Nitrobenzoxadiazole-Appended Cell Membrane Modifiers for Efficient

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Nitrobenzoxadiazole-Appended Cell Membrane Modifiers for Efficient Optoporation with Non-Coherent Light Saya Otake, Kou Okuro, Davide Bochicchio, Giovanni M. Pavan, and Takuzo Aida Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.8b00270 • Publication Date (Web): 15 May 2018 Downloaded from http://pubs.acs.org on May 16, 2018

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Bioconjugate Chemistry

Nitrobenzoxadiazole-Appended Cell Membrane Modifiers for Efficient Optoporation with Non-Coherent Light Saya Otake,† Kou Okuro,*,† Davide Bochicchio,‡ Giovanni M. Pavan,‡ and Takuzo Aida*,†, # †

Department of Chemistry and Biotechnology, The University of Tokyo, 7-3-1 Hongo, Bunkyo-

ku, Tokyo 113-8656, Japan ‡

Department of Innovative Technologies, University of Applied Sciences and Arts of Southern

Switzerland, Galleria 2, Via Cantonale 2c, CH-6928, Manno, Switzerland #

Riken Center for Emergent Matter Science, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan.

Corresponding Authors: [email protected], [email protected]

ABSTRACT:

FL

NBD-BAMPEG2k bearing a nitrobenzoxadiazole (NBD) unit and an oleyl

terminus conjugated via a poly(ethylene glycol) (PEG) spacer (Mn = 2,000), was designed to fluorescently label cell membranes by docking its hydrophobic oleyl terminus. During laser scanning microscopy in a minimal essential medium (MEM), human hepatocellular carcinoma Hep3B cells labelled with

FL

NBD-BAMPEG2k appeared to undergo optoporation at their plasma

membrane. We confirmed this unprecedented possibility by a series of cellular uptake experiments using negatively charged and therefore membrane-impermeable quantum dots (QDs; Dh = 4.7 nm).

ACS Paragon Plus Environment

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Bioconjugate Chemistry 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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Detailed studies indicated that the photoexcited NBD unit can generate singlet oxygen (1O2), which oxidizes the constituent phospholipids to transiently deteriorate the cell membrane. Reference membrane modifiers

FL

NBD-Oleyl and

FL

NBD-BAMPEG8k having shorter or longer hydrophilic

spacers between the NBD and oleyl units showed a little or substantially no optoporation. For understanding these results, one must consider the following contradictory factors: (1) The photosensitized 1O2 generation efficiently occurs only when the NBD unit is in aqueous media, and (2) the lifetime of 1O2 in aqueous media is very short (3.0–3.5 µs).

As supported

experimentally and computationally, the hydrophilic spacer length of FLNBD-BAMPEG2k is optimal for compromising these factors. Further to note, the optoporation using FLNBD-BAMPEG2k is not accompanied with cytotoxicity.

INTRODUCTION Optoporation1,2 is a method to transiently generate pores in cellular membranes by the action of light for activating the intracellular delivery of cell membrane-impermeable substances such as nucleic acids,3–6 proteins,7–10 and nanoparticles.11–13 Optoporation can take advantage of the high spatiotemporal resolution and tissue permeability of light for realizing the site-selective intracellular uptake of guests in vivo.14,15 However, because of its potentially low efficiency, optoporation requires nanosecond or sub-nanosecond focused pulsed lasers, even when sensitizers such as gold16,17 and carbon18 nanoparticles are used. This is unfavorable for practical therapeutic applications. If optoporation using organic dyes that can sensitize the generation of singlet oxygen (1O2) is developed, a variety of lower-energy light sources such as continuous wave lasers, fluorescent lamps, and even light emitting diodes (LEDs) are usable.19 1O2 has been utilized for photodynamic cancer therapy (PDT), where 1O2 is generated within the cellular membrane and


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supposed to oxidize constituent phospholipids20,21 to kill cancer cells.22,23 In contrast, optoporation should not be accompanied by acute cell death and certainly requires a different mechanism from PDT to transiently generate pores in cellular membranes. Here we report the first successful optoporation of cells at their plasma membrane using a nitrobenzoxadiazole (NBD)-based organic dye FLNBD-BAMPEG2k (Figure 1). NBD is a versatile fluorescent motif. Initially, we aimed at developing NBD-based dyes for fluorescently labelling living cells and synthesized

FL

NBD-Oleyl (Figure 1). For intense fluorescence emission, this

molecule has a NBD unit attached to an oleyl terminus that possibly docks into cellular membranes. As a reference, we also synthesized FLNBD-BAMPEG2k (Figures 1 and 2a), which carries NBD at one end of water-soluble poly(ethylene glycol) (PEG; Mn = 2,000) end-functionalized with an oleyl unit. Oleyl-ended PEGs are known to anchor onto cellular membranes at their oleyl terminus and called BAM (biocompatible anchors for membrane).24,25 By means of laser scanning microscopy, we confirmed that both properly labelled human hepatocellular carcinoma Hep3B cells. However, in the case of using FLNBD-BAMPEG2k, the cells under illumination efficiently took up membraneimpermeable exogenous substances such as negatively charged quantum dots (QDs; Figure 2d). Being motivated by this intriguing observation, we investigated the behaviors of

FL

NBD-

BAMPEG2k in comparison with those of its longer-PEG version (FLNBD-BAMPEG8k; Figure 1) and FL

NBD-Oleyl, and confirmed that photoexcited FLNBD-BAMPEG2k specifically gives rise to highly

efficient and non-toxic 1O2-mediated optoporation of Hep3B cells at their plasma membrane (Figures 2c and 2d).


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Figure 1. Molecular structures of cell membrane modifiers:

Page 4 of 22

FL

NBD-BAMPEG2k and

FL

NBD-

BAMPEG8k carrying a fluorescent nitrobenzoxadiazole (NBD) unit and an oleyl terminus conjugated via a poly(ethylene glycol) (PEG) spacer (the PEG Mn = 2,000 and 8,000 for FLNBDBAMPEG2k and FLNBD-BAMPEG8k, respectively); FLNBD-Oleyl carrying a NBD unit and an oleyl terminus conjugated via a triethylene glycol (TEG) spacer; TEG-BAMPEG2k carrying a TEG chain and an oleyl terminus conjugated via a PEG spacer (Mn = 2,000). An oleyl group conjugated with a PEG chain serves as a biocompatible anchor for the membrane (BAM).24,25

Figure 2. Schematic illustration of the proposed mechanism of optoporation using

FL

NBD-

BAMPEG2k as a sensitizer. (a, b) FLNBD-BAMPEG2k binds to the plasma membrane by docking its oleyl terminus into the hydrophobic interior of the membrane. Computational analysis suggested


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that the NBD unit in

FL

NBD-BAMPEG2k can dynamically move back and forth between the

hydrophobic layer of the plasma membrane and the extracellular aqueous phase. (c) Upon photoirradiation, the NBD unit, when located in the extracellular aqueous phase, fluoresces minimally but efficiently generates singlet oxygen (1O2), whereas the NBD unit, when located inside the membrane, intensely fluoresces but generates 1O2 much less efficiently. The 1O2 generated near the membrane oxidizes the constituent phospholipids to increase their polarity and disorder the membrane transiently. (d) Guest quantum dots (QDs) are taken up into living cells through the disordered membrane.

RESULTS AND DISCUSSION FL

NBD-BAMPEG2k (Figure 1) was synthesized according to the following procedures.26

First, Boc-ethylenediamine and NBD-Cl were conjugated by the nucleophilic substitution reaction to produce NBD-labeled Boc-ethylenediamine; the yield was 47%. This was followed by the removal of the Boc group using a dioxane solution of HCl (4 M), wherein the yield was 82%. The resultant amine was then conjugated with PEG (Mn = 2,000) end-functionalized with an Nhydroxysuccinimidyl (NHS) ester and an oleyl group (SUNBRIGHT OE-020CS, NOF Corp.) by the condensation reaction to give

FL

NBD-BAMPEG2k; the yield was 74%.

FL

NBD-BAMPEG8k

(Figure 1) was likewise synthesized using PEG (Mn = 8,000) end-functionalized with an NHS ester and an oleyl group (SUNBRIGHT OE-080CS, NOF Corp.) with a total yield of 25% (3 steps).26 FL

NBD-Oleyl (Figure 1) was synthesized by the conjugation of Boc-protected 3,6-dioxa-1,8-

octanediamine with NBD-Cl and the removal of the Boc group using a dioxane solution of HCl (4 M), followed by the condensation with oleoyl chloride (total yield was 40%, 3 steps).26 TEG-


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BAMPEG2k (Figure 1) was synthesized by the condensation reaction between 3,6,9trioxadecylamine and SUNBRIGHT OE-020CS in 64% yield.26 All the compounds were unambiguously characterized by 1H and

13

C NMR spectroscopy and MALDI-TOF mass

spectrometry.26 Hep3B cells (1.0 × 104 cells) were incubated at 37 °C for 10 min in a minimum essential medium (MEM) containing 10% fetal bovine serum (FBS) and FLNBD-BAMPEG2k (50 µM). When the resultant sample was subjected to confocal laser scanning microscopy (CLSM; λext = 488 nm), Hep3B cells displayed a fluorescence emission assignable to NBD (Figure 3a), indicating the successful immobilization of

FL

NBD-BAMPEG2k onto the cellular membrane (Figure S12a).26 In

order to confirm whether the optoporation indeed occurs or not, a mixture of Hep3B cells and FL

NBD-BAMPEG2k was irradiated for 20 min using a continuous wave laser at 488 nm under

conditions described above, and the reaction mixture was rinsed with Dulbecco’s phosphate buffered saline (D-PBS) to remove

FL

NBD-BAMPEG2k for avoiding spectral interference. Then,

the resultant cells were incubated for 30 min with QDs (50 nM; Dh = 4.7 nm) in MEM (10% FBS) at 37 °C. We confirmed an intense fluorescence emission due to QDs (λext = 405 nm) from the cell interior (Figures 4a and 4g), indicating the successful cellular uptake of QDs. In sharp contrast, an analogous cell sample, prepared without photoirradiation under conditions otherwise identical to the above, did not display any appreciable fluorescence emission due to QDs (Figure S13b).26 The presence of NBD in the modifier is essential since negligible cellular uptake of QDs resulted when TEG-BAMPEG2k without NBD was used instead of FLNBD-BAMPEG2k (Figures 4d and 4g). Notably, even when the incubation with QDs was carried out at 4 °C, the cells modified with FL

NBD-BAMPEG2k (50 µM) took up QDs (Figure 4e). Since energy-dependent cell processes such

as endocytosis are known to be inhibited at 4 °C,27 the cellular uptake of QDs occurs most likely


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via direct penetration through the cell membrane (Figure 2d). Noteworthy, even when a xenon lamp at 490–600 nm was used instead of the continuous wave laser at 488 nm, the optoporation using

FL

NBD-BAMPEG2k for the incorporation of QDs proceeded apparently with a comparable

efficiency (Figure S15).26 No cytotoxicity was observed for Hep3B cells modified with

FL

NBD-

BAMPEG2k (50 µM) before and even after the non-coherent photoirradiation at 490–600 nm (Figure S17).26

Figure 3. (a–c) Confocal laser scanning microscopy (CLSM; λext = 488 nm) images of Hep3B cells after the incubation at 37 °C for 10 min in MEM (10% FBS) containing (a) FLNBD-BAMPEG2k (50 µM), (b) FLNBD-BAMPEG8k (50 µM), and (c) FLNBD-Oleyl (50 µM). Scale bar = 50 µm. (d, e) Molecular dynamics (MD) snapshots with the coarse-grained MARTINI force field of a


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molecular model of a bilayer membrane composed of 1-palmitoyl-2-oleoyl-sn-glycero-3phosphocholine (POPC) containing (d) and (e)

FL

NBD-BAMPEG2k ([FLNBD-BAMPEG2k]/[POPC] = 1/32)

FL

NBD-Oleyl ([FLNBD-Oleyl]/[POPC] = 1/32). (f) The average numbers of

FL

NBD-

BAMPEG2k (50 µM), FLNBD-BAMPEG8k (50 µM), and FLNBD-Oleyl (50 µM) bound to a single cell.

Figure 4. CLSM (λext = 405 nm) images of Hep3B cells incubated in MEM (10% FBS) for 10 min at 37 °C in the presence of (a, e) FLNBD-BAMPEG2k (50 µM), (b, f) FLNBD-Oleyl (50 µM), (c)


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FL

NBD-BAMPEG8k (50 µM), and (d) TEG-BAMPEG2k (50 µM). Micrographs were recorded after

a 30-min incubation in MEM (10% FBS) containing QDs (50 nM) at (a–d) 37 °C and (e, f) 4 °C. Prior to CLSM, Hep3B cells were irradiated with a continuous wave laser at 488 nm for 20 min at room temperature in MEM (10% FBS) and then rinsed with D-PBS. Scale bar = 50 µm. (g) Average fluorescence (FL) intensities of Hep3B cells evaluated from the micrographs (a)–(f), and a micrograph of Hep3B cells after a 30-min incubation at 37 °C in MEM (10% FBS) containing QDs (50 nM; Figure S13a),26 using the ImageJ software.

No report has been made on whether the non-radiative decay of photoexcited NBD in aqueous media28 is accompanied by the generation of

1

O2.

9,10-Anthracenediyl-

bis(methylene)dimalonic acid (ABDA), a water-soluble anthracene derivative with a broad absorption band at 360–400 nm is a representative indicator for 1O2 (Figures 5a and 5b).29 In order to confirm the possibility of generating 1O2 in the optoporation process, we continuously exposed a ABDA (1 mM)-containing PBS (pH 7.4) buffer solution of FLNBD-BAMPEG2k (20 µM) to xenon light at 490–600 nm at 25 °C, whereupon the absorbance at 360–400 nm decreased as a consequence of the conversion of ABDA into its peroxide (Figures 5a and 5b, 0–10 min). This observation demonstrates, for the first time, that photoexcited NBD sensitizes the generation of 1

O2. According to a previously reported method,30 the quantum yield for the generation of 1O2

[Φ(1O2)NBD] by FLNBD-BAMPEG2k was evaluated to be 0.182 from the absorption spectral change profile at 380 nm (Figure 5c, green). Photoexcited NBD is known to preferentially undergo radiative decay in aprotic environments.28 Accordingly, in an aprotic medium such as DMSO, the peroxidation of ABDA upon photoirradiation in the presence of FLNBD-BAMPEG2k proceeded less


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efficiently (Figure 5c, light green) with an estimated Φ(1O2)NBD value of 0.040, which is much smaller than that in PBS [Φ(1O2)NBD = 0.182].

Figure 5.

(a) Singlet oxygen (1O2)-mediated peroxidation of 9,10-anthracenediyl-

bis(methylene)dimalonic acid (ABDA). (b) Changes in absorption spectra of a mixture of ABDA (1 mM) and FLNBD-BAMPEG2k (20 µM) at 25 °C in PBS (pH 7.4) upon photoirradiation at 490– 600 nm (0–10 min). (c) Absorbance change profiles at 380 nm of a mixture of ABDA (1 mM) and

FL

NBD-BAMPEG2k (20 µM) in PBS (pH 7.4, green) and DMSO (light green) at 25 °C upon

photoirradiation at 490–600 nm.

As briefly described above, the decay profile of photoexcited NBD strongly depends on its environment: NBD intensively fluoresces in an aprotic environment but preferentially undergoes a non-radiative decay in a protic environment, to generate 1O2 under aerobic conditions. As shown


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in Figures 3a and 5b, FLNBD-BAMPEG2k can fluorescently visualize Hep3B cells explicitly, and at the same time, sensitize the generation of 1O2 efficiently, thereby raising a question about how FL

NBD-BAMPEG2k can address such contradictory requests. So, we constructed a coarse-grained

(CG) molecular model where FLNBD-BAMPEG2k is dispersed in a bilayer membrane composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)31 and carried out molecular dynamics (MD) simulations using the MARTINI force field.32–34 The simulations suggested that the NBD unit in

FL

NBD-BAMPEG2k, anchored by docking its hydrophobic oleyl terminus into the cell

membrane, moves back and forth dynamically between the extracellular aqueous phase and the hydrophobic interior of the bilayer membrane (Figure 3d). This may account for how

FL

NBD-

BAMPEG2k copes with the excellent fluorescent labeling and 1O2 generation capabilities (Figures 2b and 2c). We also conducted MD simulations of an analogous membrane model featuring FL

NBD-Oleyl instead of

FL

NBD-BAMPEG2k. As expected,

FL

NBD-Oleyl is suggested to remain

entirely embedded in the bilayer membrane (Figure 3e). From the fluorescence intensity (λext = 488 nm, λem = 550 nm) of the cell lysate obtained after the removal of the external media, the average number of the FLNBD-Oleyl bound to a single cell was estimated to be 4.0 × 1010 (Figure 3f), which is 4.6-fold larger than that of

FL

NBD-BAMPEG2k (8.7 × 109 per cell; Figure 3f).

Accordingly, FLNBD-Oleyl served as an even better fluorescent visualizer than FLNBD-BAMPEG2k for Hep3B cells (Figure 3c) but barely induced the optoporation of the plasma membrane (Figures 4b, 4f, and 4g). For further reference to confirm the mechanism, we also synthesized

FL

NBD-BAMPEG8k

(Figure 1) bearing a much longer (Mn = 8,000) water-soluble spacer between the NBD and oleyl units. The cells treated with FLNBD-BAMPEG8k (50 µM) exhibited negligible fluorescence (Figure 3b), although the amount bound to the cells (8.0 × 109 per cell; Figure 3f) was almost comparable


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to that of FLNBD-BAMPEG2k (8.7 × 109 per cell; Figure 3f). Thus, due to its long hydrophilic PEG spacer,

FL

NBD-BAMPEG8k bound to the cell membrane likely exposes its NBD moiety

continuously to the extracellular aqueous phase, and therefore is supposed to actively sensitize the generation of 1O2. However, no cellular uptake of QDs was observed when

FL

NBD-BAMPEG8k

was used for the optoporation of Hep3B cells (Figure 4c and 4g). Considering that the lifetime of 1

O2 in aqueous environments is very short (3.0–3.5 µs),35–37 the NBD unit linked to the long PEG

spacer (Mn = 8,000) is considered to be located away from the membrane, certainly at a distance greater than the diffusible range of 1O2 (60–100 nm in aqueous media),37 so that 1O2 may decay before reaching the membrane to cause its optoporation.

CONCLUSIONS We demonstrated the first dye-sensitized optoporation of living cells at their plasma membrane mediated by 1O2 using non-coherent xenon light. The key player is FLNBD-BAMPEG2k, which has a photoactive NBD unit and an oleyl terminus as a membrane-docking unit conjugated via a hydrophilic PEG spacer. Because of its optimal length,

FL

NBD-BAMPEG2k dynamically

distributes its NBD unit between an extracellular aqueous phase and the plasma membrane (Figure 2b). Consequently,

FL

NBD-BAMPEG2k enables both 1O2-mediated optoporation and fluorescent

visualization of the plasma membrane (Figures 2c and 2d). When the hydrophilic spacer is much longer (FLNBD-BAMPEG8k) or much shorter (FLNBD-Oleyl) than that of virtually no optoporation occurs, although

FL

NBD-BAMPEG2k,

FL

NBD-Oleyl enables fluorescent labeling of Hep3B

cells. Differently from dye sensitizers for PDT, which are designed to generate 1O2 within the membrane to kill cancer cells, FLNBD-BAMPEG2k generates 1O2 in an extracellular aqueous phase


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in proximity to the plasma membrane. This may lead to the virtually no toxicity observed for the 1

O2-mediated optoporation of Hep3B cells. For therapeutic applications, intracellular delivery of

antibody drugs38–40 using the FLNBD-BAMPEG2k-mediated optoporation technique is an interesting subject worthy of further investigation.

MATERIALS AND METHODS

Materials. SUNBRIGHT OE-020CS and SUNBRIGHT OE-080CS were purchased from NOF Corporation. Human hepatocellular carcinoma Hep3B cells (HB-8064) were purchased from ATCC.

Minimal essential medium (MEM) was purchased from Thermo Fisher Scientific.

Trypsin-EDTA (0.25%), CdTe core-type quantum dots functionalized with CO2H, and 9,10anthracenediyl-bis(methylene)dimalonic acid (ABDA) were purchased from Sigma-Aldrich. Dulbecco’s phosphate buffered saline (D-PBS) and Cell Lysis Buffer M were purchased from Wako Pure Chemical Industries. Fetal bovine serum (FBS) was purchased from GE Healthcare. Cytotoxicity LDH Assay Kit-WST was purchased from Dojindo. Rose Bengal (acid red 94) was purchased from Tokyo Chemical Industry. Phosphate buffered saline (pH 7.4) was purchased from Nacalai Tesque.

Cell Membrane Modification. Hep3B cells (1.0 × 104 cells/well; 8-chambered glass substrate) were incubated at 37 ºC for 24 h in MEM (10% FBS, 200 µL) and then rinsed with D-PBS (100 µL × 2). Then, the resultant cells were incubated at 37 ºC for 10 min in MEM (10% FBS) containing

FL

NBD-BAMPEG2k (50 µM),

FL


FL

NBD-Oleyl (50 µM), or

TEG-BAMPEG2k (50 µM).


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Quantification of Modifiers Bound to Cells. Hep3B cells (5.0 × 103 cells/well; 96-well plate) were incubated at 37 ºC for 24 h in MEM (10% FBS, 100 µL) and then rinsed with D-PBS (50 µL × 2). Then, the resultant cells were incubated at 37 ºC for 10 min in MEM (10% FBS) containing FL

NBD-BAMPEG2k (50 µM), FLNBD-BAMPEG8k (50 µM), or FLNBD-Oleyl (50 µM). The medium

was removed from the cell sample, and the cells were lysed using Cell Lysis Buffer M (50 µL). The cell lysate was incubated on ice for 5 min and then subjected to fluorescence spectroscopy (λext = 488 nm, λem = 550 nm). For the calibration, fluorescence intensities (λext = 488 nm, λem = 550 nm) of

FL

NBD-BAMPEG2k (0.5–10 µM),

FL

NBD-BAMPEG8k (0.5–10 µM), and

FL

NBD-Oleyl

(0.5–10 µM) in Cell Lysis Buffer M (50 µL) were measured.

Optoporation for Intracellular Delivery of Quantum Dots (QDs). Hep3B cells modified with FL


FL


FL

NBD-Oleyl (50 µM), or TEG-

BAMPEG2k (50 µM) by the procedure described above were irradiated with a continuous wave laser at 488 nm for 20 min and then rinsed with D-PBS (100 µL × 3). The resultant cells were incubated at 37 °C for 30 min in MEM (10% FBS) containing QDs (50 nM) and then subjected to confocal laser scanning microscopy (λext = 405 nm). The average fluorescence intensity of the cells was calculated from the micrographs using the ImageJ software.

Singlet Oxygen (1O2) Detection. The absorption spectra of a mixture of ABDA (1 mM) and FL

NBD-BAMPEG2k (20 µM) in PBS (pH 7.4) buffer at 25 °C was measured (0–20 min) upon

continuous exposure to light at 490–600 nm. Likewise, the absorption spectra of a mixture of ABDA (1 mM) and Rose Bengal in PBS (pH 7.4) buffer at 25 °C was measured (0–5 min) upon


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continuous exposure to light at 546 ± 5 nm. Similar experiments using DMSO instead of PBS buffer were carried out under conditions otherwise identical to the above. The quantum yield for the generation of 1O2 [Φ(1O2)NBD] by

FL

NBD-BAMPEG2k was calculated using the following

equation: Φ(1O2)NBD = Φ(1O2)RB·(kNBD/kRB)·(IRB/INBD), where Φ(1O2)RB is the quantum yield for the generation of 1O2 by Rose Bengal [in PBS: Φ(1O2)RB = 0.75, in DMSO: Φ(1O2)RB = 0.16],41 and IRB and INBD are total absorption intensities of Rose Bengal and FLNBD-BAMPEG2k at 546 ± 5 nm and 490–600 nm, respectively.

Coarse-Grained Molecular Dynamic Simulation. A coarse-grained (CG) molecular model of FL

NBD-BAMPEG2k was constructed using the CG MARTINI force field.32–34 Likewise, a molecular

model of a bilayer membrane composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) was constructed. Then, the lipid bilayer composed of 2048 POPC molecules was equilibrated in water. To the resultant lipid bilayer, 64

FL

NBD-BAMPEG2k molecules were

dispersed, and the system was equilibrated at 310 K in the NpT ensemble for 1 µs. For the simulations, the GROMACS molecular dynamics suite42 was used. For production runs, the md integrator with a time step of 20 fs, the v-rescale thermostat43 with a time constant of 2 ps, and the Parrinello-Rahman barostat44 with a time constant of 8 ps were used.

Cytotoxicity Assay. Cytotoxicity was evaluated based on the enzymatic activity of lactose dehydrogenase (LDH). Hep3B cells (5.0 × 103 cells/well; 96-well culture plate) were incubated in MEM (10% FBS, 100 µL) at 37 ºC for 24 h. The cells were rinsed with D-PBS (50 µL × 2) and incubated for 10 min at 37 °C in MEM (200 µL) containing

FL

NBD-BAMPEG2k (50 µM). Then,

the cells were exposed to light at 490–600 nm for 20 min and incubated for 3 h at 37°C. The


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Page 16 of 22

medium (100 µL) of the resultant cell sample was added to Working Solution (100 µL) of LDH Assay Kit-WST. After a 30-min incubation at room temperature, Stop Solution (50 µL) of LDH Assay Kit-WST was added to the mixture, and the reaction mixture was subjected to absorption spectroscopy at 490 nm. The LDH was normalized to that of the lysate obtained from untreated Hep3B cells.

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS This work was supported by Grant-in-Aid for Young Scientists (B) (26810046) to K.O. and partially supported by Grant-in-Aid for Specially Promoted Research (25000005) to T.A.

ASSOCIATED CONTENT Supporting Information. Synthesis of FLNBD-BAMPEG2k, FLNBD-BAMPEG8k, FLNBD-Oleyl, and TEG-BAMPEG2k; 1H NMR,

13

C NMR, and MALDI-TOF mass spectral data; confocal laser

scanning micrographs; electronic absorption spectra; and related experimental procedures (PDF)

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Xiong, R., Samal, S. K., Demeester, J., Skirtach, A. G., De Smedt, S. C., and Braeckmans, K. (2016) Laser-assisted photoporation: fundamentals, technological advances and applications. Adv. Phys. X 1, 596–620.

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