability of BDTE inside a micellar core to regulate in vivo generation of singlet-oxygen by ZnTPP with observable tissue responses.

4. CONCLUSION The use of photochromic switches to prevent systemic side effects due to environmental light conditions is one of the emerging targets in photodynamic therapy45that needs to be solved in order to allow clinical application of photodynamic therapy without causing considerable discomfort to patients. In this study, we incorporated a FRET pair of quenched Zinc−tetraphenylporphyrin (ZnTPP) as a photosensitizer and 1, 2-bis (5-(4-carbonxyphenyl)-2-


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methylthien-3-yl) cyclopent-1-ene (BDTE) as a photochromic switch in PEG-b-PCL micellar nanocarriers. Closing and opening of the switch could be carried out repetitively with minor fatigue of the switch through different thicknesses of porcine tissue. Discrepancies were observed in ZnTPP/BDTE feeding ratios and laser light-activation intensities required to generate singlet-oxygen with biological effects towards mammalian cells and bacteria in vitro and tissue cells in vivo in comparison with EPR evaluations of singlet-oxygen generation. These discrepancies were attributed to the extreme sensitivity towards singlet-oxygen detection based on TEMP-1O2 adduct formation in relation with the amount of singlet-oxygen required to generate biological effects and high absorption of the activation wavelength by fat tissue. However, open-switch form ZnTPP/BDTE5 upon high intensity activation by 405 nm laser light clearly caused tissue damage that could not be observed for closed form micelles, demonstrating the ability of BDTE inside a micellar core to regulate in vivo generation of singlet-oxygen by ZnTPP. Herewith, photodynamic therapy has come one step closer to clinical application for the treatment of superficial tumors or sub-cutaneous infectious biofilms.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: XXXXXXXXXXXX and comprises: Scheme S1

Synthesis route of BDTE.

Scheme S2

Synthesis route of PEG-b-PCL.

Table S1

Cumulative percentage release of ZnTPP or BDTE from different micelles.

Figure S1

NMR spectra of BDTE and intermediate products obtained during its synthesis.


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Figure S2

ESI mass spectrum of BDTE.

Figure S3

1

Figure S4

Calibration for ZnTPP and BDTE in UV/Vis spectroscopy.

Figure S5

Micellar diameters as a function of time in water.

Figure S6

Singlet-oxygen sensor green monitoring of singlet oxygen generation in a cycle of

H NMR spectra of PEG-b-PCL copolymer in CDCl3.

closing and re-opening of the photoswitches in different ZnTPP/BDTE-X micelles.

AUTHOR INFORMATION Corresponding Authors *Linqi Shi, Email address: [email protected] Tel.: +86-22-23506103, Fax: +86-2223503510, *Henk J. Busscher, Email address: [email protected], Tel.: +31-050-3633148, Fax: +3150- 3633159. ORCID Linqi Shi: 0000-0002-9534-795X Henk J. Busscher: 0000-0002-3644-5533 Author Contributions The manuscript was written through contributions of all authors.


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Notes HJB is also director of a consulting company SASA BV. The authors declare no potential conflicts of interest with respect to authorship and/or publication of this article. Opinions and assertions contained herein are those of the authors and are not construed as necessarily representing views of the funding organization or their respective employer(s). ACKNOWLEDGEMENTS This study was financially supported by the National Natural Science Foundation of China (nos. 21620102005, 91527306 and 51390483) and National Natural Science Foundation of Tianjin, China (no. 17JCYBJC16900).


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For Table of Contents Only


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REFERENCES (1) Ackroyd, R.; Kelty, C.; Brown, N.; Reed, M. Photochem. Photobiol. 2001, 74, 656-669. (2) Dougherty, T. J.; Gomer, C. J.; Henderson, B. W.; Jori, G.; Kessel, D.; Korbelik, M.; Moan, J.; Peng, Q. J. Natl. Cancer Inst. 1998, 90, 889-905. (3) van Straten, D.; Mashayekhi, V.; de Bruijn, H. S.; Oliveira, S.; Robinson, D. J. Cancers (Basel) 2017, 9. (4) Maisch, T. Photochem. Photobiol. Sci. 2015, 14, 1518-1526. (5) Dolmans, D. E. J. G. J.; Fukumura, D.; Jain, R. K. Nat. Rev. Cancer 2003, 3, 380-387. (6) Bacellar, I. O. L.; Tsubone, T. M.; Pavani, C.; Baptista, M. S. Int. J. Mol. Sci. 2015, 16, 20523-20559. (7) O'Connor, A. E.; Gallagher, W. M.; Byrne, A. T. Photochem. Photobiol. 2009, 85, 10531074. (8) Ormond, A. B.; Freeman, H. S. 2013, 6, 817-840. (9) Leonard, K. A.; Nelen, M. I.; Simard, T. P.; Davies, S. R.; Gollnick, S. O.; Oseroff, A. R.; Gibson, S. L.; Hilf, R.; Chen, L. B.; Detty, M. R. J. Med. Chem. 1999, 42, 3953-3964. (10) Bonnett R. Chem. Soc. Rev., 1995, 24, 19-33. (11) Zhang, Y.; He, L.; Wu, J.; Wang, K.; Wang, J.; Dai, W.; Yuan, A.; Wu, J.; Hu, Y. Biomaterials 2016, 107, 23-32.


36

Page 37 of 40 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

(12) Hou, L.; Zhang, X.; Pijper, T. C.; Browne, W. R.; Feringa, B. L. J. Am. Chem. Soc. 2014, 136 , 910-913. (13) Liu, G. X.; Xu, X. F.; Chen, Y.; Wu, X. J.; Wu, H.; Liu, Y. Chem. Commun. 2016, 52, 7966-7969. (14) Park, J.; Jiang, Q.; Feng, D. W.; Zhou, H. C. Angew. Chem., 2016, 55, 7188-7193. (15) Chen, D.; Yang, D.; Dougherty, C. A.; Lu, W.; Wu, H.; He, X.; Cai, T.; Van Dort, M. E.; Ross, B. D.; Hong, H. ACS Nano 2017, 11, 4315-4327. (16) Zhang, Y.; Feng, X.; Yuan, S.; Zhou, J.; Wang, B. Inorg. Chem. Front. 2016, 3, 896-909. (17) Gaucher, G.; Dufresne, M. H.; Sant, V. P.; Kang, N.; Maysinger, D.; Leroux, J. C. J. Control. Release. 2005, 109, 169-188. (18) Uhrich, K. E.; Cannizzaro, S. M.; Langer, R. S. Chem. Rev. 1999, 99, 3181-3198. (19) Jin, N.; Morin, E. A.; Henn, D. M.; Cao, Y.; Woodcock, J. W.; Tang, S.; He, W.; Zhao, B. Biomacromolecules 2013, 14, 2713-2723. (20) Liu, Y., van der Mei, H. C., Zhao, B., Zhai, Y., Cheng, T., Li, Y., Zhang Z., Busscher, H. J., Ren, Y., Shi, L. Adv. Funct. Mater, 2017, 27, 1701974. (21) Liu, Y.; Busscher, H. J.; Zhao, B.; Li, Y.; Zhang, Z.; van der Mei, H. C.; Ren, Y.; Shi, L. ACS Nano 2016, 10, 4779-4789. (22) Gao, H. J.; Xiong, J.; Cheng, T. J.; Liu, J. J.; Chu, L. P.; Liu, J. F.; Ma, R. J.; Shi, L. Q. Biomacromolecules 2013, 14, 460-467.


37


Page 38 of 40

(23) DeRosa, M. C.; Crutchley, R. J. Chem. Rev. 2002, 233, 351-371. (24) Tian, H.; Yang, S. Chem. Soc. Rev. 2004, 33, 85-97. (25) Gao, H.; Cheng, T.; Liu, J.; Liu, J.; Yang, C.; Chu, L.; Zhang, Y.; Ma, R.; Shi, L. Biomacromolecules 2014, 15, 3634-3642. (26) Wu, C. F.; Szymanski, C.; McNeill, J. Langmuir 2006, 22, 2956-2960. (27) Das, K. C.; Das, C. K. Res. Commun. 2002, 295, 62-66. (28) Kuo, C. T.; Thompson, A. M.; Gallina, M. E.; Ye, F.; Johnson, E. S.; Sun, W.; Zhao, M.; Yu, J.; Wu, I. C.; Fujimoto, B.; DuFort, C. C.; Carlson, M. A.; Hingorani, S. R.; Paguirigan, A. L.; Radich, J. P.; Chiu, D. T., O. Nat. Commun. 2016, 7, 11468. (29) Das, K. C.; Misra, H. P.; J. Bio. Chem., 1992, 267, 19172-19178. (30) Das, K. C., Misra, H. P. Mol. Cel. Biochem. 1992, 115, 179-185. (31) Visser, J.; Katsonis, N.; Vicario, J.; Feringa, B. L., Langmuir 2009, 25, 5980-5985. (32) Rizzo, M. A.; Springer, G. H.; Granada, B.; Piston, D. W. Nat. Biotechnol. 2004, 22, 445449. (33) Hsu, C. Y.; Chen, C. W.; Yu, H. P.; Lin, Y. F.; Lai, P. S. Biomaterials 2013, 34, 12041212. (34) Herder, M.; Schmidt, B. M.; Grubert, L.; Patzel, M.; Schwarz, J.; Hecht, S. J. Am. Chem. Soc. 2015, 137, 2738-2747. (35) Wang, Y. Q.; Tang, J.; Liu, X. D. Biophys. J. 2015, 108, 321a.


38

Page 39 of 40 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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(36) Hoshino, M; Ozawa, K; Seki, H.; Ford, P. C. J. Am. Chem. Soc, 1993, 115, 9568-9575. (37) Jori, G.; Fabris, C.; Soncin, M.; Ferro, S.; Coppellotti, O.; Dei, D.; Fantetti, L.; Chiti, G.; Roncucci, G. Lasers Surg. Med. 2006, 38, 468-481. (38) Jain, M.; Zellweger, M.; Wagnieres, G.; van den Bergh, H.; Cook, S.; Giraud, M. N., Cardiovasc Ther 2017, 35, (2). (39) Kessel D, Oleinick N L. Photochemistry and photobiology 2017, DOI: 10.1111/php.12857 (40) Kataoka, K.; Harada, A.; Nagasaki, Y. Adv. Drug Delivery Rev. 2001, 47, 113-131. (41) Chandler, D. Nature 2005, 437, 640-647. (42) Klaper, M.; Fudickar, W.; Linker, T. J. Am. Chem. Soc. 2016, 138, 7024-7029. (43) Snyder, J. W.; Skovsen, E.; Lambert, J. D. C.; Poulsen, L.; Ogilby, P. R. Phys. Chem. Chem. Phys. 2006, 8, 4280-4293. (44) Cheong W F, Prahl S A, Welch A J. IEEE J. Quantum Elect. 1990, 26, 2166-2185. (45) Clo, E.; Snyder, J. W.; Ogilby, P. R.; Gothelf, K. V. Chembiochem 2007, 8, 475-481.


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Photoswitchable Micelles for the Control of Singlet-Oxygen Generation

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