Polythiophenes with Cationic Phosphonium Groups as Vectors for Imaging, siRNA Delivery, and Photodynamic Therapy

In this work, we exploit the versatile function of cationic phosphonium-conjugated polythiophenes to develop multifunctional platforms for imaging and combined therapy (siRNA delivery and photodynamic therapy). The photophysical properties (absorption, emission and light-induced generation of singlet oxygen) of these cationic polythiophenes were found to be sensitive to molecular weight. Upon light irradiation, low molecular weight cationic polythiophenes were able to light-sensitize surrounding oxygen into reactive oxygen species (ROS) while the highest were not due to its aggregation in aqueous media. These polymers are also fluorescent, allowing one to visualize their intracellular location through confocal microscopy. The most promising polymers were then used as vectors for siRNA delivery. Due to their cationic and amphipathic features, these polymers were found to effectively self-assemble with siRNA targeting the luciferase gene and deliver it in MDA-MB-231 cancer cells expressing luciferase, leading to 30–50% of the gene-silencing effect. In parallel, the photodynamic therapy (PDT) activity of these cationic polymers was restored after siRNA delivery, demonstrating their potential for combined PDT and gene therapy.


Introduction
Cancer is a multifaceted and complex disease, which remains one of the main causes of death, forcing researchers to continuously develop new anticancer treatments with high efficiency to their corresponding ammonium analogues in gene delivery applications [55]. Indeed, the more localized positive charge distribution at the P atom is supposed to be the origin of the higher nucleic acid binding ability [56]. Studies also revealed that phosphonium-containing polymer vectors result in lower cell toxicity and increased transfection efficiency with respect to their ammonium analogues [56,57]. The photophysical properties (absorption, fluorescence emission, light-induced generation of singlet oxygen) of the synthesized regioregular cationic polythiophenes were found to be dependent on the molecular weight. Their fluorescent properties were exploited for imaging to determine their intracellular location. Finally, their potential to deliver genetic material and act as a PS in a cooperative manner was examined.

Polymer Synthesis and Characterisation
Poly(3-(6 -bromohexylthiophene)) (P3HT-Br) precursors were synthesized according to reported procedures [31,58]. The polymer characteristics (number averaged (M n ) and weight-averaged (M w ) molecular weights and the molecular weight distribution (Ð)) of P3HT-Br1 were estimated by size exclusion chromatography (SEC). An Agilent liquid chromatograph integrating an Agilent degasser (Diegem, Belgium), an isocratic HPLC pump with chloroform (CHCl 3 ) as an eluent at a flow rate of 1 mL min −1 , an Agilent autosampler (loop volume = 100 µL) with a polymer concentration in a solution of 2 mg mL −1 , an Agilent-DRI refractive index detector (Diegem, Belgium), and three columns (a PL gel 5 µm guard column and two PL gel Mixed-B 5 µm columns (linear columns for separation of M w of polystyrene (PS) ranging from 200 to 4 × 10 5 g mol −1 )) was used. For the two other poly(3-6 -bromohexyl)thiophene) precursors, SEC analyses were performed in THF at 35 • C. A Polymer Laboratories liquid chromatograph was used. This chromatograph integrates a PL-DG802 degasser, an isocratic HPLC pump LC 1120 (flow rate = 1 mL min −1 ), a Marathon autosampler (loop volume = 200 mL) with a polymer concentration in a solution of 1 mg mL −1 , a PL-DRI refractive index detector, and three columns: a PL gel 10 mm guard column and two PL gel Mixed-B 10 mm columns (linear columns for the separation of M w PS standards ranging from 500 to 10 6 Da). Calibration was perfomed in both cases with polystyrene standards.

UV-Visible Absorption and Emission Properties
Absorption spectra were measured on a UV-3600 UV-visible double beam spectrophotometer using Software UVProbe 2.33 (SHIMADZU, MARNE LA VALLEE, France). Fluorescence spectra were measured on a Fluorolog FL3-222 spectrofluorimeter using Software DataMax 2.20 (HORIBA Jobin Yvon, Longjumeau, France). The spectrofluorimeter is fitted with a 450 W Xenon lamp, a thermostated cell compartment (25 • C), a UV-visible photomultiplier R928 (HAMAMATSU Photonics, Hamamatsu, Japan) and an InGaAs infrared detector (DSS-16A020L Electro-Optical System Inc., Phoenixville, PA, USA). Excitation and emission beams are diffracted by double-ruled grating SPEX monochromators with the following features: 1200 grooves/mm blazed at 330 nm for excitation and 1200 grooves/mm blazed at 500 nm for emission, respectively. The detection of singlet oxygen emission was performed by using a double-ruled grating SPEX monochromator (600 grooves/mm blazed at 1 µm) and a long-wave pass (780 nm). All spectra were recorded by using 4 faces quartz cells. All the emission spectra (fluorescence and singlet oxygen luminescence) were recorded at the same absorbance (less than 0.2 at the excitation wavelength) by exploiting the lamp and photomultiplier corrections.
Fluorescence lifetime measurements were performed using a pulsed laser diode emitting at 407 nm (LDH-P-C-400M, FWHM < 70 ps, 1 MHz) coupled with a driver PDL 800-D (PicoQuant GmbH, BERLIN, Germany) for excitation and an avalanche photodiode SPCM-AQR-15 (EG & G, VAUDREUIL, Canada) coupled with a 550 nm long-wave pass filter as a detection system for excitation. A PicoHarp 300 module with a 4-channel router PHR-800 (both PicoQuant GmbH, Berlin, Germany) was used for acquisition. The single photon counting method was employed to record the fluorescence decays. The collection of data was performed up to 1000 counts. The data were accumulated in the maximum channel and analyzed using Time Correlated Single Photon Counting (TCSPC) software Fluofit 4.2 (PicoQuant GmbH, Berlin, Germany) based on iterative reconvolution using a Levensberg-Marquandt algorithm, which enables one to obtain multi-exponential profiles (mainly one or two exponentials in our cases).

Dynamic Light Scattering (DLS) and Zeta Potential
Hydrodynamic diameter and zeta potential were estimated using a Zetasizer Nano ZS (Malvern Instruments Ltd., Malvern, UK). The cationic polythiophenes (5 µM) and cationic polythiophenes/siRNA polyplexes at a P + /Pratio of 100 (5 µM) were prepared in water. The measurements were then performed after 30 min incubation at 37 • C to guarantee the complexation of siRNA. Particle size measurements were carried out with transparent ZEN0040 disposable micro-cuvette (40 µL) at 25 • C. Zeta potential measurements were performed at 25 • C and were carried out in DTS 1070 zeta potential cells made with 18 runs for each.

Cell Culture Conditions
Human breast adenocarcinoma MDA-MB-231, the standard cell line or this expressing luciferase and red fluorescent protein (MDA-MB-231 Luc RFP) were purchased from ATCC and AMSBIO (Abingdon, UK), respectively. Cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM), incorporating 10% fetal bovine serum and antibiotic (0.05 mg mL −1 gentamycin). The cell growth was performed in humidified atmosphere at 37 • C under 5% CO 2 .

In Vitro Cytotoxicity Assay
The in vitro cytotoxicity analyses were performed by using MDA-MB-231 cells seeded into 96-well culture plates, 2000 cells per well in 200 µL of culture medium, and allowing them to grow for 24 h. The cells were then treated with different concentrations of phosphonium-based polythiophene conjugated polyelectrolytes, and, after 3 days, a colorimetric assay of living cells (MTT, (4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, assay) was performed, as previously described [59].

In Vitro Phototoxicity Assay
In vitro phototoxicity assays were performed with MDA-MB-231 cells seeded into 96-well plates at a concentration of~2 × 10 3 cells/well in 100 µL of culture medium and allowed to grow for 24 h. The cells were then incubated for 24 h in the presence or absence of polymer (5 µM in water). After incubation with polymers, cells were submitted, or not, to light excitation (λ = 450 nm, 1.45 J cm −2 ) for 5 min. A total of 48 h after irradiation, the cytotoxicity or phototoxicity of the polymers were evaluated using an MTT assay. Briefly, the incubation of cells was performed in the presence of MTT (0.5 mg mL −1 ) during 4 h to determine mitochondrial enzyme activity. Then, the cell medium was aspirated and the MTT precipitates were dissolved in an ethanol/DMSO (1:1) solution (150 µL) and a reading of the absorbance at 540 nm was performed.

Confocal Fluorescent Imaging on Living Cells
Confocal fluorescent imaging was perfomed on MDA-MB-231 cells seeded onto glass bottom dishes (World Precision Instrument, Stevenage, UK) at a concentration of 10 6 cells cm −2 . One day after seeding, cells were incubated in the presence or absence of PTH1, PTH2 and PTH3 (10 µM) for 24 h. The incubation of cells with Hoechst 33,342 (5 µg mL −1 , Invitrogen) was performed for nuclear staining for 15 min and cells were washed 3 times with culture medium. Fluorescence pictures were recorded on confocal microscope (LSM 780 live, Carl Zeiss Microscope) under a 450 nm wavelength excitation for polythiophenes detection and a 760 nm wavelength for nuclei imaging. A high magnification (63×/1.4 OIL DIC Plan-Apo) was used for all images.

Gel Electrophoresis with siRNA
A total of 5 µL of a 20 µM siRNA, mixed with the appropriate amounts of polythiophenes (in order to reach the desired P + /Pratio) in RNase free water (final volume: 20 µL) was used to perform gel retardation assays with siRNA and 4 µL of Blue 6X loading dye (Fisher Scientific, Hampton, NH, USA) was then added. These samples were submitted to an electrophoresis performed with a 2% wt/vol agarose gel in TBE (90 mM Tris-borate/2 mM EDTA, pH 8.2) at 50 V for 1 h. The standard was a 100 bp DNA ladder (Sigma-Aldrich, Saint-Quentin-Fallavier, France). To visualize siRNA, a GelRed nucleic acid gel stain (Interchim, Montluçon, France) was used allowing to detect siRNA through an ultraviolet transilluminator (Infinity Gel documentation Imaging, Vilber Lourmat, Paris, France).

In Vitro Combined PDT and siRNA Delivery
MDA-MB-231 Luc RFP cells derived from MDA-MB-231 human breast cancer cells by the stable transfection of firefly luciferase gene and RFP for red coloration and easy detection were used. They were seeded in a multiwell plate (96 well white opaque tissue culture plates) at 2 × 10 3 cells cm −2 in 100 µL culture medium. Eight hours after seeding, the cells were incubated with or without 5 µM PTH2/siRNA and PTH3/siRNA polyplexes at a P + /Pratio of 100 during the allocated 24 h. The quantities of siRNA used for PHT2 and PTH3 complexation were 132 and 81 µM, respectively. Cells were irradiated for 5 min using a standard fluorescent microscope (Leica DM IRB) with a mercury lamp at 450 nm, magnification x4. Next day, cells were irradiated for a second time with the same process. The day after, to quantify either the cell death or the transfection efficieny of a 21-mer siRNA targeting the expression of luciferase inside MDA-MB 231 Luc RFP cells, cells were submitted to an MTT assay and to luciferase activity assay, respectively. The siRNA targeting sequence (sense: AACUUACGCUGAGUACUUCGA and anti-sense UCGAAGUACUCAGCGUAAGUU) for luciferase was purchased from Eurogentec (Serring, Belgium). The addition into a culture medium of luciferin (10 −3 M, Promega, Charbonnières-les-bains, France) was used to determine the expression of luciferase. A total of 10 min after, a plate reader CLARIOstar ® High-Performance Monochromator Multimode Microplate Reader (BMG Labtech, Ortenberg, Germany) was used to measure living cell luminescence. The cytotoxicity or phototoxicity of polymers were evaluated by MTT assay.

Statistical Studies
Statistical analyses were carried out using student t-test, to compare paired groups of data. A p-value of <0.05 was considered to be statistically significant.

Polymer Synthesis
The phosphonium-based polythiophene conjugated polyelectrolytes were synthesized according to our previous reported procedures [31,58]. Briefly, poly(3-(6'-bromohexyl)thiophene) precursors (P3HT-Br) were first synthesized by KCTCP (Scheme 1), which enables the preparation of polythiophenes with a high degree of control over the final structure and the molecular weight [50,58,60]. Indeed, due to the living chain-growth polymerization mechanism, a control on the molecular weight of the polymer can be achieved by carefully tuning the feed ratio of monomer to the nickel catalyst [50,60]. Using this method, three different well-defined polymer precursors were synthesized in view of evaluating the effect of polythiophene molecular weight on the PDT effect and gene delivery properties (Table 1). These precursors were then converted in 75-80% yield into the corresponding phosphonium-based polythiophene CPEs by reaction with trimethylphosphine (Scheme 1) [31]. The polyelectrolyte nature of these CPEs prevents direct molecular weight determination using size exclusion chromatography (SEC). Consequently, the molecular weights of these CPEs were estimated from the number of units calculated from the P3HT-Br precursors and the molar mass of the phosphonium repeating unit (321 g·mol −1 ) leading to M n = 53 kDa (PTH1), M n = 19 kDa (PTH2) and M n = 11.5 kDa (PTH3). Nanomaterials 2020, 10, x FOR PEER REVIEW 6 of 15 The phosphonium-based polythiophene conjugated polyelectrolytes were synthesized according to our previous reported procedures [31,58]. Briefly, poly(3-6'-bromohexyl)thiophene) precursors (P3HT-Br) were first synthesized by KCTCP (Scheme 1), which enables the preparation of polythiophenes with a high degree of control over the final structure and the molecular weight [50,58,60]. Indeed, due to the living chain-growth polymerization mechanism, a control on the molecular weight of the polymer can be achieved by carefully tuning the feed ratio of monomer to the nickel catalyst [50,60]. Using this method, three different well-defined polymer precursors were synthesized in view of evaluating the effect of polythiophene molecular weight on the PDT effect and gene delivery properties (Table 1). These precursors were then converted in 75-80% yield into the corresponding phosphonium-based polythiophene CPEs by reaction with trimethylphosphine (Scheme 1) [31]. The polyelectrolyte nature of these CPEs prevents direct molecular weight determination using size exclusion chromatography (SEC). Consequently, the molecular weights of these CPEs were estimated from the number of units calculated from the P3HT-Br precursors and the molar mass of the phosphonium repeating unit (321 g.mol −1 ) leading to Mn = 53 kDa (PTH1), Mn = 19 kDa (PTH2) and Mn = 11.5 kDa (PTH3).

Optical Properties of the Phosphonium-Based Polythiophene CPEs
The UV-visible absorption and emission spectra of CPEs are depicted in Figure 1 and the corresponding data are summarized in Table 2. The UV-visible absorption spectra of these CPEs show a broad absorption band attributed to a π-π* transition [63]. The maxima absorption wavelengths (λabsmax) are observed at ~465 nm for PTH2 and PTH3, while a red-shifted absorption is noticed (495 nm) for PTH1, indicating a significant extent of the π-conjugation. The emission spectra of these CPEs are also dependent on the molecular weight Scheme 1. Synthetic pathway towards phosphonium-based conjugated polyelectrolytes (CPEs) (PTH).

Optical Properties of the Phosphonium-Based Polythiophene CPEs
The UV-visible absorption and emission spectra of CPEs are depicted in Figure 1 and the corresponding data are summarized in Table 2. The UV-visible absorption spectra of these CPEs show a broad absorption band attributed to a π-π* transition [63]. The maxima absorption wavelengths (λ abs max ) are observed at~465 nm for PTH2 and PTH3, while a red-shifted absorption is noticed (495 nm) for PTH1, indicating a significant extent of the π-conjugation. The emission spectra of these CPEs are also dependent on the molecular weight since a maxima emission wavelength of 605 nm is found for PTH2 and PTH3, whereas, for PTH1, an emission broad band centered at~620 nm is observed. These results suggest that the cationic polythiophene PTH1 adopts a more ordered and planar conformation, which may be due to the polymer aggregation in water. Indeed, compared to other CPEs, PTH1 exhibits a lower solubility in water due to its much higher molecular weight. To gain insight into the aggregation behaviour of these polymers, the fluorescence quantum yields of the three cationic polythiophenes were measured using tris(bipyridine)ruthenium(II) chloride ([Ru(bpy) 3 ]Cl 2 ) in water as a reference (Φ F = 0.042) [61]. As observed in Table 2, PTH2 and PTH3 exhibit similar fluorescence quantum yields (7-8%) [48], whereas a strong decrease of the fluorescence quantum yields is noted for PTH1 (1%), which is consistent with a significant aggregation in solution as a result of longer π-conjugated backbone leading to increased interchain interactions and fluorescence self-quenching [64][65][66]. The increased interchain contacts in solution for PTH1 in comparison with PTH2 and PTH3 are also evidenced by the decrease in the singlet state emissive lifetime (0.5 ns vs. 0.7 ns) ( Table 2 and Figure S1 in the Supporting Information). These values of singlet state emissive lifetime are in good agreement with those reported for poly(3-hexylthiophene) (P3HT) [67,68]. To evaluate the degree of aggregation of these polymers, their size was then determined by Dynamic Light Scattering (DLS) leading to average diameters of around 110 and 103 nm for PTH2 and PTH3, respectively, while, for PTH1, an average diameter of around 527 nm is observed, confirming its aggregation in aqueous media ( Figures S3-S5 in the Supporting Information).

Cytotoxicity, Imaging and Photodynamic Therapy Activity
To demonstrate the in vitro biocompatibility of phosphonium-based polythiophene CPEs, their cytotoxicity against MDA-MB-231 cells were estimated using the 3-[4,5-dimethylthiazol -2-yl]-2,5-diphenyl tetrazolium bromide (MTT) method, in which soluble MTT is converted into formazan crystal by mitochondrial dehydrogenases, thus allowing one to measure the cell viability. MDA-MB-231 cells were grown in 96-well culture plates and were then treated with PTH1, PTH2 and PTH3 for 72 h. Figure 2A shows the effect of PTH1, PTH2 and PTH3 with concentrations ranging from 0 to 500 µM (relative to repeating unit) on the viability of MDA-MB-231 cells. Although these cationic polymers displayed in vitro toxicity from a concentration of 10 µM, the cell viability is still larger than 70% at a concentration of 50 µM. These values are in good agreement with those previously reported for other CPEs, such as cationic polyfluorenes [38] and poly(fluorene-co-thiophenes) [69]. From Figure 2A, the half inhibitory concentrations (IC50) values for these cationic polymers were also determined leading respectively to IC50 > 100 µM for PTH1 and PTH2 and IC50 > 250 µM for PTH3. Based on these results, cationic polymers were then used at a concentration of 5 µM for investigating PDT and gene delivery applications.
Considering PDT applications, we first evaluated the ability of phosphonium-based polythiophene CPEs to generate singlet oxygen ( 1 O 2 ) by monitoring photoluminescence of 1 O 2 at 1270 nm in aerated D 2 O using Ru(bpy) 3 as a reference (Φ ∆ = 0.53) ( Figure S2 in the Supporting Information) [62]. PTH2 and PTH3 showed relatively low 1 O 2 luminescence quantum yield: Φ ∆ = 0.12 and = 0.14, respectively, while PTH1 did not produce 1 O 2 likely due to aggregation in solution resulting in increased interchain contacts and thus intermolecular charge transfer. Indeed, aggregation is known to lead to reduced ROS generation as previously observed for conventional PSs, such as porphyrins in aqueous media [70,71]. Then, we explored their cellular uptake by MDA-MB-231 cells. The internalization of PTH1, PTH2 and PTH3 in MDA-MB-231 cells was clearly visualized by fluorescence microscopy ( Figure 2B) exploiting the fluorescence properties of phosphonium-based polythiophene CPEs. Indeed, the cationic charge of the phosphonium side groups of these polythiophene-based CPEs enables their binding to the negatively charged cell membrane and subsequently enhances their endocytosis by cells. As shown in Figure 2B, the orange fluorescence of PTH1, PTH2 and PTH3 indicated that they are mainly accumulated around the perinuclear region (located concurrently by Hoechst staining) [72]. Finally, the efficiency of CPEs as PSs towards MDA-MB-231 cells was evaluated in vitro under illumination. also determined leading respectively to IC50 > 100 μM for PTH1 and PTH2 and IC50 > 250 μM for PTH3. Based on these results, cationic polymers were then used at a concentration of 5 μM for investigating PDT and gene delivery applications.
Considering PDT applications, we first evaluated the ability of phosphonium-based polythiophene CPEs to generate singlet oxygen ( 1 O2) by monitoring photoluminescence of 1 O2 at 1270 nm in aerated D2O using Ru(bpy)3 as a reference ( = 0.53) ( Figure S2 in the Supporting Information) [62]. PTH2 and PTH3 showed relatively low 1 O2 luminescence quantum yield:  = 0.12 and = 0.14, respectively, while PTH1 did not produce 1 O2 likely due to aggregation in solution resulting in increased interchain contacts and thus intermolecular charge transfer. Indeed, aggregation is known to lead to reduced ROS generation as previously observed for conventional PSs, such as porphyrins in aqueous media [70,71]. Then, we explored their cellular uptake by MDA-MB-231 cells. The internalization of PTH1, PTH2 and PTH3 in MDA-MB-231 cells was clearly visualized by fluorescence microscopy ( Figure 2B) exploiting the fluorescence properties of phosphonium-based polythiophene CPEs. Indeed, the cationic charge of the phosphonium side groups of these polythiophene-based CPEs enables their binding to the negatively charged cell membrane and subsequently enhances their endocytosis by cells. As shown in Figure 2B, the orange fluorescence of PTH1, PTH2 and PTH3 indicated that they are mainly accumulated around the perinuclear region (located concurrently by Hoechst staining) [72]. Finally, the efficiency of CPEs as PSs towards MDA-MB-231 cells was evaluated in vitro under illumination.  To this aim, MDA-MB-231 cells were incubated for 24 h with phosphonium-based polythiophene CPEs at 5 µM and then, submitted or not to monophotonic light excitation at 450 nm for 5 min. An MTT assay was performed two days after illumination to determine the cells viability. As indicated in Figure 2C, none of the cationic polymers showed significant cytoxicity in the dark (between 4 and 12% cell death at 5 µM). After illumination at 450 nm for 5 min, PTH2 and PTH3 exhibits efficient phototoxicity, inducing around 60% cell death, whereas, in these conditions, no significant phototoxicity is noted for PTH1 due to its aggregation in aqueous media and thus its incapacity to generate 1 O 2 under illumination.

In Vitro Combined PDT and siRNA Delivery
The siRNA loading capability of PTH1, PTH2 and PTH3 was first studied. For this, an agarose-gel electrophoresis assay was performed to estimate the siRNA complexation efficiency of these cationic polythiophene according to the P + /Pmolar ratio between the positively charged phosphonium groups (P + ) in the polymer and the negatively charged phosphodiester groups (P − ). As shown in Figure 3A, as the P + /Pratio increases, the negative charges of siRNA were gradually neutralized and moved to a positive electrode (retardation effect), except for PTH1, in which, regardless of the used P + /Pratio, the siRNA migration was not inhibited. In the case of PTH2 and PTH3, the migration of siRNA was inhibited at P + /Pratio of 100 and 50, respectively. Consequently, we decided to focus only on cationic polythiophenes PTH2 and PTH3 for the further combination of in vitro PDT and gene delivery since PTH1 did not generate 1 O 2 and was not able to complex siRNA. A P + /Pratio of 100 was chosen for PTH2 and PTH3 for the further studies of the in vitro combined PDT and siRNA delivery. The size of the PTH2/siRNA and PTH3/siRNA polyplexes at a P + /Pratio of 100 was then determined by Dynamic Light Scattering leading to polyplexes with average diameters of around 82 and 92 nm, respectively ( Figures S6 and S7 in the Supporting Information), a suitable size for facilitating endocytosis [73,74]. Furthermore, the positive zeta potential of PTH2/siRNA (ζ = 15 mV) and PTH3/siRNA (ζ = 20 mV) polyplexes at a P + /Pratio of 100 allows these polyplexes to interact with the negatively charged cell membrane favorizing their cellular uptake ( Figure S5 and S6 in the Supporting Information) [40,75]. The in vitro PDT activity of PTH2/siRNA and PTH3/siRNA nanoparticles was first examined, exploiting the same conditions as described above. As shown in Figure 3B, siRNA complexation by cationic polythiophenes PTH2 and PTH3 led to the complete loss of the PDT activity of the polymers since no phototoxicity was noticed upon illumination, while, for polymers alone, around 60% cell death was observed. We speculate that this loss of PDT activity can be attributed to the formation of aggregates and thus fluorescence self-quenching and reduced ROS production [32,44].
To this aim, MDA-MB-231 cells were incubated for 24 h with phosphonium-based polythiophene CPEs at 5 μM and then, submitted or not to monophotonic light excitation at 450 nm for 5 min. An MTT assay was performed two days after illumination to determine the cells viability. As indicated in Figure 2C, none of the cationic polymers showed significant cytoxicity in the dark (between 4 and 12% cell death at 5 μM). After illumination at 450 nm for 5 min, PTH2 and PTH3 exhibits efficient phototoxicity, inducing around 60% cell death, whereas, in these conditions, no significant phototoxicity is noted for PTH1 due to its aggregation in aqueous media and thus its incapacity to generate 1 O2 under illumination.

In Vitro Combined PDT and siRNA Delivery
The siRNA loading capability of PTH1, PTH2 and PTH3 was first studied. For this, an agarosegel electrophoresis assay was performed to estimate the siRNA complexation efficiency of these cationic polythiophene according to the P + /Pmolar ratio between the positively charged phosphonium groups (P + ) in the polymer and the negatively charged phosphodiester groups (P − ). As shown in Figure 3A, as the P + /Pratio increases, the negative charges of siRNA were gradually neutralized and moved to a positive electrode (retardation effect), except for PTH1, in which, regardless of the used P + /Pratio, the siRNA migration was not inhibited. In the case of PTH2 and PTH3, the migration of siRNA was inhibited at P + /Pratio of 100 and 50, respectively. Consequently, we decided to focus only on cationic polythiophenes PTH2 and PTH3 for the further combination of in vitro PDT and gene delivery since PTH1 did not generate 1 O2 and was not able to complex siRNA. A P + /Pratio of 100 was chosen for PTH2 and PTH3 for the further studies of the in vitro combined PDT and siRNA delivery. The size of the PTH2/siRNA and PTH3/siRNA polyplexes at a P + /Pratio of 100 was then determined by Dynamic Light Scattering leading to polyplexes with average diameters of around 82 and 92 nm, respectively ( Figures S6 and S7 in the Supporting Information), a suitable size for facilitating endocytosis [73,74]. Furthermore, the positive zeta potential of PTH2/siRNA (ζ = 15 mV) and PTH3/siRNA (ζ = 20 mV) polyplexes at a P + /Pratio of 100 allows these polyplexes to interact with the negatively charged cell membrane favorizing their cellular uptake ( Figure S5 and S6 in the Supporting Information) [40,75]. The in vitro PDT activity of PTH2/siRNA and PTH3/siRNA nanoparticles was first examined, exploiting the same conditions as described above. As shown in Figure 3B, siRNA complexation by cationic polythiophenes PTH2 and PTH3 led to the complete loss of the PDT activity of the polymers since no phototoxicity was noticed upon illumination, while, for polymers alone, around 60% cell death was observed. We speculate that this loss of PDT activity can be attributed to the formation of aggregates and thus fluorescence selfquenching and reduced ROS production [32,44].  polyplexes at a P + /Pratio of 100 at 5 µM and then submitted or not to one or two monophotonic light irradiations at 450 nm for 5 min, with the second light irradiations occuring 24 h after the first one. Figure 4 shows the obtained results for in vitro combined PDT and siRNA delivery exploiting PTH2/siRNA and PTH3/siRNA polyplexes in MDA-MB-231 Luc RFP cells. As observed previously in Figure 3B, the first light irradiation did not result in cell death, as confirmed in Figure 4A (red bars). However, it enables the delivery of siRNA targeting the luciferase gene inside MDA-MB 231 Luc RFP cells leading to 35% and 52% of luciferase gene silencing for PTH2 and PTH3, respectively ( Figure 4B). The siRNA decomplexation is confirmed by the restoration of the PDT activity of PTH2/siRNA and PTH3/siRNA leading, respectively, to 44% and 49% of cell death after a second light irradiation ( Figure 4A). This second light irradiation (green bars) also results in a significant improvement in the luciferase gene silencing, mainly for PTH2/siRNA leading to a 67% decrease in luminescence ( Figure 4B). We can note that, for PTH3/siRNA, the effect on luminescence decrease is maximal after only one irradiation (48% at the first irradiation and 40% after 2 irradiations), suggesting that PTH3 can be more easily used for nucleic acid delivery. This light-triggered siRNA release will necesitate deeper investigation, and the understanding of the mechanism responsible for this effect will help us to develop highly potent polythiophenes for nucleic acid delivery under light stimulation. All in all, these results show the remarkable synergistic effect of using these cationic polymers both as a polymer vector for siRNA delivery and as a PS for PDT.
a mercury lamp at 450 nm, magnification x4. Two days after illumination, cells were submitted to an MTT assay to quantify living cells.
Finally, to evaluate the therapeutic potential of these polymers in PDT-siRNA combination therapy, we set up experiments using siRNA targeting the expression of luciferase gene in MDA-MB-231 Luc RFP cells. MDA-MB-231 Luc RFP cells were incubated for 24 h with PTH2/siRNA and PTH3/siRNA polyplexes at a P + /Pratio of 100 at 5 μM and then submitted or not to one or two monophotonic light irradiations at 450 nm for 5 min, with the second light irradiations occuring 24 h after the first one. Figure 4 shows the obtained results for in vitro combined PDT and siRNA delivery exploiting PTH2/siRNA and PTH3/siRNA polyplexes in MDA-MB-231 Luc RFP cells. As observed previously in Figure 3B, the first light irradiation did not result in cell death, as confirmed in Figure  4A (red bars). However, it enables the delivery of siRNA targeting the luciferase gene inside MDA-MB 231 Luc RFP cells leading to 35% and 52% of luciferase gene silencing for PTH2 and PTH3, respectively ( Figure 4B). The siRNA decomplexation is confirmed by the restoration of the PDT activity of PTH2/siRNA and PTH3/siRNA leading, respectively, to 44% and 49% of cell death after a second light irradiation ( Figure 4A). This second light irradiation (green bars) also results in a significant improvement in the luciferase gene silencing, mainly for PTH2/siRNA leading to a 67% decrease in luminescence ( Figure 4B). We can note that, for PTH3/siRNA, the effect on luminescence decrease is maximal after only one irradiation (48% at the first irradiation and 40% after 2 irradiations), suggesting that PTH3 can be more easily used for nucleic acid delivery. This lighttriggered siRNA release will necesitate deeper investigation, and the understanding of the mechanism responsible for this effect will help us to develop highly potent polythiophenes for nucleic acid delivery under light stimulation. All in all, these results show the remarkable synergistic effect of using these cationic polymers both as a polymer vector for siRNA delivery and as a PS for PDT. The experiments were carried out with PTH2/siRNA and PTH3/siRNA at a P + /Pratio of 100 without or after one or two irradiations (450 nm, 5 min). * Statistical significance (p < 0.05), of the irradiated condition versus non irradiated condition using Student's t-test.

Conclusions
Phosphonium-based conjugated polythiophenes with different molecular weight (going from 11.5 kDa to 53 kDa) were prepared by KCTCP and used as multifunctional platforms to complex and deliver siRNA and to generate ROS species for PDT. Upon light irradiation, cationic polythiophenes The experiments were carried out with PTH2/siRNA and PTH3/siRNA at a P + /Pratio of 100 without or after one or two irradiations (450 nm, 5 min). * Statistical significance (p < 0.05), of the irradiated condition versus non irradiated condition using Student's t-test.

Conclusions
Phosphonium-based conjugated polythiophenes with different molecular weight (going from 11.5 kDa to 53 kDa) were prepared by KCTCP and used as multifunctional platforms to complex and deliver siRNA and to generate ROS species for PDT. Upon light irradiation, cationic polythiophenes PTH2 and PTH3 with lower molecular weight were able to light-sensitize surrounding oxygen into ROS, which could disturb the endosome-lysosome membrane and thus lead to an enhanced endosomal escape and more efficient gene delivery. In contrast, these polymers are also fluorescent, allowing for the imaging of intracellular location through confocal microscopy; thus, their good internalization was confirmed. The most promising conjugated polyelectrolytes, PTH2 and PTH3, were then used as carriers for siRNA delivery. Due to their cationic and amphipathic features, these polymers were found to effectively self-assemble with siRNA, deliver siRNA targeting the luciferase gene in MDA-MB-231 cancer cells expressing luciferase and lead to 35 and 52% of the gene silencing effect. In parallel, the photodynamic activity of these cationic polymers was restored after siRNA delivery, demonstrating their potential for combined PDT and gene therapy. Nevertheless, further investigation will be needed to determine the mechanism responsible for the light-triggered release of siRNA by these cationic polythiophenes. This study illustrates that multiple functions can be achieved by exploiting the features of conjugated polyelectrolytes and suitably tailoring their molecular structures (molecular weight, nature of the ionic group, etc.). To go further, modifying the polymer structure by introducing notably multiple cationic side groups per monomeric units will be necessary to improve their ability to bind and condense nucleic acid and their solubility in aqueous medium, which will thus positively impact their optical properties. In addition, two-photon excited photodynamic therapy and siRNA delivery experiments will be conducted in the future to extend the scope of this multifunctional platform.  Figure S4: Particle Size distribution of PTH2 in water (5 µM) at 25 • C, Figure S5: Particle Size distribution of PTH3 in water (5 µM) at 25 • C, Figure S6: Particle size distribution (up) and zeta potential (down) of PTH2/siRNA polyplex in water (5 µM) at a P + /Pratio of 100 at 25 • C, Figure S7: Particle size distribution (up) and zeta potential (down) of PTH3/siRNA polyplex in water (5 µM) at a P + /Pratio of 100 at 25 • C. Figure S8: (A) MDA-MB-231 cells were incubated with PTH1, PTH2, PTH3 during 72 h. Cells were irradiated using confocal microscope with a chameleon laser beam at 800 nm, magnification x10, during 3 x 1.57 seconds. Two days after irradiation, cells were submitted to a MTT assay to quantify the cell death. (B) MDA-MB-231 cells were incubated with or without PTH1, PTH2, PTH3 during 24 h. Cells were incubated with Hoechst 15 min. They were imaged at 800 nm for polythiophenes and 760 nm for Hoechst using confocal microscope.