Synthesis of Structurally Defined Cationic Polythiophenes for DNA

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Synthesis of Structurally Defined Cationic Polythiophenes for DNA Binding and Gene Delivery Chi Zhang, Jinkai Ji, Xiaoyan Shi, Xiaoyu Zheng, Xuewei Wang, and Fude Feng ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b17948 • Publication Date (Web): 11 Jan 2018 Downloaded from http://pubs.acs.org on January 12, 2018

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ACS Applied Materials & Interfaces

Synthesis of Structurally Defined Cationic Polythiophenes for DNA Binding and Gene Delivery Chi Zhang†‡§, Jinkai Ji†§, Xiaoyan Shi‡, Xiaoyu Zheng‡, Xuewei Wang†, Fude Feng*† †

Department of Polymer Science & Engineering, School of Chemistry & Chemical Engineering, Nanjing University, Nanjing 210023, P. R. China

‡

School of Chemistry & Chemical Engineering, Shangqiu Normal University, Shangqiu 476000, P. R. China

Email: [email protected]

KEYWORDS: water-soluble conjugated polymers, regioregular polythiophenes, Kumada catalyst-transfer polycondensation, gene delivery, photochemical internalization

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ABSTRACT: Water-soluble conjugated polymers (WCPs) have prospective applications in the field of bioimaging, disease diagnosis and therapy. However, the use of WCPs with controllability and regio-regularity for bioapplications have scarcely been reported. In this work, we synthesized polythiophenes

containing

ester

side

chains

(P3ET)

via

Kumada

catalyst-transfer

polycondensation (KCTP) and confirmed a quasi-“living” chain-growth mechanism. In addition, we obtained cationic regioregular polythiophenes (cPTs) by aminolysis of P3ET with varied chain lengths, and studied DNA binding capability and gene delivery performance. Benefiting from photo-controlled generation of intracellular reactive oxygen species (ROS), the cationic polythiophenes successfully delivered DNA into tumor cells without additional polymer species.

1. Introduction In the past ten years, water-soluble conjugated polymers (WCPs) have gained great interest in bioapplications such as bioimaging, disease diagnosis, and therapy.1-5 Currently, the synthetic methods of WCPs mainly include palladium-catalyzed coupling reactions (Suzuki,6 Heck,7 and Sonogashira8), Wessling reaction,9 and FeCl3-catalyzed oxidative polymerization.10 These methods follow the step-growth polymerization mechanism, which has disadvantages with regard to polymer structures such as uncontrollable molecular weights, wide molecular weight distribution, difficult end-group functionalization, poor regio-regularity (cannot guarantee headto-tail couplings, H-T), and the disability of forming diblock or multiblock copolymers. Therefore, the WCPs prepared straightforward by the above-mentioned methods are encountered with structural deficiency issues that impact on optical properties and intermolecular interactions. Thus, controllable polymerization methods to achieve structurally well-defined WCPs are urgently needed.


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Kumada catalyst-transfer polycondensation (KCTP) is a new but rapidly developing tool for the preparation of well-defined conjugated polymers (CPs).11,12 In 2004 ~ 2005, McCullough13,14 and Yokozawa15,16 et al reported that the Ni- catalyzed KCTP of regioregular poly(3-hexylthiophene) (P3HT) proceeds via a chain-growth mechanism. Moreover, McCullough revealed the quasi“living” nature of KCTP for the synthesis of P3HT.14 Recently, the KCTP method has been developed in the synthesis of numerous diblock and multiblock copolythiophenes bearing alkyl groups (PATs).17-20 The PATs have been widely used in optoelectric areas including electrochromic devices, field-effect transistors, and organic light-emitting diodes, rather than in biological areas because of the insolubility in water. It is problematic to apply KCTP reactions for preparation of WCPs as the Grignard metathesis (GRIM) of thiophene monomers is not successful if the monomers bear charged ionic side chains for water solubility. Alternatively, ester groups of polythiophenes can easily undergo hydrolysis or aminolysis reaction to generate anions or cations, respectively. However, it remains challenging for availability of polythiophene esters compared to PATs via KCTP method. Up to date, there are a few pioneering KCTP approaches on synthesis of polythiophenes bearing ester side chains. The synthesis of regioregular poly(alkyl thiophene-3-carboxylates) was reported by the Ni-catalyzed process using dibromo monomers.21 However, this method employed Grignard reaction at 40 °C and provided polymers with low monomer conversion. Improvement was made by Catala et al who used 2-bromo-3-hexyloxycarbonylmethyl-5-iodo-thiophene as the monomer to prepare P3ET with high regioregularity under milder conditions, affording polyelectrolyte after ester hydrolysis.22 Although the authors performed kinetic analysis by gel permeation chromatography (GPC) measurements of reaction mixture to show a quasi-controlled character of monomer polymerization, it is not so clear with respect to the living polymerization mechanism as



no detailed evidence of forming block copolymers by the sequential addition of different monomers and lack of end groups characterization. Furthermore, the discrepancies of GPC-based molecular weight from actual number of polythiophenes should be considered.23 So a lot of work remain to do on the investigation of living polymerization mechanism using thiophene ester monomers. WCPs functionalized with positive charges have DNA binding affinity as well as capability of oxygen photosensitization, which allows them to be to be used in gene delivery studies. 24-26 Our previous work revealed introduction of cPTs in gene delivery systems led to multiple outcomes including modulation of polyplex size and zeta potential, increase of polyplex stability, promotion of endo-lysosome membrane disruption, light-induced generation of reactive oxygen species (ROS), and significant enhancement of gene delivery to tumor cells.27 However, these studies suffer from drawbacks of structurally deficient WCPs prepared by step-growth polymerization. Studies need be carried out on the interactions between regioregular polythiophenes with DNA and gene delivery performance of regioregular polythiophenes.

2. Experimental Section 2.1 Materials and Methods. All of chemicals were purchased from commercial sources and used as received unless otherwise stated. NMR spectra were measured on a Bruker DPX-400 spectrometer, using the residual proton resonance of the deuterated solvents as the internal standard. GPC analysis was run on a Shimadzu LC20 AD liquid chromatography system equipped with a RID-20A differential refractive index detector against monodisperse polystyrene standards. THF was used as the eluent at a flow rate of 1.0 mL·min−1. MALDI-TOF MS measurement was performed on a Bruker Autoflex III MALDI-TOF mass spectrometer using dithranol as a matrix.


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UV-vis spectra were measured on a Shimatzu UV-2600 photospectrometer. Steady state fluorescence spectra were determined on a Hitachi F-7000 fluorimeter. Atomic force microscopy (AFM) images were collected on a Bruker DimensionICON (Bruker, USA). Particle sizes and ζpotential values of polyplexes nanoparticles were measured on Zetasizer nanoseries (Nano zs90, Malvern Instruments Ltd., U.K.). The in vitro transfection images were acquired by an inverted fluorescence microscope (Nikon TE2000-U, Japan). The irradiation light source is a LED lamp (pE-4000, CoolLED Ltd., U.K.). 2.2 Synthesis of Monomers. Details of synthesis and characterization of monomers M1 ~ M6 were provided in the Supporting Information (Figures S1-S6). 2.3 General Procedure for the Polymerization of Monomer M1 and M2. Monomer M1 or M2 (5 mmol) was dissolved in THF (25 mL) and cooled to 40 °C under an argon atmosphere. To this solution was added i-PrMgBr (1.0 M in THF, 5 mmol) via a syringe over a period of 5 min. The mixture was stirred under argon for 1 h at 40 °C. After quick warming up to 20 °C, 20 mL of the solution was added in a new flask containing 54.2 mg of Ni(dppp)Cl2 (0.1 mmol). The mixture was stirred at 20 °C for 1 h and then quenched by addition of 0.5 mL of 1.0 M aq. HCl. After filtration, solvent was removed under reduced pressure. The residue was redissolved in chloroform, successively washed by aq. NH4Cl, brine and water, and dried over anhydrous MgSO4 to yield an orange solution. After evaporation, multiple methanol precipitation, filtration and vacuum drying treatments, the polymer products were obtained. 2.4 General Procedure for the Polymerization of Monomer M5. A two-neck flask (50 mL) containing 25.3 mg of (4-bromophenylethynyl) trimethylsilane (0.1 mmol) was filled with Ar before addition of THF (10 mL). i-PrMgCl (2 M in THF, 0.1 mmol) was added via a syringe, and the mixture was refluxed for 2 hours (solution A). A three-neck flask (100 mL) containing M5



(0.1, 0.3, 0.6, and 0.8 mmol, respectively) was placed under argon before addition of certain amounts of THF. After cooling at 0 °C, i-PrMgCl (2 M in THF) was added via a syringe. The reaction medium was maintained at 0 °C under stirring for 20 min. After quick warming up to 20 °C, the solution was added to a new flask containing 5.42 mg of Ni(dppp)Cl 2 (0.01 mmol) via a syringe. The mixture was stirred at 20 °C for 0.5 h. Then solution A was added via a cannula after cooling to room temperature. 15 min later, solvent was removed under reduced pressure. The residue was dissolved in chloroform, successively washed by aq. NH4Cl, brine and water, and dried over anhydrous MgSO4 to yield an orange solution. After evaporation, multiple methanol precipitation, filtration and vacuum drying treatments, the polymer products were obtained. 2.5 Synthesis of P3ET-b-P3HT. Two round-bottomed two-neck flasks were flame-dried under reduced pressure and filled with Ar. Then 2-bromo-5-iodo-3-hexylthiophene (2.5 mmol) was placed in one of the flasks under Ar. After adding THF (25 mL) into the flask via a syringe, iPrMgCl (2 M in THF, 2.5 mmol) was added via a syringe, and the mixture was refluxed for 2 hours (solution A). M5 (1.5 mmol) and THF (25 mL) placed in the other flask under Ar. After cooling at 0 °C, i-PrMgCl (2 M in THF, 1.5 mmol) was added via a syringe. The reaction medium was maintained at 0 °C under stirring for 20 min. After a fast warming at 20 °C, Ni(dppp)Cl2 (0.1 mmol) was added. The mixture was stirred at 20 °C for 1 h, aliquot of this solution was taken to measure the Mn and PDI values. Then, solution A was added to this solution via a cannula, and the resulting solution was stirred for 1 h, then an aliquot was taken to check the Mn and PDI values of copolymer. The reaction was quenched by adding 1.0 M aq. HCl to the solution. The THF in crude polymer solution was removed by evaporation. The residue was redissolved in chloroform, successively washed by aq. NH4Cl, brine and water, and dried over anhydrous MgSO4 to yield an


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orange solution. After evaporation, multiple methanol precipitation, filtration and vacuum drying treatments, the polymer products were obtained. 2.6 Polymerization Kinetics. At certain polymerization time points (30, 120, 240, 480, and 1200 s) using M5 as monomer ([5]0/[Ni]0 = 80:1), a fraction of samples were taken from the reaction mixture and quenched with 1.0 M aq. HCl, and concentrated under vacuum. The samples were marked as K-1, K-2, K-3, K-4, and K-5, respectively. 1H NMR and GPC measurements were carried out for all them. 2.7 General Procedure for the Synthesis of Cationic Poly{N-[N′-(2-aminoethyl)-2aminoethyl]-2-(thiophen-3-yl)acetamide}s (cPTs). N-[N′-(2-aminoethyl)-2-aminoethyl] (DET) modification of structurally defined polythiophenes was achieved by aminolysis reaction according to the reported procedure with minor modifications.28 To a solution of DET (1.71 g, 16.5 mmoL) in N-methyl-2-pyrrolidone (NMP) (10 mL), a solution of P3ET (32 mg, 0.165 mmol of monomer units) in NMP (20 mL) was added at 0 °C under argon. After the addition was completed, the mixture was heated to 50 °C. The reaction was continued for 48 h, and cooled to 0 °C. The mixture was added dropwise to 1.0 M aq. HCl (60 mL) at 0 °C. The solution was subsequently dialyzed against 0.01 M aq. HCl for 24 h and de-ionized water for another 24 h, followed by lyophilization to obtain cPTs. 2.8 Ethidium Bromide (EB) Exclusion Assay. EB exclusion assay was performed according to the previously reported procedure.29 Ethidium bromide (5 μg·mL-1) and green fluorescent protein plasmid DNA (1.6 μg·mL-1) were mixed in pure water. After 15 min incubation at 25 °C, various amounts of cPTs were gradually added to the mixture until N/P 4 (N/P ratio refers to the molar ratio between the aminonitrogen atoms of cPTs and the anionic phosphate moieties of



pDNA). The decay of EB fluorescence intensity at 600 nm was recorded with excitation at 550 nm. As control, EB exclusion assay was carried out for bPEI-25 KDa. 2.9 Preparation of Polyplexes Nanoparticles. The green fluorescent protein plasmid DNA (pDNA) were complexed with four cPTs separately in de-ionized water by self-assembling at different N/P ratios to form the polyplexes nanoparticles (NPs) as follows. cPTs were diluted in de-ionized water to different concentrations for desired charge ratio and then a solution of pDNA with the same volume of cationic polymer solutions in de-ionized water was added gently. The resulting NPs dispersion was incubated at room temperature for 15 ~ 20 min before further use. 2.10 Agarose Gel Electrophoresis. To determine the pDNA condensing ability of cPTs, NPs were prepared at various N/P ratios and incubated at 25 °C for 20 min. The amount of the pDNA was maintained constant at 0.5 μg per sample and naked pDNA was used as a control. Then the NPs was mixed with loading buffer and applied to a 0.8% agarose gel containing 0.5 μg·mL-1 EB in TAE buffer. The electrophoresis was carried out at 120 V for 60 min and the gels were imaged by a Tanon gel documentation system. To check whether light irradiation of polyplexes induced damage of DNA or not, we performed a heparin competitive displacement assay. Polyplexes (N/P 5) containing 0.5 μg of pDNA received light irradiation (300 mW·cm-2, 1 min), and were incubated with heparin sodium salt at a final concentration of 10 IU per μg of pDNA for 20 min at 25 °C for 20 min before agarose gel electrophoresis. 2.11 Dynamic Light Scattering (DLS) and ζ-Potential Measurements. The particle sizes and zeta potentials of polyplex nanoparticles were examined by a DLS instrument fixed with a 90° scattering angle at room temperature. All of NPs were prepared at N/P 5 in pure water at 25 °C and the measurements were performed in triplicate (3 × 30 times). For hydrodynamic diameter


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test, the NP solutions were diluted to a final volume of 1.2 mL containing a final pDNA concentration of 3.2 μg·μL-1. The ζ-potential values of NPs were obtained in water solution containing 1 mM KCl at 25 °C. 2.12 Imaging by Atomic Force Microscopy (AFM). AFM was used for imaging the shape of four different NPs prepared at N/P 5. A volume of 50 μL of aqueous NP solution (pDNA 10 μg·mL1

) was loaded onto the freshly cleaved mica substrate. After 30 min at room temperature, excess

NP solution was removed and mica surface was dried under a nitrogen gas flow at room temperature before imaging. The test mode was set in ScanAsyst mode and the average scanning speed was 1.0 – 1.5 Hz. 2.13 Intracellular Reactive Oxygen Species (ROS) Assay. A well-known probe (2,7dichlordihydrofluorescein diacetate, DCFH-DA) was used for the detection of intracellular ROS level. The intracellular ROS assay was carried out according to the previously reported procedure.30 HeLa cells were seeded into 96-well plates (1.1 × 104 per well) and incubated with NPs (prepared at N/P 5) for 4 hours, then DCFH-DA were added with a final concentration of 20 μM and incubated in the dark for a further 20 min. For ROS test groups, HeLa cells were irradiated with light (λ > 400, 300 mW·cm-2, 2 min) after washed with PBS and immediately imaged by Inversed Fluorescent Microscope. As control, HeLa cells were further incubated in dark conditions. 2.14 Cell Viability Assay by MTT. The cytotoxicities of polyplexes were evaluated by MTT assay on HeLa cells. The cells were seeded into black 96-well plates (1 × 104 per well) and incubated in complete DMEM containing 10% fetal bovine serum at 37 °C under 5% CO2 for 24 h. The medium was replaced with fresh growth medium containing polyplexes (N/P 5). As control, polyplex-free medium was used. Half cells were irradiated by light (λ > 400 nm, 300 mW·cm-2) for 1 min or 2 min, and other cells were cultured in dark. After incubation for 24 h, the cells were



washed with PBS three times. Then 100 μL of fresh culture medium contained MTT (0.5 mg·mL1

) was added into each well and incubated for 4 h at 37 °C. The supernatant was discarded, and

then DMSO (150 μL·well-1) was added and the 96-well plates were shaken for 20 min to dissolve the formazan. The optical density of the solution in each well was detected by microplate reader at 562 nm. The cell viability was expressed as the ratio of optical density at 562 nm relative to the control group. 2.15 In vitro Transfection of HeLa Cells. HeLa cells were seeded in 96-well plates at a density of 1.5 × 104 per well and incubated in 200 μL DMEM medium. The cells were washed with PBS after 24 h of incubation, and a mixture (100 μL) of serum-free medium and NPs (N/P 5) was added. The concentration of pGFP was maintained at 2 μg·mL-1 per well and cells treated with bPEI-25 KDa/pGFP polyplexes were used as control. The cells were cultivated for 4 h, and then another 100 μL of fresh culture medium containing 20% FBS was added into each well. Half of the experimental groups were treated by light irradiation (λ > 400 nm, 300 mW·cm-2, 1 min or 2 min) and other cells were incubated in dark conditions. After transfection for further 36 h, fluorescence images were obtained under Inversed Fluorescent Microscope.

3. Results and Discussion Scheme 1. Schematic presentations for chemical structures and synthetic routes: (a) chemical structures of monomers M1 ~ M6, (b) GRIM of monomers M1 ~ M6, and (c) synthetic routes of P1 ~ P6, P3ET-b-P3HT, and S1 ~ S4.


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3.1 Magnesium-Halogen Exchange Reaction. Magnesium-halogen exchange reaction was applied using various monomers by addition of Grignard reagents in stoichiometry. Ester groups on GRIM have significant impact on reaction selectivity. GRIM reaction mixture was quenched with aq. HCl and checked by 1H NMR. As shown in Scheme 1a and 1b, for all the dibromo monomers (M1 ~ M3), GRIM reactions were processed at low temperature (appr. 40 °C), as intermediates were not stable for longer than 5 min at temperatures above 0 °C. According to 1H NMR data, after Grignard exchange reaction, M1 mainly formed 2-magnesiumchloride-3hexylcarboxylate-5-bromo-thiophene at ~60% conversion (Figure S7). M2 formed two regiochemical isomers, 2-magnesiumchloride-3-ethylacetate-5-bromo-thiophene and 2-bromo-3-



ethylacetate-5-magnesiumchloride-thiophene in a molar ratio of 70/30 (Figure S8). M3 was also converted to two regiochemical isomers, suggesting that GRIM was not affected by steric hindrance and occurred randomly at the 2- or 5-position of the monomer (Figure S9). However, strict head to tail polymerization of M2 and M3 in the following polymerization was not available, which led to polymer products lacking regioregularity. GRIM reaction for M4 ~ M6 was carried out at 0 °C. As shown in the 1H NMR spectra, these monomers mainly underwent magnesium-halogen exchange at 5-position of the corresponding monomer (Figures S10-S12), and the products were stable at room temperature in a period over 20 min. It seems that ester groups impacted on GRIM reaction of M1 ~ M6, different from the use of the alkyl substituted thiophene monomers that were studied by McCullough et al.31,14 GRIM reaction of M1 occurred at the 2-position of thiophene ring due to the stabilization of resulted organomagnesium species by chelation effect of ester group.32 Such chelation effect was weakened by insertion of methylene group between thiophene ring and carbonyl group, and led to random GRIM of M2 and M3 at the 2- and 5-positions of thiophene ring. For M4 ~ M6, the magnesium-halogen exchange activity of iodide at the 5-position was much higher than that of bromide, and GRIM reaction occurred dominantly at the 5-position which was typically the site for GRIM reaction of alkyl substituted thiophene monomers. Hence, the introduction of ester substitution has profound impact on the GRIM selectivity. The closer the ester groups positioned relative to the thiophene ring, more profound the chelation effect of the ester groups was observed. In contrast, steric hindrance did not matter too much. The effect of different Grignard reagents was investigated using monomer M5. It was found that Grignard reagents is another key factor in GRIM reaction of bromo- or iodo- substituted monomers (such as M4 ~ M6). i-PrMgCl·LiCl did not work well on promoting activation of


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monomer M5. With the same stoichiometry, 90% of monomers were activated when using iPrMgCl, contrast to only 68% activated monomers when using i-PrMgCl·LiCl (Figure S13). Based on previous reports, ester group is susceptible to nucleophilic reaction towards common Grignard reagents at temperature above 0 ℃ during GRIM.31-34 We performed GRIM reaction of M5 at 0 ℃, with i-PrMgCl showing quantitative iodine-magnesium exchange. However, according to the activating effect of LiCl,35 we speculate that reaction between Grignard reagent i-PrMgCl·LiCl and ester group may occur. To confirm this hypothesis, GRIM of monomer M5 were performed at -20 ℃ and 15 ℃ with i-PrMgCl·LiCl, respectively. As is shown in Figures S14 and S15, 58% of M5 were activated at 15℃, and quantitative iodine-magnesium exchange was found at -20 ℃. It is evident that with the activating effect of LiCl, i-PrMgCl·LiCl are more reactive toward ester group at the same temperature in comparison to i-PrMgCl. Under the same condition, Grignard exchange reaction did not take place when t-BuMgCl was used. Hence, to prepare P3ETs under milder conditions, Magnesium-halogen exchange reaction was carried out at 0 ℃ using i-PrMgCl as the Grignard reagent for M5. 3.2 Molecular Weights Characterization. To investigate the controllability of the polymerization reaction, a series of polymers (P3 ~ P6) were prepared using M5 at different r values (r is defined as [monomer]0/[Ni]0) for comparison with P1 and P2 prepared from M1 and M2, respectively (Scheme 1c and Table 1). The 1H NMR spectra of the polymers (Figures S16S21) indicated that M5 and M1 provided P3 ~ P6 and P1 with head-to-tail (H-T) regioregularity up to ~ 90%, whereas M2 gave P2 without regioregularity. The observations were well consistent with the Grignard exchange reaction results.

Table 1. Outlined Results of Polymerizations



Polymer

Monomer

r

P1 P2 P3 P4 P5 P6

M1 M2 M5 M5 M5 M5

50:1 50:1 10:1 30:1 60:1 80:1

a)

(Theor.)a) 10,581 8,481 2,213 6,115 12,013 15,915

Mn [g mol-1] 1 GPC H NMR method methodb) 3,241 — 9,257 — 2,326 2,133 6,609 5,857 9,724 11,756 12,640 15,343

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MALDI-TOF MS method — — 2,213 — — —

PDI 1.24 1.17 1.12 1.27 1.35 1.32

Calculated from amount of monomer based on one Ni catalyst per chain plus the molecular weights for

one Br (80 g·mol-1) and one phenylethynyltrimethylsilane (173 g·mol-1);

b)

Determined according to the

ratios of the peak integrals at 7.18 ppm and 0.24 ppm in the 1H NMR spectra (Figures S18-S21).

Polymers were characterized by GPC technique using polystyrene as standard to obtain molecular weights and polydispersity index (PDI) values. Polythiophenes had narrow PDIs between 1.12 and 1.35, showing greater Mn of P3 ~ P6 as r value increased (Table 1 and Figure S22). Considering the significant structural difference between polythiophene and standard sample, GPC is not an accurate method for estimation of polymer molecular weights.23 1H NMR is a useful tool in acquiring molecular weight information of polymers which were installed with a reference substitute as an end cap.36 For this purpose, (4-magnesiumchloridephenylethynyl) trimethylsilane was applied to terminate the chain growth in the end of KCTP polymerization, leading to trimethylsilyl end functionalization. As shown in Figures S18-S21, peaks at 0.24 ppm in the 1H NMR spectra of P3 ~ P6 are assigned to the proton resonance of trimethylsilyl groups. MALDITOF MS analysis was also carried out to gain more structural details. As shown in Figure 1, the m/z values measured by MALDI-TOF MS spectra of P3 match well the sum of n ×196 (repeating unit of P3), 80 (Br) and 173 (phenylethynyltrimethylsilane). Meanwhile, a small quantity of polymer chains without trimethylsilyl end groups were visualized by the presence of Br/H and


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Br/Br terminuses. Plot of 1H NMR-based Mn versus r (Figure 2a) shows a linear increase of the molar mass close to the theoretical line established by presuming one Ni catalyst molecule per chain. The MALDI-TOF data (Figure 1 and Table 1) demonstrate that the Mn of P3 agrees well with that calculated by 1H NMR method, suggesting the excellent reliability of 1H NMR method for estimation of polymer molecular weights.

Figure 1. (a) MALDI-TOF spectrum of P3, and (b) Selected region of MALDI-TOF spectrum with signals assigned.

3.3 Kinetic Studies. To determine whether polymerization for P3ET followed a chain-growth mechanism, polymerization kinetics of M5 to give P6 (r = 80) was carefully investigated (Figures S23-S28). When r was smaller than 80, polymerization reaction was too fast to determine. This might be the similar issue as described in the previous report that kinetic studies of 2-bromo-3hexyloxycarbonylmethyl-5-iodo-thiophene polymerization (r = 97) only provided relationship between the conversion and reaction time.22 As shown in Figure 2b and 2c, both monomer conversion percentage and Mn increased gradually with KCTP polymerization time. In addition, Mn exhibited a good linear relationship with monomer conversion rate (Figure 2d). These kinetics data are in good agreement with a chain-growth polymerization mode, akin to KCTP polymerization of P3HT reported by McCullough et al.14 Notably, polymerization rate of M5 was



very fast at the early stage, and slowed down at 240 s as a result of lowering monomer concentration when monomer conversion was already close to 60%.

Figure 2. (a) Plot of Mn as a function of r for polymerization of M5, (b) Conversion of M5 (r = 80) at varying polymerization time, (c) Mn at varying polymerization time using M5 (r = 80) as monomer, and (d) Plot of Mn as a function of monomer conversion for polymerization of M5 (r = 80). Mn values were estimated by 1H NMR method for (a), and GPC method for (b) & (c).

3.4 Chain Extension by Sequential Monomer Addition. To further confirm the living polymerization mechanism assuming that Ni catalyst stayed in the terminus of growing polymer chains, 2-bromo-5-iodo-3-hexylthiophene was utilized as a second monomer after the first monomer was exhausted. Accordingly, a diblock copolymer P3ET-b-P3HT was expected (Scheme 1c). As shown in Figure 3a, addition of 2-bromo-5-iodo-3-hexylthiophene to the P3ET KCTP polymerization mixture led to a unimodal distribution and reduced retention time from 10.2 min for P3ET to 9.6 min in GPC traces, which suggests that P3HT grew into a new polymer with a larger molecular weight by consuming the second monomer. The new polymer was characterized


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by 1H NMR (Figure 3b), and the appearance of proton resonances originated from both of thiophene esters and hexylthiophene units confirmed successful integration of the second monomer into polymer chains, evidencing “living” nature of nickel terminated P3ET. This method can be used to synthesize diblock or multiblock copolymers in a controllable manner, and allows additional functionalization such as polymer assemblies and preparation of WCPs.

Figure 3. Characterization of P3ET-b-P3HT: (a) GPC traces, and (b) 1H NMR spectrum in CDCl3.

3.5 Photophysical Characterizations of P1 ~ P6. As a light harvesting material, polythiophenes are known to have unique optical properties. P1 ~ P6 were characterized by UVvis absorption and steady state fluorescence. As shown in Figure 4a and 4b, P2 ~ P6 in THF have similar absorption and emission bands, which were centered at 420 nm and 540 nm, respectively, because of their comparable chain lengths to the effective conjugation length of polythiophene backbone. Bearing a linkage of carboxylic ester group to each thiophene unit, P1 exhibited blue shifted maximum wavelengths by ~ 10 nm in both absorption and emission.



Figure 4. (a) UV-vis absorption, and (b) Fluorescence spectra of polythiophenes P1 ~ P6 in THF.

3.6 Photophysical Characterizations of Structurally Defined cPTs. cPTs S1 ~ S4 were yielded by aminolysis of P3ETs with excess DET followed by acidification and purification treatments (Scheme 1c). P3ETs with different molecular weights (3.7, 5.3, 8.4 and 11.3 KDa, respectively) and PDIs (1.33, 1.23, 1.31 and 1.29, respectively) were used as precursors. The primary amine groups were protonated to ensure water solubility according to the pKa (10.05 ± 0.10 at 25 °C) of DET chains.37 Disappearance of protons of t-butyl group in 1H NMR spectra confirmed complete conversion to the desired amine (Figure S29). The single proton resonance peaked at 6.93 ppm in 1H NMR spectra of S1 agrees well with well-controlled regioregularity. Photophysical characterizations of S1 ~ S4 were carried out in water, including UV-vis absorption and steady state fluorescence. S1 ~ S4 exhibit maximum absorption at ~ 450 nm and emission at ~ 640 nm (Figure 5a and 5b), red shifted relative to the corresponding P3ETs in THF (Figure 4). Besides red shifts in both absorption and emission spectra, S1 ~ S4 displayed low quantum yield values at 0.004 to 0.011 ranges as compared to the previously reported nonregioregular linear polythiophenes, likely due to more profound polythiophene stacking induced by the strong hydrogen bonding between DET side chains.27


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Figure 5. (a) UV-vis absorption, and (b) Fluorescence spectra of S1 ~ S4 in water.

3.7 DNA Binding by Cationic Polythiophenes. DNA binding by S1 ~ S4 was investigated according to well-established EB exclusion assay. Addition of cationic polymers into a mixture of EB and DNA would replace EB from DNA and result in a decrease of EB fluorescence. As shown in Figure 6a, S1 ~ S4 exhibited more efficient fluorescence quenching than the gold standard gene carrier bPEI-25 KDa. CE50 values, defined as the charge excess required for 50% reduction in EB fluorescence intensity, were estimated to evaluate DNA binding affinity.38 S1 ~ S4 afforded CE50 values at 0.5 to 0.75 ranges which were much smaller than bPEI having a CE50 of 1.85 (Figure S30), suggesting these cationic polythiophenes possess strong DNA binding affinity that favors DNA protection, in consistency with gel electrophoresis results which showed no DNA leakage from polymer/DNA polyplexes prepared at N/P no less than 3, 4, 4, and 5 for S1 ~ S4, respectively (Figure 6b). Meanwhile, as evidenced by the difference of CE50 values in the EB exclusion assay and N/P required for complete DNA protection in electrophoresis analysis, S1 and S4 had the highest and lowest DNA condensing capability, respectively, likely attributed to the extended strand conformation of rigid polythiophene backbones that hinder DNA binding by polymers.39 It seems that regioregularity plays a role in DNA complexing, as DNA could be condensed well by S1 ~ S4 rather than the non-regioregular polythiophenes.40



Figure 6. (a) EB exclusion assay results, and (b) Agarose gel electrophoresis of S1 ~ S4/pDNA complexes. The numbers in green indicate the N/P ratios.

3.8 Characterization of Polyplex Nanoparticles. Polyplexes prepared at N/P 5 exhibited 100 to 130 nm in hydrodynamic diameters as determined by dynamic light scattering (DLS) and +25 to +30 mV in zeta potentials (Figure 7a), consistent to typical self-assemblies from cationic polymers and pDNA (Figure 7b). The polyplexes were visualized as nanoparticles under atomic force microscope (AFM) (Figure 7c), in diameters of 100 ~ 150 nm that were comparable to the DLS data. As polythiophene chain length increased, the heights of particles were increasingly growing from ~ 20 nm for S1/pDNA to ~ 40 nm for S4/pDNA, reflecting that particles composed of short polythiophenes were more flexible in shape. The relatively small particle size and positive charge of these NPs would facilitate cell uptake.41,42


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Figure 7. (a) Hydrodynamic diameters and zeta potentials of polyplexes in aqueous solution, (b) Schematic illustrations of the formation of polyplex nanoparticles, and (c) AFM images of polyplex nanoparticles at N/P 5. Heights of polyplex nanoparticles were shown to the right of each AFM image.

3.9 Detection of Intracellular Reactive Oxygen Species (ROS). The intracellular ROS level of S1 ~ S4/pDNA polyplexes nanoparticles by using commercial ROS-responsive probe 2,7Dichlorodihydrofluorescein diacetate (DCFH-DA). As control, HeLa cells pre-treated by NPs without light irradiation showed undetectable fluorescence (Figure S31). In contrast, by light



irradiation (λ > 400 nm, 300 mW·cm-2, 2 min), the NPs-treated cells emitted bright green fluorescence (Figure 8a), indicative of efficient internalization of polyplex particles as well as ROS generation that induced oxidation of DCFH-DA into fluorescent DCF. The result demonstrates that regioregular polythiophene NPs are able to elevate intracellular ROS level by oxygen photosensitization (Figure 8b), in a similar way to non-regioregular polythiophene-containing multicomponent NPs.27 The photosensitization capability allows for the occurrence of photochemical internalization (PCI) effect.

Figure 8. (a) DCF fluorescence from polyplexes-treated HeLa cells in response to ROS generation (λ > 400 nm, 300 mW·cm-2, 2 min), and (b) Schematic illustration of 1O2 photosensitization by exciting cationic polythiophenes.

3.10 DNA Stability and Polyplex Cytotoxicity after PCI Treatment. To confirm whether the ROS generated by PCI treatment could damage DNA, a heparin competitive displacement assay was performed. PEI/pDNA and S1 ~ S4/pDNA polyplexes were irradiated (λ > 400 nm, 300 mW·cm-2, 1 min), followed by incubation with excess heparin,43,44 allowing pDNA strands to release from polyplexes and be detected by electrophoresis analysis. Damaged DNA strands are


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expected to separate from naked original pDNA. As shown in Figure 9a, non-heparin treated pDNA in each of polyplexes was fully retarded, indicating complete complexation of pDNA by polymers. Migration of heparin-treated pDNA samples was visualized in the same pattern with naked pDNA that acted as control, which clearly reveals that pDNA in irradiated polyplexes retained integrity without detectable photodamage and preserved the supercoiled structure. This observation agrees with the previous report that cationic polymers-complexed DNA was in a compacted state and could be protected from benzophenone-induced photodamage.45 It seems that pDNA complexed by polythiophenes was successfully survived from ROS attack. We speculate that polythiophenes, the most tightly bound with supercoiled pDNA, are in a deactivated state like aggregates because of neutralization with pDNA. Those polythiophene chains in the periphery of pDNA are more likely to contribute to ROS generation upon excitation. To evaluate the cytotoxicity of S1 ~ S4/pDNA polyplexes, we examined cell viabilities by MTT assay after 24-h incubation of HeLa cells with each of polyplexes. All of non irradiation-treated cells exhibited ~90% cell viabilities. Cell viabilities were slightly reduced to ~80% upon 1-min PCI treatment (Figure 9b). It is safe to apply small light influx to generate ROS without causing significant cell death or deactivation of nucleic acids.46,47 However, phototoxicity increased when light exposure was extended to 2 min (Figure S32). According to our previous report that peripheral non-regioregular polythiophenes with weak capability of inducing ROS generation can achieve enough PCI effect,27 to avoid cell phototoxicity, it is not necessary to prolong light exposure to improve transgene performance.



Figure 9. (a) DNA stability against heparin displacement. S1 ~ S4/pDNA complexes (N/P 5) were incubated with heparin (10 IU per μg of pDNA) after 1- min light irradiation. “+” represents light and heparin-treated polyplexes. “−” represents control without light or heparin treatment. (b) In vitro cytotoxicity of polyplexes with or without PCI treatment (λ > 400 nm, 300 mW·cm-2, 1 min).

3.11 In vitro Gene Delivery. We previously showed that attributed to the poor DNA condensing capability, non-regioregular polythiophenes, regardless of linear or branched backbones, presented poor gene transfer performance and could only be used as a gene delivery enhancer by PCI-mediated disruption of endolysosomal membranes.27 Without PCI treatment, HeLa cells exhibited negligible GFP fluorescence after transfection with pGFP using S1 ~ S4 as gene carriers (Figure 10a). Cell uptake should not be a problem, as the particles have enough positive zeta potentials (Figure 7a). Generally, endolysosome entrapment is the top barrier for intracellular translocation of nanoparticles into nuclei.48 Interestingly, GFP-positive cells were clearly detected after light treatment for 1 min (Figure 10b) or 2 min (Figure S33), correlated to the elevated intracellular ROS level by photoexciting polythiophenes (Figure 8a). The occurrence of PCI effect suggests that S1 ~ S4/pGFP particles were trapped in endolysosomes before light irradiation and underwent successful endolysosomal escape after light treatment (Figure 10c). Light treatment itself did not affect GFP expression level as indicated by the transfection


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experiment with bPEI-25 KDa/pGFP which acted as positive control (Figure 10b). As a result, the photoproduct of exited polythiophenes, ROS, plays a crucial role in the overall gene transfer performance of S1 ~ S4. The endolysosome membrane disruption did not require high ROS level by prolonging light exposure that would risk inducing phototoxicity of cells (Figure 9b and Figure S32).

Figure 10. (a-b) GFP expression in the HeLa cells, (a) without PCI treatment and (b) with PCI treatment (λ > 400 nm, 300 mW·cm-2, 1 min). (c) Schematic illustration of PCI effect in promoting gene delivery to cells.

4. Conclusions In summary, we synthesized a series of P3ETs via KCTP method. GRIM studies indicate that both chelation effect from ester groups and reaction activity of halogens at the 5-position of thiophene rings influence the selectivity of magnesium-halogen exchange reaction. Kinetic studies demonstrate a chain-growth polymerization mechanism. KCTP method affords P3ETs with controlled molecular weight and narrow PDIs, and allows the synthesis of P3ET-b-P3HT block copolymer by the sequential addition of monomers, on a basis of the quasi-“living” nature of polymerization. Cationic regioregular polythiophenes S1 ~ S4 were obtained by aminolysis of P3ETs with different molecular weights. Compared to previously reported non-regioregular polythiophenes, S1 ~ S4 exhibited enhanced DNA binding and condensing capability. The gene



delivery performance of S1 ~ S4 was significantly improved by introducing PCI effect with polythiophene backbones as photosensitizer to overcome endolysosome barrier while maintaining integrity of complexed DNA, which may alleviate the concern of ROS-assisted transgene enhancement by shortly elevating ROS level in endolysosome compartments. Using the opposite strategy, on the one hand this PCI effect-based method differs from the gene delivery system capable of ROS scavenging from thioether cores in nanomicelles, recently reported by Wang et al,49 in that more active and short-lived singlet oxygen rather than hydrogen peroxide is applied for rapid endolysosomal membrane disruption by selectively exciting polythiophenes. On the other hand, PCI effect requires careful optimization on the balance between gene delivery efficacy and phototoxicity, to minimize oxidative stress caused by excess transient ROS in cells.49

ASSOCIATED CONTENT Supporting Information. Experimental procedures, additional Figures S1 – S33. This material is available free of charge via the Internet at http://pubs.acs.org/.

AUTHOR INFORMATION Corresponding Author *Email: [email protected]

ORCID Fude Feng: 0000-0002-5348-5959 Author Contributions §These authors contributed equally.


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Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT We’re grateful to Dr. Yifan Ruan (Nanjing University) and State Key Laboratory of Analytical Chemistry for Life Science (Nanjing University) for help with AFM measurements. We thank National Natural Science Foundation of China (Grant No. 21474046 and 21601119), the 1000 Young Talent Program and the Program for Changjiang Scholars and Innovative Research Team in University for financial support.

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Synthesis of Structurally Defined Cationic Polythiophenes for DNA

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