Two-Photon PhotosensitizerâPolymer Conjugates for Combined

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Two-photon photosensitizer-polymer conjugates for combined cancer cell death induction and two-photon fluorescence imaging: structure/photodynamic therapy efficiency relationship Cristina Cepraga, Sophie Marotte, Edna Ben Daoud, Arnaud Favier, Pierre-Henri Lanoe, Cyrille Monnereau, Patrice Baldeck, Chantal Andraud, Jacqueline Marvel, Marie-Therese Charreyre, and Yann Leverrier Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.7b01090 • Publication Date (Web): 11 Oct 2017 Downloaded from http://pubs.acs.org on October 12, 2017

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Two-photon photosensitizer-polymer conjugates for combined cancer cell death induction and two-photon fluorescence imaging: structure/photodynamic therapy efficiency relationship

Cristina Cepraga,1,2,3 Sophie Marotte,1,4 Edna Ben Daoud,1,4 Arnaud Favier,1,2* Pierre-Henri Lanoe,3 Cyrille Monnereau,3 Patrice Baldeck,3 Chantal Andraud,3 Jacqueline Marvel,4 Marie-Thérèse Charreyre,1,2* Yann Leverrier4*

1) Univ Lyon, École Normale Supérieure de Lyon, CNRS, Laboratoire Joliot-Curie, F-69364 Lyon, France 2) Univ Lyon, INSA-Lyon, Université Claude Bernard, CNRS, Laboratoire Ingénierie des Matériaux Polymères, F-69621 Villeurbanne, France. 3) Univ Lyon, École Normale Supérieure de Lyon, Université Claude Bernard, CNRS, Laboratoire de Chimie, Site Monod, 46 allée d’Italie, F-69364, Lyon, France. 4) Univ Lyon, INSERM, ENS de Lyon, CNRS, Université Claude Bernard, Centre International de Recherche en Infectiologie (CIRI), U1111, F-69007 Lyon, France.

(PHL) Present address: Université Pierre et Marie Curie, Institut Parisien de Chimie Moléculaire F- 75005 Paris, France. Marie-Thérèse Charreyre and Yann Leverrier share senior co-authorship.

Keywords: photosensitizer-polymer conjugate, zwitterionic polymer, two-photon absorption chromophore, optical imaging probe, two-photon fluorescence microscopy, photodynamic therapy. 1 ACS Paragon Plus Environment

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ABSTRACT One of the challenges of photodynamic therapy is to increase the penetration depth of light irradiation in the tumor tissues. Although two-photon excitation strategies have been developed, the two-photon absorption cross-sections of clinically used photosensitizers are generally low (below 300 GM). Besides, photosensitizers with high cross-section values are often non water-soluble. In this research work, a whole family of photosensitizer-polymer conjugates was synthesized via the covalent binding of a photosensitizer with a relatively high cross-section along a biocompatible copolymer chain. The resulting photosensitizer-polymer conjugates were water-soluble and could be imaged in cellulo by two-photon microscopy thanks to their high two-photon absorption cross-sections (up to 2600 GM in water, in the NIR range). In order to explore the structure/photodynamic activity relationship of such macromolecular photosensitizers, the influence of the polymer size, photosensitizer density and presence of charges along the polymer backbone was investigated (neutral, anionic, cationic and zwitterionic conjugates were compared). The macromolecular photosensitizers were not cytotoxic in the absence of light irradiation. Their kinetics of cellular uptake in B16F10 melanoma cell line were followed by flow cytometry over 24h. The efficiency of celldeath upon photo-activation was found to be highly correlated to the cellular uptake in turn correlated to the global charge of the macromolecular photosensitizer which appeared as the determining structural parameter.

INTRODUCTION Photodynamic therapy (PDT) has emerged as a promising alternative to radio- and chemotherapy of cancers.1,2 It is less invasive than surgery, does not induce toxicity for organs and can be applied to areas not eligible for surgery. PDT is based on the combined use of a photosensitizer (PS), oxygen and light irradiation. Photo-activation of the PS, followed by energy transfer to molecular oxygen, induces the generation of highly toxic reactive oxygen species such as singlet oxygen able to kill cancer cells. Beyond direct cancer cell death induction, PDT can also cause a local inflammation and activation of the immune system that could participate to the tumor destruction.3 Various PS families have been studied and several molecules are used in clinical trials.1,4 It is generally considered as an advantage if the PS can serve both as therapeutic agent for PDT and as imaging agent for fluorescence microscopy.4,5 Then, it can enable i) visualization of 2 ACS Paragon Plus Environment

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the targeted zone before treatment, thus confirm the presence of the PS and the level of uptake within the cancer tissue and ii) monitoring of treatment response. To combine sensitive imaging properties with selective destruction of cells, an ideal PS should exhibit a significant fluorescence emission. Although porphyrin derivatives (widely used in PDT) are fluorescent, they possess small Stokes shifts that limit their application for fluorescence imaging.1 In addition, classical optical imaging is limited by the penetration depth of light into tissues due to strong absorption and scattering of light. To overcome these limitations, irradiations in the biological transparency window are preferred (wavelength range of 700-900 nm). Then, twophoton excitation (TPE) strategies (using NIR photons) have been developed to image deeper tissues.2,6,7 Moreover, since TPE takes place only at the laser focus (1 µm3), a very precise control of the excitation volume is possible (spatial targeting). TPE is also advantageous for future developments of PDT in clinics.8,9 However, most clinically approved PS (porphyrins, phthalocyanines) possess relatively low two-photon absorption (TPA) cross-sections, in the range of 1-300 Göppert-Mayer (GM).10 Then, the design of new photosensitizers with high TPA cross-sections has been a challenge over the last 15 years.4,11,12 One drawback of chromophores (including PS) that exhibit high TPA cross-sections is often their hydrophobic conjugated structure that can lead to aggregation in aqueous media and alteration of photophysical properties.1,13 Therefore, various strategies have been proposed to overcome this problem such as: i) to encapsulate the PS inside mesoporous silica nanoparticles,14 polymer micelles15,16 or organic nanoparticles;9,17 ii) to grow dendritic structures or hydrophilic polymer chains from the PS core;18-20 iii) to covalently bind the PS along water-soluble polymer chains either biocompatible21,22 or biodegradable.23 We have recently applied this last strategy to the design of an original macromolecular PS21 via the covalent anchoring of several molecular PS based on a dibromobenzene (DBB) core (exhibiting a relatively high TPA cross-section, in the order of 400 GM in chloroform) onto a water-soluble polymer chain based on poly(N-acryloylmorpholine). This biocompatible polymer possesses properties similar to PEG.24,25 The resulting PS-polymer conjugate (harboring an average number of 4.4 PS per chain, for a chain of 12 900 g.mol-1) was fully water-soluble, did not induce any cell toxicity in the absence of light irradiation and led to melanoma cell death upon photo-activation in vitro (at 2 µM equivalent PS). Those promising results challenged us to explore the structure/PDT efficiency relationship of a whole family of PS-polymer conjugates, based on that same PS. We decided to investigate 3 ACS Paragon Plus Environment

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the influence of the polymer size (molecular weight of the chain), photosensitizer density and absence/presence of charges along the polymer backbone (either negative or positive charges or both). It has been reported that the presence of both hydrophilic and hydrophobic groups in the PS structure plays an important role in tumor uptake.1 Moreover, a mixture of negative (carboxylate) and positive (imidazolium) charges along polymer arms surrounding the PS resulted in an enhanced cell uptake of the star PS in comparison with a neutral counterpart.26 Our objectives were to identify the best photosensitizer-polymer conjugates among the neutral, anionic, cationic and zwitterionic ones in this family, regarding the following properties i) high water-solubility; ii) high two-photon absorption cross-sections for twophoton imaging of cellular uptake using low laser power; iii) rapid (i.e. within a few hours) and high cellular uptake; iv) non-cytotoxicity per se in the absence of light irradiation; v) high efficiency of photo-induced cell death upon PS photo-activation.

EXPERIMENTAL SECTION Materials. All chemicals (including 4-(2-aminoethyl)morpholine (AEM, 99%) and (2-aminoethyl) trimethylammonium chloride hydrochloride (AETMAC, 99%)) and solvents were purchased from Sigma Aldrich or ACROS at the highest purity available and used without further purification.

Random

and

block

copolymers,

poly(N-acryloylmorpholine-co-N-

acryloxysuccinimide) (P(NAM-co-NAS)) and P(NAM-co-NAS)-b-PNAM,

with 60/40

NAM/NAS molar ratio in the P(NAM-co-NAS) block, were synthesized according to previously reported RAFT polymerization procedure (Table 1).27

Photosentisizer-polymer conjugate synthesis DBB-polymer conjugates were synthesized following a previously reported procedure.21 The reaction medium was precipitated in diethyl ether in order to purify the polymer-dye conjugate. In the case of the AETMAC-containing conjugates, introduction of AETMAC was carried out before post-treatment of the remaining activated ester functions. A given volume of

AETMAC

solution

(24.4

mg.mL-1)

in

a

dimethylformamide/water

mixture

(DMF/H2O:90/10:V/V) was added to 1 mL solution of polymer-dye conjugate in DMF (25 mg.mL-1). The volume was chosen depending on the targeted QA value. Finally, 2 equivalents of triethylamine (TEA) per AETMAC were added and the reaction proceeded at 40°C for 24h. Post-treatment of the remaining activated ester functions was performed either by 4 ACS Paragon Plus Environment

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aminoethylmorpholine capping28 or by hydrolysis.21 All conjugates were purified by dialysis against deionized water and dried by lyophilization.

Characterization Methods. NMR. Spectra (1H) were recorded in deuterated chloroform (CDCl3) at 298K on a BRUKER® AC 200 operating at 200.13 MHz. Data in parts per million (ppm) are reported relative to tetramethylsilane as internal standard. SEC/UV. Binding yields were determined (before post-treatment) by size exclusion chromatography (SEC) using UV detection at 400 nm. SEC apparatus consisted in Varian ProStar Dynamax 800 system fitted with Styragel HR4E linear column (7.8x300 mm²). Eluent was DMF with LiBr (0.05 mol.L-1). Samples were prepared by diluting 50 µL of reaction medium in 1 mL of eluent and injected via a 30 µl sample loop. Data acquisition and treatment was performed using Galaxie software.

UV/Visible absorption. Spectra were recorded on a Jasco V-670 spectrophotometer at room temperature using quartz cells with 10 mm optical paths. Extinction coefficients ε were calculated using Beer-Lambert law at 390 nm.

Fluorescence spectroscopy. Fluorescence emission spectra were recorded on a Horiba-Jobin Yvon Fluorolog-3 spectroﬂuorimeter, at room temperature using a quartz cell with 10 mm optical path. Excitation wavelength was 390 nm. Fluorescence quantum yields, Q, were calculated according to the approximate Equation (1) for diluted solutions having an optical density lower than 0.1, where A is the absorbance at the excitation wavelength λ, n is the refractive index and D is the integrated luminescence intensity. “r” and “x” stand for reference and sample, respectively. Reference was Coumarin 153 (Q=0.45 in methanol). n2 D A x = r × x× x 2 D Q A r x nr r

Q

(1)

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Singlet oxygen generation Generation of singlet oxygen from the DBB-polymer conjugates was recorded in CHCl3 solution by detection of 1O2 phosphorescence at 1270 nm. The instrumental setup was similar to the one used for fluorescence spectroscopy (excitation wavelength = 390 nm). The singlet oxygen emission spectrum was recorded between 1225nm and 1325nm using SPEX® DSSIGA020L InGaAs detector which operates at liquid nitrogen temperature to minimize thermal noise. The slit apertures were chosen much larger than for fluorescence measurements: entrance slit=14, exit slit=40; integration time=1s. Quantum yield of singlet oxygen generation (Φ∆) was calculated using the Stern-Volmer model according to the approximate Equation (2) for diluted solutions having an optical density lower than 0.1, where Φ∆_r/Φ∆

is the ratio of the singlet oxygen luminescence quantum yield, Ar/A is the ratio of

absorbance at the excitation wavelength and Lr/L is the ratio of the luminescence (intensity or area). “r” stands for reference (phenalenone, Φ∆_r =1 in chloroform). Φ∆ = Φ∆_r ×

Ar L × A Lr

(2)

Two-Photon Absorption (TPA) spectroscopy Two-photon Absorption (TPA) cross-section spectra were obtained between 700-950 nm by up-conversion fluorescence using a Ti:sapphire femtosecond laser (Coherent Verdi-V5 pump coupled with a Femtoseconde MIRA 900 cavity) with an intensity power fixed at 50 mW and a S2000 Ocean Optics Spectrometer. The excitation beam was collimated over the cell (10mm) with a pulse frequency of 80MHz. Incident beam intensity was adjusted to 50mW in order to ensure an intensity-squared dependence of the ﬂuorescence over the whole spectral range. Detector integration time was set at 1s. Measurements were performed at room temperature in chloroform or water and at a concentration of 10-4 mol.L-1 in chromophore. Calibration was performed by comparison with the published two-photon absorption spectrum of Coumarin-307 (σTPA = 0.56 in ethanol) between 700 and 900 nm.29 2 I FlTwoPhoton ∝ σ TPA × I laser × Q × c × K laser

(3)

TPA cross-section (σTPA) values were obtained using Equation (3), where IFl represents the two-photon fluorescence intensity; Ilaser the LASER intensity at 50mW, Klaser a constant depending on the measurement set-up; Q the fluorescence quantum yield, c the concentration of chromophore (c=10-4 mol.L-1) and n the refractive index. The measurements were conducted in an intensity regime where the fluorescence signal showed a quadratic 6 ACS Paragon Plus Environment

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dependence on the intensity of the excitation beam, as checked for all samples. The uncertainty in the measured cross-sections is about 15% in CHCl3 and 20% in water.

Biological evaluation Cell cultures and reagents Murine IL-3 dependent Baf3 cells30 and B16-F10 melanoma cells were cultivated in DMEM medium (Invitrogen Life Technologies) supplemented with 6% heat-inactivated Fetal Bovin Serum (Lonza, Belgium) and 10 mg.L-1 gentamicin (Invitrogen). Baf3 cells are non-adherent cells and were grown in suspension with 5% WEHI 3B cell-conditioned medium as a source of IL-3. B16-F10 cells are adherent cells and were grown as sub-confluent monolayer. Cells were cultured at 37°C and 7% CO2 in a humidified atmosphere. Cells were pre-incubated for 30 minutes with zVAD-fmk (250 µM, BACHEM) before irradiation. Cells were incubated with Staurosporine (STS, 1µM, Sigma Aldrich) for 24 h. For the various DBB-polymer conjugates, comparison of cell uptake and photo-induced cell death was performed using an equivalent DBB concentration, assuming that DBB emitted the same luminescence whatever the conjugate.

Cell mortality analysis by flow cytometry Baf3 cells are non-adherent and were collected by pipetting. B16-F10 cells as adherent cells loosen their attachment or detach from the substratum during apoptosis. Therefore, cell mortality among B16-F10 cells was analyzed by pelleting floating cells and adherent cells harvested by trypsinization. To measure cell mortality, cells were collected and incubated with propidium iodide (PI, Sigma, 1 mg.L-1), a standard flow cytometry viability probe. At least 5 000 cells were analyzed immediately by flow cytometry (Canto flow cytometer (Becton Dickinson Bioscience) or MACSQuant flow cytometer (Miltenyi Biotec)). This method enables to distinguish between viable cells with an intact plasma membrane that exclude PI and non-viable cells that are permeable to PI. Alternatively, cell mortality was assessed using the scattering properties of the cells. Forward scatter (FSC) determines the size of the cells from the scattered light in the direction of the laser path. Side scatter (SSC) determines the granularity of the cells from the scattered light at 90 degrees to the laser path. Dead cells can be distinguished from live cells as they have lower FSC and higher SSC. Cytometry data were analyzed using FlowJo Software (Tree Star, Inc., USA) after excluding debris. 7 ACS Paragon Plus Environment

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Cell uptake. To measure photosensitizer uptake, 2×105 Baf3 cells.mL-1 were incubated at 37 °C with a given DBB-polymer conjugate for the indicated period of time. Cells were analyzed using a Canto flow cytometer (Becton Dickinson Bioscience) or a MACSQuant flow cytometer (Miltenyi Biotec) with a 405 nm laser and a 450 nm emission filter to detect incorporation of DBB derivatives. At least 5 000 cells were recorded per assay. Cytometry data were analyzed using FlowJo Software (Tree Star, Inc., USA) after excluding debris.

Photo-induced cell death. To assay the phototoxicity of the different DBB-polymer conjugates in cell culture systems, 2×105 Baf3 cells.mL-1 were seeded into 96-well plates and cultured for 24 h in the presence of a given conjugate at the indicated concentration. B16-F10 were seeded at 6×104 cells.mL-1 into 24-well plates, grown over-night and incubated for 3 h or 24 h with a given conjugate. Cells were irradiated at 8 J.cm-2 (Baf3 cells) or 16 J.cm-2 (B16-F10 cells) at 365 nm (Bio-Sun, Vilber Loumart, Marne la vallée, France). Cell mortality was assessed 1 h or 5 h later by flow cytometry.

In cellulo mono- and two-photon fluorescence imaging. B16-F10 cells were plated on glass coverslips (2×104 cells/coverslip) and allowed to adhere overnight. Cells were then incubated for 24h with 13K4-H (10-9 mol.L-1 DBB). DRAQ5 (10 µM, Alexis Biochemicals) was added for the last 3 minutes of culture to stain the nuclei. Cells were washed with PBS and fixed with 4% paraformaldehyde (Sigma-Aldrich). The cell surface was stained using anti-CD44-FITC (clone IM7, 0.5 µg.mL-1, BD Biosciences) in PBS for 2 h at room temperature. Specimens were mounted (Antifade Kit, Molecular Probes) and examined using an inverted LSM 710 Zeiss confocal microscope (Carl Zeiss, Germany) with 63X/1.4 objective. LSM 710 confocal uses variable spectral detection system. 488 nm and 633 nm lasers were used for anti-CD44-FITC and DRAQ5 excitation respectively. Imaging of 13K4-H was performed using one-photon excitation (488 nm) or two-photon excitation (750 nm) with the tunable infrared laser Ti:Sapphire Chameleon Ultra II (690 nm-1040 nm). Images were processed with ImageJ software, a public domain image processing and analysis program.


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RESULTS AND DISCUSSION Synthesis of various DBB-polymer conjugates A whole family of PS-polymer conjugates were synthesized using macromolecular structures closely related to a previously reported polymer architecture.21 The latter, based on a short statistical copolymer backbone of about 13 000 g.mol-1 and harboring an average number of 4.4 DBB PS per chain, was used here as a reference (13K4-H, Table 2). It had shown promising properties in terms of cell internalization and efficiency of photo-induced cell death. In a next step, in order to systematically investigate structure/properties relationships, we decided to explore the influence of several structural parameters on the final biological properties of the conjugate (Figure 1): (i) the size of the polymer chain, (ii) the architecture of the backbone (statistical versus block copolymer), (iii) the number of covalently bound PS per chain, (iv) the absence or the presence of a variable number of charged groups per chain (positive and/or negative). To reach such objectives, three kinds of polymer backbones were used (Table 1), either statistical P(NAM-co-NAS) or block P(NAM-co-NAS)-b-PNAM copolymer backbones, with molecular weight varying from 12 900 to 35 000 g.mol-1. An increasing number of DBB PS were anchored on the activated ester units (NAS) along the copolymer chains (from 3 to 10 DBB per chain, Table 2). Binding yield was 70-80 %. In some cases, a quaternary ammonium (QA), 2-aminoethyl-trimethylammonium chloride hydrochloride, was then bound to introduce positive charges (from 3 to 30 per chain). Finally, a post-treatment of the remaining activated ester units was carried out, either via a capping strategy (using aminoethyl morpholine, AEM) resulting in mainly neutral side-groups, or via a hydrolysis strategy resulting in the presence of negative charges (potential carboxylate groups (COO-), from 24 to 56 per chain). One of the conjugates, 35K3-H-QA20%, harboured both positive and negative charges along the


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chain. The characteristics of the ten different DBB-polymer conjugates (neutral, cationic, anionic or zwitterionic) prepared according to this general guideline are reported in Table 2.

Figure 1: The various DBB-polymer conjugate architectures synthesized from the P(NAMco-NAS) and P(NAM-co-NAS)-b-PNAM, statistical and block copolymer backbones, respectively, after post-treatment via a capping (AEM) or a hydrolysis (H) strategy. x and y = average degree of polymerization of NAM and NAS units, respectively, in the first block; nC = average number of DBB photosensitizers per chain; QA= average number of quaternary ammonium groups per chain; z = average degree of polymerization of NAM units in the second block.

The DBB-polymer conjugates were first characterized by SEC with a UV-Vis detector (determination of the binding yield). After purification by precipitation (to remove the free dye) and dialysis, the conjugates were analyzed by 1H NMR spectroscopy. Spectra of the hydrolyzed conjugates were similar to that of the reference sample 13K4-H, whereas spectra of the AEM-capped conjugates confirmed a whole capping (Figure S1 in ESI).


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Table 1 Molar mass and dispersity data for the copolymer samples used to prepare the DBBpolymer conjugates.

Polymer sample

Mn a

(g.mol-1)

Mw/Mn

a

Xn

NAM/NAS molar ratio (nNAS)b

P1 Stat

P(NAM-co-NAS) 12 900

1.05

85

60/40 (34)

P2 Block

P(NAM-co-NAS)-b-PNAM 21 600 + 7 800 = 29 400

1.20

195

60/40 (56)

1.18

235

60/40 (60)

P3 Block

P(NAM-co-NAS)-b-PNAM 23 000 + 12 000 = 35 000

a

Determined by aqueous SEC/Light Scattering Detector

b

Average number of NAS units per chain


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Table 2 Physico-chemical characteristics of the DBB-polymer conjugates.

Polymer probe (Mn) g.mol-1

13K4-AEM (16 700) 13K4-H (13 800) 13K10-AEM (20 400) 13K10-H (18 100)

P1 12 900

Block (28 800)

(33 800)

35K3-AEM-QA5% (37 900) 35K3-AEM-QA20% (38 300) 35K3-AEM-QA50% (39 100)

35K3-H-QA20% (33 200)

81 %

c

(global charge)

80 %

c

AEM capping

/

4.1

4.4

12 900

29 400

4.6

d

c

12 900

Posttreatment

nCOO- and nQAb/chain

4.4

81 %

P3

35K4-H

Average number of DBB/ chain

12 900

P2

29K5-H

Block

Polymer DBB backbone binding Mn yielda g.mol-1

4.8

9.8 9.5

hydrolysis AEM capping

80 %

9.8

hydrolysis

83 %

4.6

hydrolysis

nCOO- = 30 (-30) / nCOO- = 24 (-24) nCOO- = 51 (-51) nCOO- = 56

95 %

4.0

hydrolysis

35 000

70%

2.8

QA: 5%NAS + AEM

nQA = 3

35 000

70%

2.8

QA:20%NAS + AEM

nQA = 12

35 000

70%

2.8

QA:50%NAS + AEM

nQA = 30

2.8

QA:20%NAS +hydrolysis

35 000

35 000

70%

(-56) (+3)

(+12)

(+ 30) nQA = 12 nCOO- = 45 (-33)

a

determined by SEC/UV nCOO- and nQA = average number of carboxylate and quaternary ammonium groups, respectively, per chain c determined by 1H NMR spectroscopy d determined by UV-Vis

b


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Photophysical characterization of the DBB-polymer conjugates The photophysical properties of the various DBB-polymer conjugates could be characterized in water thanks to the hydrophilicity of the polymer chain (free DBB is not water-soluble). In particular, we focused on their absorption and fluorescence emission spectra (Figure 2), molar extinction coefficient, fluorescence quantum yield and brightness (Table 3). The two-photon absorption spectra could also be determined in water from 700 to 900 nm (Figure 3). In the case of the AEM-capped conjugate harboring 4.4 DBB per chain (13K4-AEM), it was possible to carry out the characterization both in water and in chloroform. Moreover, for some representative conjugates, fluorescence quantum yields were also determined in dioxane (Table 3) known as a solvent which polarity best mimics that of intracellular media.31 First, comparison of AEM-capped conjugates with free DBB in the same solvent (13K4-AEM and 13K10-AEM, in chloroform) indicated a higher brightness of the DBB-polymer probes than that of free DBB (up to 6 fold), although quantum yield moderately decreased (from 0.23 for free DBB to 0.18 and 0.16) when labeling density increased, which we attribute to chromophore self-quenching.32 Moreover, the two-photon absorption was also higher for the DBB-polymer conjugates than for free DBB (Figure S2 in ESI). Comparison of a same AEMcapped conjugate (13K4-AEM) in different solvents (chloroform and water) indicated a much smaller brightness in water as a consequence of a drastic decrease of the quantum yield (from 0.18 to 0.01) resulting from solvent induced aggregation of the hydrophobic dyes. However, a significantly higher TPA cross-section (σTPA) value ca 1400 GM was measured in water instead of 600 GM in chloroform. Second, a possible influence of the post-treatment (AEM-capping vs. hydrolysis) on the photophysical properties could be evaluated with 13K4-AEM and 13K4-H since both kinds of probes could be studied in water. In fact, not any influence of the presence of the carboxylate side-groups (instead of ethyl morpholine groups) was observed, neither on absorption and emission spectra (λmax Abs = 384 nm and λmax Em = 510 nm, Figure 2), nor on molar extinction coefficient, fluorescence quantum yield, brightness and σTPA values (Table 3).


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1.2

13K4-H 13K10-H 13K4-AEM

1

Normalized spectra

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0.8 0.6 0.4 0.2 0 300

350

400

450

500

550

600

650

700

Wavelength (nm)

Figure 2: Absorption and fluorescence emission spectra in water of 13K4-H (blue), 13K10-H (green) and 13K4-AEM (pink) DBB-polymer probes. The sharp peak centered at 450 nm corresponds to a Stokes Raman peak of water. Third, we investigated the influence of the photosensitizer density (average number of bound DBB molecules related to the total monomer units per polymer chain). In fact, in a previous study with another kind of TPA chromophore, we noticed a significant influence of the labeling density on the brightness of the corresponding polymer probe in water.33 Here, we could carry the study with the hydrolyzed DBB-polymer conjugates (all analyzed in water). When the number of chromophores per chain increased from 4 to 10 for a same polymer length (DBB density of 5.2 and 11.5%, respectively, for 13K4-H and 13K10-H), both brightness and TPA cross-section increased, in the same proportion (Table 3 and Figure 3). The TPA cross-sections in water were above 1000 GM and reached up to 2600 GM (at 740 nm) for 13K10-H. These values are very high in comparison with the cross-sections of commercial photosensitizers in water (generally between 1-300 GM).10 Influence of the photosensitizer density could also be evaluated with other hydrolyzed conjugates when the polymer chain length increased for a similar number of DBB per chain (i.e. decreasing labeling density from 5.2 to 3.3 and 2.7%, for 13K4-H, 29K5-H and 35K4-H, respectively). While absorption and emission spectra in water remained similar (Figure S3 in ESI), fluorescence quantum yield increased, both in water (0.01 to 0.05 and 0.06) and in dioxane (0.10 to 0.11 and 0.13), as expected.34 The highest brightness in water (9100) was provided by 35K-4H conjugate that exhibited a labeling density below 3%.


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Table 3 Photophysical characteristics of the DBB-polymer probes.

Polymer probe

Free DBB

13K4-AEM (16 700) 13K4-AEM

solvent

CHCL3

CHCl3

water

(16 700) 13K4-H

water

(13 800) 13K10-AEM

CHCl3

(20 400) 13K10-H

water

(18 100) 29K5-H Block (28 800)

water

35K4-H Block (33 800)

water

Epsilon

Brightness /

DBB/ chain

COO-/ chain

(density, %)a

(density, %)a

/

/

0.23 (0.18)d

60,000

/

0.18

230,000

/

< 0.01

190,000

< 1900

(-) 30

< 0.01

(35.3)

d

(0.10)

180,000

< 1800

/

0.16

480,000

< 0.01

335,000

< 3300

175,000

8700

4.4 (5.2) 4.4 (5.2) 4.4 (5.2) 9.8 (11.5)

-1

-1

Qb

at

M .cm

±20%

λmax Abs

(amplifycation factor)

±20%

9.8

(-) 24

(11.5)

(28.2)

4.6

(-) 51

0.05

(3.3)

(36.4)

(0.11)d

4.0

(-) 56

0.06

(2.7)

(37.3)

(0.13)d

σTPA GMc ±20% (λ Exc)

13,300

400

(1)

(730 nm)

41,200

500

(3.1)

(730 nm) 1400 (740 nm) 1400 (740 nm)

76,300

1600

(5.7)

(730 nm)

150,000

2600 (740 nm) /

/ 9100

a

density: Average number of DBB photosensitizers (or COO-) related to the total monomer units per polymer chain (for the block copolymers: total monomer units in the random block). b fluorescence quantum yield c σTPA = TPA cross-section ; 1 GM = 10-50 cm4.s.photon d fluorescence quantum yield determined in dioxane


Biomacromolecules

3000

13K4-H

TPA Cross Section (GM)

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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2500

13K10-H 13K4-AEM

2000 1500 1000 500 0

720 740 760 780 800 820 840 860 880 900 920

Wavelength (nm)

Figure 3: Two-photon absorption cross-section spectra for several DBB-polymer conjugates, 13K4-H (dark blue), 13K4-AEM (blue) and 13K10-H (green), in water.

Finally, the conjugates were tested for singlet oxygen generation: the singlet oxygen emission spectrum (Figure S4 in ESI) indeed exhibited the expected peak in the infrared range, with a maximum wavelength at 1275 nm. Singlet oxygen generation quantum yield reached rather high values, 47% for 13K4-AEM and 13K10-AEM conjugates, in comparison with 33% for free DBB. We attribute this slight increase to the presence of a NH2 group on the free photosensitizer, known to be involved in radiationless singlet-oxygen vibrational quenching;35 this deactivation pathway is suppressed in the polymer-bound photosensitizer, resulting in a singlet oxygen generation efficiency close to that of an alkyl substituted DBB analogue (53%), as studied in a previous article.15

Photo-induced cell death efficiency with the various DBB-polymer conjugates The different DBB-polymer conjugates with various sizes, labelling densities, side-groups and global charges have been designed to assess influence of these structural parameters on 1) cytotoxicity, 2) cellular uptake, and finally 3) ability to induce the death of cancer cells upon photo-activation. Two series of polymer probes were evaluated: the first group (13K4-H, 13K10-H, 13K4-AEM) has been designed to study the influence of DBB density and type of post-treatment (AEM-capping vs. hydrolysis); the second group (13K4-H, 29K5-H, 35K4-H, plus the four QA-containing conjugates) has been designed to study the influence of polymer size as well as nature and density of charges on the biological activity.


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Cytotoxicity To evaluate the impact of the polymer conjugates in cellulo, we used mouse Baf3 cells since these cells are very sensitive to a wide spectrum of cell death inducing treatments36 and B16F10 melanoma cell line as these highly invasive and metastatic cells represent a widely-used tumor model.37,38 The results indicated that, in the absence of photo-activation, the DBBpolymer conjugates were not toxic (Figure 4). Hence, after incubation of Baf3 or B16-F10 cells with the various polymer conjugates for 24 h, not any sign of increased mortality was observed in comparison with cells alone (i.e. untreated). In contrast, positive controls indicated that cell death could be efficiently induced when Baf3 and B16-F10 cells were either starved of Interleukin-3 (Starvation, Figure 4A) or incubated with apoptosis-inducing drug staurosporine (STS, Figure 4B), respectively.

B

Baf3 cells 100 80 60 40 20 0

Untreated Starvation 13K4-H 13K10-H 13K4-AEM

B16-F10 cells 100

Cell Viability (%)

A

Cell Viability (%)

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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80 60 40 20 0

Untreated STS 13K4-H 13K10-H 13K4-AEM 29K5-H 35K4-H 35K3-AEM-QA5% 35K3-AEM-QA20% 35K3-AEM-QA50% 35K3-H-QA20%

Figure 4: Cytotoxicity study. Baf3 cells (A) or B16-F10 cells (B) were incubated for 24 h with the indicated conjugates at a concentration equivalent to 2 x 10-6 mol.L-1 DBB. As a control, Baf3 cells apoptosis was induced by 24 h growth factor starvation and B16-F10 apoptosis was induced by 24 h treatment with staurosporine (STS, 1µM). Cells were analyzed using a Canto flow cytometer. Cell mortality was assessed by FSC/SSC analysis. Results show the mean mortality ± SD of at least 3 independent experiments.

Cellular uptake and photo-induced cell death The kinetics of cellular uptake of the DBB-polymer conjugates was first evaluated with conjugates of the same size (same polymer backbone of 12 900 g.mol-1, Figures 5A and B). Thus, Baf3 and B16-F10 cells were incubated for various lengths of time (1, 3 or 24h) with the 13K conjugates (equivalent of 10-5 mol.L-1 DBB chromophore) and their fluorescence was recorded by flow cytometry. Increase of fluorescence intensity over time was indicative of a 17 ACS Paragon Plus Environment

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continuous uptake. 13K4-H probe (4.4 DBB and 30 carboxylate groups per chain) afforded the highest uptake whatever the incubation time (1, 3 or 24h). Increase of DBB density along the chain (13K10-H, 9.8 DBB and 24 carboxylate groups per chain) or capping by AEM instead of hydrolysis (13K4-AEM, 4.4 DBB and not any carboxylate group per chain) impacted negatively the ability of the polymer probes to enter both Baf3 and B16-F10 cells. Cell uptake with 13K4-AEM remained moderate. At that step, it could not be discriminated if the key parameter for a better cell uptake was a higher number of negative charges along the chain and/or a lower DBB density and/or a shift of the hydrophilic/hydrophobic balance towards more polarity, since these three parameters are correlated. For the next study of cell death induction upon photo-activation, an incubation period of 24h (plateau) was chosen to avoid kinetics effects on conjugate comparison.


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Cell uptake

Baf3 cells (Mean Fluorescence Intensity)

A

Cells only 13K4-H 13K10-H 13K4-AEM

50000 40000 30000 20000 10000 0 0

6

12

18

24

Time (h)

Cell uptake

(Mean Fluorescence Intensity)

B

B16-F10 cells

70000

Cells only 13K4-H 13K10-H 13K4-AEM

60000 50000 40000 30000 20000 10000 0 0

6

12

18

24

Time (h) Baf3 cells + 13K4-H 7.3

Propidium Iodide uptake

Baf3 cells only Propidium Iodide uptake

C

87.1

Cell size D

Cell size

Baf3 cells

E

100

100

80

80

Cell Death (%)

Cell Death (%)

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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60 40

60 40

20

20

0

0

Time (h): 1 5

1 1 5 5

1 1 5 5

1 1

5 5

B16-F10 cells

Time (h):

5

5

5

5


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Figure 5: Cellular uptake and photo-induced cell death with the 13K conjugates. (A,B) Kinetics of conjugate uptake. Baf3 cells (A) and B16-F10 cells (B) were incubated with conjugates containing the equivalent of 10-5 mol.L-1 DBB for the indicated period of time. Uptake was evaluated by flow cytometry analysis. Results are expressed as the average of the mean fluorescence intensity ± SD of 2 independent experiments. Cells cultured in the absence of conjugates (cells only) were used as a control. (C) Measure of cell death by flow cytometry. Baf3 cells were incubated for 24 h with 13K4-H conjugate containing the equivalent of 2x10-6 mol.L-1 DBB (Baf3 cells + 13K4-H). Untreated cells (Baf3 cells only) were used as a reference. Both treated and untreated cells were submitted to irradiation. After 5 h, cells were stained with Propidium Iodide (PI) followed by flow cytometry analysis. Density dot plots display two parameters: fluorescence (logarithmic Y-axis) that is proportional to PI incorporation inside cells and cell size (X-axis). The colors range from blue (low cell density) to red (high cell density). Percentages of dead cells in the marked regions are indicated. (D,E) Induction of cell death upon photo-activation. Baf3 cells (D) were incubated for 24 h with increasing amount of various conjugates: 13K4-H (red), 13K10-H (orange) and 13K4AEM (purple) (corresponding to the equivalent of 4x10-7 and 2x10-6 mol.L-1 DBB). B16-F10 melanoma cells (E) were incubated for 24 h with increasing amount of 13K4-H conjugate (red, corresponding to the equivalent of 2x10-8, 4x10-7and 2x10-6 mol.L-1 DBB). Untreated cells (black bars) were used as reference. After the incubation period, cells were submitted to irradiation (16 J.cm-2). The percentage of cell mortality was assessed 1 h or 5 h after irradiation by staining with PI followed by flow cytometry analysis. Results show the mean mortality ± SD of 2 to 8 independent experiments.

Then, the ability of the 13K conjugates to induce cell death following photo-activation was determined by flow cytometry (typical dot plots are shown in Figure 5C). Photo-induced cell death was already detected 1h after irradiation. However, as it was significantly more intense after 5h (Figure 5D), further evaluations were all carried out 5h following photo-activation. Efficiency appeared dose-dependent, with an overall similar trend for Baf3 and B16-F10 cells (Figures 5D and E). Interestingly, the hydrolyzed conjugates induced significantly more Baf3 cell death than their AEM-capped analogues (Figure 5 D). A clear correlation could be drawn between the efficiency of photo-induced cell death and the cellular uptake (Figure 5A). The two hydrolyzed conjugates of the same size but with different DBB densities (5.2 and 11.5% for 13K4-H and 13K10-H, respectively) displayed a similar photo-induced cell death 20 ACS Paragon Plus Environment

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efficiency for a given concentration of DBB (ie with a double concentration of 13K4-H as compared to 13K10-H). Therefore, it seems that within that range, photo-induced cell death efficiency is exclusively related to DBB concentration, and not directly to DBB density along the polymer chain. Conversely, this result suggests that for a same molar amount of 13K10-H and 13K4-H probes, efficiency should be higher with 13K10-H. This hypothesis was confirmed in an additional experiment (data not shown), where the two polymer probes were compared in term of polymer probe concentration (instead of DBB concentration): for a same probe concentration of 2x10-7 mol.L-1, the photo-induced cell-death was 72% +/-1 for 13K10H compared to 50% +/-16 for 13K4-H.

Cell death pathway

Cells can undergo at least two modes of cell death following light irradiation of the photosensitizer: apoptosis or necrosis.39 These two mechanisms can be distinguished by performing caspase inhibition experiments. Caspases are indeed key enzymes that are activated during the apoptotic program but are not usually required for necrotic cell death. Caspases activity can be inhibited using caspases inhibitors such as zVAD-fmk that efficiently inhibits classical apoptotic pathways, i.e. starvation-induced apoptosis in Baf3 cells (Figure 6A), while leaving necrotic processes unaffected.40 Moreover, zVAD-fmk also inhibits staurosporine-induced apoptosis in B16-F10 cells (Figure 6B, black and grey bars). In order to define the cell death pathway induced by our polymer probes, both types of cells were incubated with 13K4-H for 24 h and then with zVAD-fmk for 30 min, before light irradiation. Photo-induced cell death was efficiently inhibited in Baf-3 cells, indicating a cell death pathway mainly involving apoptosis (Figure 6A). On the contrary, in B16-F10 cells, photo-induced cell death was barely inhibited by zVAD-fmk, suggesting a necrotic form of cell death (Figure 6B). These results indicate that photo-activated 13K4-H polymer probe can induce different cell death pathways depending on the cell type.


Biomacromolecules

A

B

Baf3 cells 100

B16-F10 cells 100

80

13K4-H 2x10-6 + zVAD

60 40

Cell Death (%)

13K4-H 2x10-6

Cell Death (%)

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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STS

80

STS + zVAD

60

13K4-H 1x10-6

40

13K4-H 1x10-6 + zVAD

20

20

0

0

13K4-H 2x10-6 13K4-H 2x10-6 + zVAD

Figure 6: 13K-4H–triggered cell death pathways. Baf3 cells (A) and B16-F10 cells (B) were incubated with 13K4-H (equivalent of 10-6 or 2x10-6 mol.L-1 DBB) for 24 h and then with pan-caspase inhibitor (zVAD-fmk, 125 µM) for 30 min. 5 h after photo-activation, cell death was assessed by flow cytometry. Results show the mean mortality ± SD of 2 to 4 independent experiments.

One and two-photon cell imaging

We next tested whether DBB-polymer probes could be visualized in cellulo by one- and twophoton fluorescence microscopy. We used B16-F10 melanoma cells that are adherent cells and therefore easily examined on microscopy slides. Upon one-photon excitation at 405 nm, 13K4-H polymer probe emission was detected throughout the cytoplasm of the cells (not in the nucleus) and concentrated in the perinuclear region (Figure 7, top panels). 13K4-H probe was also successfully visualized upon two-photon excitation, with a maximal signal and a minimal auto-fluorescence emission for 750 nm excitation wavelength (NIR range) (Figure 7, bottom panels). A fluorescence emission spectrum was recorded in cellulo with a very good signal-to-noise ratio (Figure S5 in ESI). That emission profile differs from the one of the polymer probe in water, the maximum emission wavelength being significantly blue-shifted (440 nm instead of 510 nm in water). It is however very similar to the emission of the probe in dioxane, a solvent known to exhibit polarity characteristics close to biological micro-environments.31 Such phenomenon has already been observed for an anthracene-derived TPA probe.21 It is an indication that, inside the cell, the DBB-polymer probes are mostly located in low polarity regions (lipid membranes, proteins). Consequently, the very good quality of the images recorded in cellulo (Figure 7) can certainly be explained by the much higher fluorescence


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quantum yield of the DBB-polymer probe in such a low polarity environment (like dioxane, φF = 0.10, Table 3) than in water (φF < 0.01 ).

Therefore, our DBB-polymer probes can be used not only to photo-induce cancer cell death but also to image these cells before treatment (to attest the internalization of the photosensitizer). The ability of these DBB-polymer probes to enable cell imaging under twophoton excitation in the NIR (750 nm and above) can be very beneficial in view of future imaging of living tissue, given the aforementioned advantages of two-photon fluorescence microscopy over confocal microscopy in terms of light penetration depth.

No 13K4-H Merge

With 13K4-H 13K4-H

Merge

Onephoton λ=405 nm

Twophoton λ=750 nm

Figure 7: One-photon and two-photon fluorescence images of DBB-polymer conjugate. Adherent B16-F10 melanoma cells were cultured for 24 h in the presence of 13K4-H polymer probe (equivalent of 10-6 mol.L-1 DBB). Plasma membrane (green) and nuclei (blue) were visualized in all cases using anti-CD44 antibody (488 nm) and DRAQ5 DNA dye (633 nm), respectively. The conjugate (red) was detected using one-photon (405 nm, top panels) or twophoton excitation (750 nm, bottom panels) by laser-scanning fluorescence microscopy. Scale bar is 10 µm.


Biomacromolecules

Evaluation of the DBB-polymer probes possessing quaternized ammonium groups

We next evaluated the impact of modifying the size of the polymer chain and the number/nature of the charges along the chain on the cellular uptake and photo-induced cell death. Three types of polymer backbones of increasing size were compared, corresponding to molecular weights of 12 900 (13K), 29 400 (29K) and 35 000 (35K) g.mol-1. Moreover, for the same polymer backbone of 35K, five different charge distribution were compared: either exclusively anionic charges (35K4-H, bearing 56 carboxylate groups) or exclusively cationic charges (35K3-AEM-QA5%, -QA20% and -QA50%, respectively bearing 3, 12 and 30 quaternary ammonium groups) or a mixture of anionic and cationic charges (35K3-HQA20%, zwitterionic copolymer bearing 45 carboxylate groups and 12 quaternary ammonium groups). B16-F10 melanoma cells were incubated for 24 h with increasing concentrations of each conjugate (from 7.4x10-8 to 2x10-6 mol.L-1 eq. DBB) and their fluorescence was recorded by flow cytometry (Figure 8).

B16-F10 cells (Mean Fluorescence Intensity)

Cell uptake

A 14000 12000 10000 8000 6000

Untreated

4000

13K4-H

2000

29K5-H

0

35K4-H 35K3-AEM-QA5%

100

B

Cell Death (%)

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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35K3-AEM-QA20%

80

35K3-AEM-QA50%

60

35K3-H-QA20%

40 20 0

Figure 8: Cellular uptake and photo-induced cell death with conjugates of various sizes and charges. B16-F10 cells were incubated with increasing concentrations of DBB-polymer probe (corresponding to the equivalent of 7.4x10-8, 2.2x10-7, 6.7x10-7, 2x10-6 mol.L-1 DBB) for 24 h.


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(A) Uptake was evaluated using a MACSQuant flow cytometer. Results are expressed as the average of the mean fluorescence intensity ± SD of 2 independent experiments. (B) Cells were submitted to irradiation (16 J.cm-2) and the percentage of cell mortality was assessed 5 h later by staining with propidium iodide followed by flow cytometry analysis (MACSQuant). Results show the mean mortality ± SD of 2 independent experiments.

Comparison between the hydrolyzed probes of increasing size did not show a drastic influence on their cellular uptake, in spite of a significantly higher uptake for the longer probe (35K4-H) of lower DBB density (2.7 instead of 5.2% for 13K4-H). Accordingly, the photoinduced cell death was much more efficient for this longer probe (> 95% dead cells compared to 30% for the shorter 13K4-H polymer probe, at the intermediate concentration of 6.7x10-7 mol.L-1 equivalent DBB). This behavior was confirmed at the lower concentration of 2.2x10-7 mol.L-1 equivalent DBB (>20% dead cells instead of 10%). Now, for a same polymer size (35K), influence of the nature of the charges along the chain appeared as the determining factor. The anionic polymer probe (35K4-H) and the zwitterionic one (35K3-H-QA20%, with a negative global charge) displayed a higher efficiency of photoinduced cell death than the three cationic probes (> 95% dead cells compared to 35-60%, at the intermediate concentration of 6.7x10-7 mol.L-1 equivalent DBB). Such high efficiency of photo-induced cell death is in line with their higher cellular uptake, confirming the correlation between these two parameters. At the lower concentration (2.2x10-7 mol.L-1 eq. DBB), the zwitterionic conjugate appeared as the best candidate, with ca 50% dead cells compared to 20% for the anionic conjugate and 10% for the three cationic conjugates. These results are in agreement with existing literature: in a quantitative structure-activity study concerning various bacteriochlorin-photosensitizer derivatives bearing different types of side-groups, the positively charged ones corresponded to the less efficient PS in eukaryotic cells.41

Further evidence regarding the correlation between photo-induced cell death and polymer probe internalization can be seen in Figure 9 which reports both cellular uptake and cell death efficiency versus the (calculated) global charge of the polymer probe (represented along a linear axis) for the five probes of the same size. The more negatively charged the polymer

probe (on a scale from 0 to 60 negatives charges per conjugate), the better its internalization in B16-F10 melanoma cells and the higher the cell death efficiency. Such trend was not an obvious finding since the plasma membrane is negatively charged.


Biomacromolecules

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

cell uptake

12000

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100 % cell death 90

10000

80 70

8000

60 6000

50

4000 2000

40

uptake 1h

30

uptake 24h

20

% Cell death

10 0 -60

-40

-20

0 0

20

40

Global charge along the polymer conjugate

Figure 9: Cellular uptake (after 1h and 24h incubation) and photo-induced cell death efficiency (after 24h incubation, corresponding to the equivalent of 6.7x10-7 mol.L-1 DBB) versus the global number of charges along the polymer chain (for the five different 35K DBBpolymer conjugates).

Finally, we wanted to evaluate if it would be possible to significantly reduce the incubation time of the polymer probes while keeping a high level of photo-induced cell death efficiency. Then, internalization of the five polymer probes of 35K were followed over 24h. The kinetics (Figure S6A in ESI) confirmed the effect of charges on the cell uptake. The cationic conjugates were slowly internalized in comparison with the anionic ones. Moreover, whatever the incubation period (1h, 3h or 24h), the anionic and the zwitterionic conjugates were better internalized than the cationic ones. Since after 3h incubation, cellular uptake was almost similar than after 24h, especially for the best candidates (anionic and zwitterionic probes), a photo-induced cell death assay was therefore conducted after 3h incubation only (Figure S6B in ESI). In the case of the zwitterionic conjugate (35K3-H-QA20%), it was still possible to reach 100% cell death at 2x10-6 mol.L-1 equivalent DBB. More significantly, at the lower concentration of 6.7x10-7 mol.L-1, while all other polymer conjugates (with increasing density of positive charges) led to little-to-no photo-induced cell death, more than 63% cell death induction was achieved with the zwitterionic conjugate, strongly emphasizing the influence of the polymer charges on the cell death efficiency. Such experiment underlines the possibility, by playing on the nature 26 ACS Paragon Plus Environment

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and density of charges along the macromolecular PS, to reduce the incubation time from 24h to 3h before photo-activation of the polymer probe and achieve efficient cell death. Overall, it confirms the clear superiority of the zwitterionic DBB-polymer conjugate in comparison with the three cationic conjugates.

CONCLUSIONS This in-depth study which focused on the synthesis, optical characterization and biological evaluation of a whole family of photosensitizer-polymer conjugates of different architectures highlighted several key points towards an ideal design of efficient cargos for two-photon absorption photosensitizers. First, binding the hydrophobic TPA photosensitizer along PNAM-derived biocompatible polymers led to water-soluble macromolecular photosensitizers that could be introduced into cell cultures without the need of any organic solvent. Second, these PS-polymer conjugates exhibited high two photon absorption cross-sections in water, up to 2600 GM at 740 nm. Third, the whole conjugates were non-cytotoxic in the absence of light irradiation. Moreover, internalization of the conjugates was reproducibly observed throughout the cytoplasm of the cells, concentrated in the perinuclear region. Among the neutral, anionic, cationic and zwitterionic PS-polymer conjugates, the anionic and the zwitterionic ones appeared as the best candidates upon photo-activation for induction of death of melanoma cells. In all cases, the cell uptake and the photo-induced cell death were dose-dependent and closely correlated to each other. In addition, they were quasi-linearly dependent upon the global charge of the PS-polymer conjugate: the more negative the conjugate, the higher the cell uptake and efficiency of cell death, which was not an obvious finding since the plasma membrane is negatively charged. The cell death pathway appeared different according to the cell line: for the baf-3 cells, the cell death pathway was mostly involving apoptosis. On the contrary, for the B16-F10 melanoma cell line, a necrotic form of cell death was suggested. Finally, since it was possible to visualize, in the low polarity regions inside the cells, these polymer probes by fluorescence imaging under two-photon excitation, they might be applied as macromolecular photosensitizers for photodynamic therapy of tumor tissues.


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ASSOCIATED CONTENT Supporting information 1

H NMR spectrum of DBB-polymer conjugate, two-photon absorption cross-section spectra

and singlet oxygen emission spectra in CHCl3, absorption spectra in water, fluorescence emission spectra in water and in cellulo, internalization kinetics and photo-induced cell-death after 3h incubation of several DBB-polymer probes with melanoma cell line.

AUTHOR INFORMATION Corresponding authors *(A.F.) E-mail: [email protected] *(Y.L.) E-mail: [email protected] *(M.-T.C.) E-mail: [email protected] Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS We acknowledge the contribution of the imaging facility (PLATIM) and the cytometry platforms (Biosciences Gerland - Lyon Sud, UMS3444, US8). We acknowledge the contributions of Arij Harzallah for synthesis of the random copolymer of 12 900 g.mol-1, Agnès Crépet (Laboratoire d’Ingénierie des Matériaux Polymères) for technical support in SEC/MALLS measurements and Jean Bernard for technical support in two-photon absorption measurements. We also acknowledge Dr. Thibault Gallavardin and Jean-Christophe Mulatier (Laboratoire de Chimie, ENS de Lyon) for the synthesis of the DBB dye and for help with the fluorescence spectroscopy measurements. We thank the ANR financial support (NanoPDT, n°ANR-09-NANO-027) as well as LyonBiopole. This work was supported by several grants from INSERM, the ARC, the Ligue du Rhône, the Région Rhône-Alpes, Université Claude Bernard Lyon 1. CC acknowledges a PhD grant from the French Ministry of Research and Education. 28 ACS Paragon Plus Environment

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