Low-Bandgap Conjugated Polymer Dots for Near-Infrared

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Low-Bandgap Conjugated Polymer Dots for Near-Infrared Fluorescence Imaging Charu V. Rohatgi, Takaaki Harada, Eleanor F. Need, Marta Krasowska, David A Beattie, Gareth Dickenson, Trevor A. Smith, and Tak W. Kee ACS Appl. Nano Mater., Just Accepted Manuscript • DOI: 10.1021/acsanm.8b01014 • Publication Date (Web): 15 Aug 2018 Downloaded from http://pubs.acs.org on August 17, 2018

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ACS Applied Nano Materials

Low-Bandgap Conjugated Polymer Dots for Near-Infrared Fluorescence Imaging Charu V. Rohatgi,†,@ Takaaki Harada,†,‡,@ Eleanor F. Need,¶ Marta Krasowska,§,k David A. Beattie,§,k Gareth D. Dickenson,⊥ Trevor A. Smith,#,⊥ and Tak W. Kee∗,† †Department of Chemistry, University of Adelaide, Adelaide, South Australia 5005, Australia ‡Present address: Okinawa Institute of Science and Technology Graduate University, Onna, Okinawa 904-0495, Japan ¶Cancer Biology Group, Level 1 Basil Hetzel Institute for Translational Health Research, Freemasons Foundation Centre for Men’s Health, Queen Elizabeth Hospital, University of Adelaide, Adelaide, South Australia 5011, Australia §Future Industries Institute, University of South Australia, Mawson Lakes, South Australia 5095, Australia kSchool of Information Technology and Mathematical Sciences, University of South Australia, Mawson Lakes, South Australia 5095, Australia ⊥School of Chemistry, University of Melbourne, Melbourne, Victoria 3010, Australia #ARC centre of Excellence in Exciton Science, University of Melbourne, Melbourne, Victoria 3010, Australia @Contributed equally to this work E-mail: [email protected]

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Abstract Low-bandgap conjugated polymers attract significant research interests because of their broad light absorption spectra in the red and near-infrared regions, making them desirable materials for solar photovoltaics. To date, low-bandgap conjugated polymers yield some of the best power conversion efficiencies offered by polymer solar cells. In addition to their applications as solar photovoltaic materials, nanoparticles of these polymers may be potentially beneficial for cell imaging owing to their red and nearinfrared absorption features, which is required for significant light penetration into biological samples. In this work, conjugated polymer dots (CPdots) of PCPDTBT, PSBTBT, PTB7, PCDTBT and PBDTTPD are prepared in aqueous solution using nanoprecipitation. The maximum fluorescence wavelengths of these CPdots range from 800 to 1000 nm. The CPdots exhibit an average zeta potential of −30 mV, giving rise to colloidal stability of these nanoparticles. Dynamic light scattering results show that the CPdots have a hydrodynamic diameter of approximately 100 nm. Furthermore, analyses of atomic force microscopy images of the low-bandgap donor-acceptor CPdots show an average height of approximately 20 nm. The CPdots are introduced to live THP-1 cells, a human monocytic cell line, and the internalization of CPdots by these cells is observed. Confocal fluorescence microscopy images of cells labeled with the lowbandgap CPdots show the presence of these bright nanoparticles in the cells. In short, we demonstrate the preparation of low-bandgap CPdots as an aqueous dispersion and their applications in cell imaging.

Keywords Nanoparticles, quantum yield, colloids, cell imaging, macrophages

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Introduction Fluorescence imaging techniques with high sensitivity, low cost and temporal resolution have become indispensable for biomedical imaging applications. 1,2 Fluorescent probes emitting in the far-red and near-infrared (NIR) spectral regions (650 – 1000 nm) have gained considerable attention. These probes not only provide an emission spectral window that offers high tissue penetration and low autofluorescence, 3–5 but they also minimize various limiting factors including self-absorption and scattering, 6 which are common issues associated with the use of conventional fluorescent probes that emit in the visible range in biological media. Various fluorescent materials emitting in the far-red and NIR regions that have been employed include organic fluorophores, 7,8 fluorescent proteins, 9 upconversion nanoparticles, 10,11 semiconductor quantum dots, 12,13 and single walled carbon nanotubes. 14 Although with advantages over conventional fluorescent probes, these materials suffer from their own intrinsic limitations. For instance, some organic fluorophores and fluorescent proteins have poor photo-stability. Semiconductor quantum dots have cytotoxicity and chemical instability, even though they possess various remarkable properties in the biological environment including photo-stability, size-tunable emission and high emission quantum yield. 15 These limitations have motivated research groups to direct their attention towards developing novel far-red and NIR fluorescent probes with high molar absorptivity, high brightness, resistance to photo-bleaching and low cytotoxicity, which are desirable characteristics for bio-imaging applications. Conjugated polymers have π-conjugated backbones and exhibit large molar absorptivities, excellent light harvesting properties and signal amplifying abilities. 16,17 These properties have resulted in applications in optoelectronic devices, 18–22 bio-sensing, 23,24 drug delivery, 25–27 photodynamic therapy, 27,28 and bio-imaging. 6,27–44 In particular, nanoparticles of conjugated polymers are contributing significantly to bio-imaging applications. To date, several strategies have been developed to prepare conjugated polymer nanoparticles or dots (CPdots) with tunable fluorescent color for cell imaging. 6,29,30,32–37 These CPdots have not only shown good photo-stability but also excellent biocompatibility in biological media. Kim 3



et al. constructed cyanovinylene-backboned CPdots as nanoprobes and demonstrated their application in biomedical in vivo imaging. 6 Liu et al. reported the preparation of far-red and NIR fluorescent CPdots based on the conjugated polymer PFBDBT and applied them for targeted cancer cell detection and imaging. 35 These authors showed that PFBDBT CPdots possess a maximum absorption at 536 nm and emission in the far-red spectral region with wavelengths longer than 650 nm. Their work highlighted significant potential of PFBDBT CPdots for biological imaging application, inhibiting autofluorescence related interferences. To further expand the applications of CPdots with emission beyond the far-red spectral region, a new class of donor-acceptor conjugated polymers with a low bandgap, which are tailored by combining electron-rich (donor) and electron-deficient (acceptor) moieties, have shown great potential in overcoming issues related to conventional fluorescent probes. The energy levels and absorption properties of these low-bandgap conjugated polymers can be tuned by controlling the intramolecular charge transfer between the donor and acceptor moieties. 45 These low-bandgap conjugated polymers possess a band gap of 1.1 to 1.7 eV, enabling them to absorb further into the NIR spectral region, i.e., 700 to 1100 nm. Recently, MacNeill et al. demonstrated the preparation of low-bandgap CPdots using the polymers PCPDTBT and PCPDTBSe. 46,47 The PCPDTBT CPdots exhibit effective absorption of 808-nm light produced by a NIR laser owing to an absorption maximum at 750 nm, resulting in photo-thermal ablation of cancer cells without affecting surrounding normal cells. 46,47 The example above clearly shows the importance to develop far-red and NIR fluorescent conjugated polymer probes with a long wavelength absorption and emission and high fluorescence quantum yields to further improve the potential of CPdots in cell and tissue imaging applications. 6 In addition, several research groups have made significant progress in the applications of low-bandgap CPdots in biosensing and imaging. In 2014, Chen et al. described the synthesis of dithienylbenzoselenadiazole-based NIR-fluorescing CPdots with ultrahigh brightness and excellent photostability, improving the practicality of low-bandgap CPdots for various biological imaging and analytical applications. 44 In 2016, Qian et al. developed

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a light-activated hypoxia-responsive conjugated polymer-based nanocarrier to produce 1 O2 and induce hypoxia to promote release of its cargoes in tumor cells to enhance the antitumor efficacy of the nanocarrier. 41 In the same year, Zhu et al. reported a one-pot synthetic approach to construct multilayered NIR fluorescent CPdots with enhanced fluorescence and optimized biodistribution for in vivo molecular imaging. 42 Their study provided a straightforward approach for the application of NIR fluorescent CPdots in molecular imaging. In 2017, a review article by Qian et al. summarized the recent advances in the applications of CPdots in theranostics. 27 These applications include in vivo NIR, two-photon, photoacoustic, and multimodal imaging, and photodynamic and photothermal therapy. In the same year, there was another report on the use of low-bandgap CPdots as phototheranostics. Guo et al. reported a high singlet oxygen quantum yield of 40% and photothermal conversion efficiency of 37% using a type of CPdots, 40 which is a step forward in CPdot-based nanoagents for cancer therapy. Most recently in 2018, a report by Feng et al. detailed the applications of CPdots in brain imaging. 39 By exploiting the light-induced electron transfer between CPdots and dopamine, Feng et al. were able to track the dopamine level in the mice midbrain under normal or drug-activated condition and evaluate the long-term effect of addictive substances to the brain. The studies highlighted above represent some of the recent developments in the burgeoning use of CPdots in a number of practical applications. In this article, we report the preparation of various low-bandgap CPdots using the conjugated polymers PCPDTBT, PSBTBT, PTB7, PCDTBT, PBDTTPD and their photophysical characteristics relevant for cell imaging applications. In particular, it appears that our study is the first report of the applications of PTB7 and PBDPPTD dots in cell imaging. Figure 1 shows the chemical structures of the low-bandgap conjugated polymers used in this study. These low-bandgap CPdots can be prepared using nanoprecipitation, a method described previously, 48 with minor modifications. These CPdots are compact with an average diameter of ∼100 nm. CPdots of this size are highly desirable for sub-cellular imaging as larger particles may have issues including poor mass transfer, tissue penetration and non-

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specific adsorption. 36 Owing to a compact size, the low-bandgap CPdots in this study exhibit cellular uptake by endocytosis. The significance of this study is that the CPdot brightness, lack of cell autofluorescence during imaging and cellular uptake of CPdots indicate that the low-bandgap CPdots are novel probes with great potential for fluorescence cell imaging.

Figure 1: Chemical structures of the low-bandgap conjugated polymers used in this study, namely PCPDTBT, PSBTBT, PTB7, PCDTBT, PBDTTPD. A schematic diagram shows the preparation of low-bandgap CPdots using nanoprecipitation.

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Experimental Section Materials The low-bandgap conjugated polymers used in this study are poly[2,6-(4,4-bis-(2-ethylhexyl)4H-cyclopenta [2,1-b;3,4-b]dithiophene)-alt-4,7(2,1,3-benzothiadiazole)] (PCPDTBT, MW 40,500, polydispersity 1.9), poly[(4,4-bis(2-ethylhexyl)dithieno[3,2-b:2,3-d]silole)-2,6-diyl-alt(2,1,3-benzothiadiazole)-4,7-diyl] (PSBTBT, MW 24,150, polydispersity 2.1), poly[[4,8-bis[(2ethylhexyl)oxy]benzo[1,2-b:4,5-b’]dithiophene-2,6-diyl][3-fluoro-2-[(2-ethylhexyl)carbonyl] thieno[3,4-b]thiophenediyl]] (PTB7, MW 97,500, polydispersity 2.1), poly[N -9’-heptadecanyl2,7-carbazole-alt-5,5-(4’,7’-di-2-thienyl-2’,1’,3’-benzothiadiazole)] (PCDTBT, MW 61,000, polydispersity 2.5), and poly[[5-(2-ethylhexyl)-5,6-dihydro-4,6-dioxo-4H-thieno[3,4-c]pyrrole1,3-diyl][4,8-bis[(2-ethylhexyl)oxy]benzo[1,2-b:4,5-b’]dithiophene-2,6-diyl]] (PBDTTPD, MW 38,500, polydispersity 2.2). Figure 1 shows the structures of these conjugated polymers, which were obtained from 1-Material Inc. (St-Laurent, Quebec, Canada) and used as received. Tetrahydrofuran (THF, Scharlau) was distilled before use. Water used in all experiments was purified using a 18 MΩ Millipore Milli-Q Reagent Water System. For atomic force microscopy (AFM), silicon wafers coated with 22 nm amorphous titania layer were obtained from Philips Research Laboratories (The Netherlands). The wafers were cut into 1 × 1 cm2 substrates, which were then sonicated in ethanol for 15 min (AR, ChemSupply, Australia), rinsed with water, dried in a stream of dried nitrogen (99.999%, BOC, Australia) and exposed to UV radiation (λ = 254 nm) for 30 min prior to the AFM experiment. For cell imaging, human acute monocytic leukemia cell line (THP-1) was obtained from the American Type Culture Collection (VA, USA). RPMI 1640 cell culture medium with phenol red was used as received from Invitrogen (Mulgrave, VIC, Australia). Phosphate buffer saline (PBS), fetal bovine serum (FBS), 2-mercaptoethanol and phorbol-12-myristate-13acetate (PMA) were obtained from Sigma-Aldrich (Castle Hill, NSW, Australia). The 4’,6diamidino-2-phenylindole (DAPI) was used to dye the nucleus of THP-1 cells. The THP-1

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cell line was maintained in the RPMI medium supplemented with 10 % FBS and 0.05 mM 2-mercaptoethanol.

Preparation of Low-bandgap CPdots Dispersions of low-bandgap CPdots were prepared according to the method described by Wu et al. with some minor alterations. 49 Each low-bandgap conjugated polymer was dissolved in freshly distilled THF at a concentration of 0.1 mg mL−1 . The stock solution was then diluted to a concentration of 20 ppm. A 2-mL portion of this polymer solution was then injected into 8 mL of water under vigorous stirring, resulting in a final concentration of 4 ppm low-bandgap CPdot dispersion. The THF was removed under reduced pressure with gentle heating (40 ◦C), and the suspension was then filtered through a 0.2-µm cellulose acetate filter (Sartorius Stedim Biotech). Each prepared low-bandgap CPdot solution was then concentrated to 6 ppm.

Spectroscopic Characterization of Low-bandgap CPdots Steady-state absorption spectra were collected on a Cary 5000 UV-vis-NIR spectrophotometer (Varian). The NIR emission spectra were recorded using a system assembled from components comprising a Jobin-Yvon iH320 f/4.1 spectrometer equipped with an InGaAs detector (DSS-IG02/20T, Electro-Optical Systems IGA-020-TE2, PS/TC-1 controller). The 532 nm excitation laser beam (LambdaPro, UG-20mW) was optically chopped (HMS 222) and the output of the detector fed into a lock-in amplifier (EG&G, 5210) referenced to the optical chopper. The analogue output of the lock-in amplifier was digitized using a voltage input module (Jobin Yvon, SpectraAcq2) and acquired using the commercial software SynerJY, version 3.0.0.19. Spectra were corrected (as far as possible) for the wavelength dependence of the detection system using a Quartz Tungsten-Halogen Lamp (ThorLabs, QTH10) for which the calibration curve is provided on-line. 50 Fluorescence quantum yield (φf ) measurements were made on each low-bandgap CPdot solution with absorbance values up to 0.02. The NIR 8


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dye IR-26 was used as a reference standard with a known φf of 0.11% in 1,2-dichloroethane. 51 Dynamic light scattering (DLS) measurements were performed using Malvern Zetasizer Nano S equipped with a He-Ne (633 nm) laser source, for determining zeta potential and hydrodynamic diameter of the low-bandgap CPdots. All zeta potential results were collected using a disposable cuvette at 25 ◦C.

Atomic Force Microscopy of Low-bandgap CPdots The topography and height of low-bandgap CPdots adsorbed onto titania surfaces from aqueous CPdot solutions were characterized using AFM. The topography of bare titania surfaces was also characterized and the results are shown in the Supporting Information. A MultiMode 8 instrument (Bruker, USA) with Nanoscope V controller (Bruker, USA) was used to perform imaging in tapping mode in a liquid environment (in situ). Tapping mode allows for scanning of the soft material without causing image artefacts. 48,52,53 A standard quartz liquid cell sealed with a silicone o-ring and a scanner E (up to 10 µm scan size in the X and Y directions, and up to 2.5 µm vertical displacement) were used in the AFM experiment. The CPdot solution was injected to the cell using a glass Hamilton syringe (USA). The sample was left for 30 minutes (to allow adsorption) prior to scanning. Several high resolution (512 × 512) 2 × 2 µm2 scans were collected using a DNP-10 (Bruker, USA) Vshaped Si3 N4 probe (a nominal spring constant of 0.24 N m−1 , a nominal resonant frequency of 56 kHz, and a symmetric Si3 N4 tip). The scan rate employed was 0.99 Hz or lower. The AFM topography images were analyzed using WSxM 5.0 Develop 9.0 Edition (Nanotec, Spain) software. 54 In order to remove the image tilt, the AFM images were fitted with a first-order plane fit.

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Confocal Fluorescence Microscopy of Cells Labeled with Low-bandgap CPdots Suspensions of THP-1 cells (4.2 × 105 cells/well in a 8-well chamber slide) were differentiated to adherent model macrophage cells by treatment with 100 nM of PMA for three days in the RPMI 1640 medium (10 % FBS and 0.05 mM 2-mercaptoethanol). 55,56 Aqueous stock solutions of low-bandgap CPdots were purified by filtration using 0.2-µm filters. The model macrophage cells on the slides were washed with PBS and treated with the CPdots with a concentration of 3 – 8 ppm for 18 h, and each well contained 10%v/v water as vehicle control. The cells were then washed twice with PBS to remove extracellular CPdots, undifferentiated THP-1 cells, excess PMA and RPMI 1640. The cells were fixed with a 1 % formaldehyde solution in PBS and their nuclei were stained with DAPI. Cell images were acquired with a laser scanning confocal fluorescence microscope (Leica TCS SP5). The excitation sources were a PicoQuant PDL 800-B pulsed diode laser with a repetition rate of 40 MHz and a diode-pumped solid-state (DPSS) laser at 561 nm . The excitation light was focused onto the sample using a Leica HCX PL APO CS 40.0× N.A. 1.25 oil-immersion objective with a 220 µm working distance, and the imaging field was zoomed in by a factor of 2. The emission was collected by the same objective, separated from the excitation sources using appropriate dichroic mirrors and dispersed using a built-in spectrometer. Each image was acquired using line and frame averaging of 8 and 4, respectively. For confocal fluorescence imaging of the DAPI-bound cell nuclei, the excitation and emission wavelengths were λex = 405 nm and λem = 410 nm–550 nm, respectively. For low-bandgap CPdots, the excitation and emission wavelengths were λex = 561 nm and λem = 650 nm–800 nm, respectively. Because the absorbance of PCPDTBT CPdots at 561 nm is weak, fluorescence images of the vehicle control and PCPDTBT-treated cells were also acquired using 633-nm excitation. In addition to fluorescence images, bright field images of the cells were also acquired in the same region.

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Results and Discussion Spectral and Photophysical Properties of Low-bandgap CPdots Figure 2 shows the UV-Vis absorption and fluorescence spectra of the low-bandgap CPdots. The wavelengths at which the low-bandgap CPdots exhibit maximum UV-Vis absorbance and fluorescence intensity are shown in Table 1. The PCPDTBT, PSBTBT and PTB7 CPdots have light absorption in the red to far-red regions with λmax at 700, 660 and 610 nm, respectively, along with the presence of a shoulder near 770, 780 and 670 nm, respectively. In particular, PCPDTBT and PSBTBT CPdots show substantial light absorption in the NIR spectral region (λ ≥ 800 nm), as shown in Figure 2. Light absorption in the NIR spectral region was utilized for the photo-excitation and subsequent photo-thermal ablation of cancer cells. 46 In a recent study, MacNeill et al. reported that photo-excitation of PCPDTBT and PCPDTBSe (in which sulfur was replaced with selenium) in the NIR region leads to the formation of a bi-polaron which then undergoes several decay processes, generating heat and resulting in the destruction of cancer cells. 46 The low-bandgap CPdots prepared using PCDTBT and PBDTTPD exhibit absorption maxima at 550 and 543 nm, respectively. These absorption wavelengths of these CPdots are convenient for fluorescence microscopy because laser excitation wavelengths including 488 and 532 nm are widely available in common fluorescence microscopes. The absorption spectrum of each low-bandgap CPdot sample exhibits a broadened and blue-shifted absorption spectrum relative to that of the corresponding isolated low-bandgap conjugated polymer chains in solution (THF), which are shown in Figure S1 in the Supporting Information. Blue-shifted absorption spectra of CPdots have been observed previously for conjugated polymers including MEH-PPV and PDOF. 48,49 The spectral blue shift has been attributed to a decrease in conjugation length of the chromophoric units in the CPdots compared to those in isolated chains due to presence of bending of the polymer backbone in the CPdots. 49 The fluorescence spectra of low-bandgap CPdots are also shown in Figure 2, along with

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Figure 2: UV-Vis absorption spectra (red) and fluorescence spectra (blue) of (a) PCPDTBT, (b) PSBTBT, (c) PTB7, (d) PCDTBT, (e) PBDTTPD CPdots.

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Table 1: Photophysical and Colloidal Properties of Low-Bandgap CPdots Low-Bandgap CPdots

PCPDTBT

PSBTBT

PTB7

PCDTBT

PBDTTPD

Absorption / Fluorescence λmax (nm)

700 / 865

660 / 980

610 / 795

550 / 735

543 / 730

Fluorescence Quantum Yield (φf , %)

1.46 ± 0.05

0.70 ± 0.03

0.57 ± 0.02

7.18 ± 0.26

2.68 ± 0.10

Zeta Potential (ζ, mV)

-29.8 ± 1.6

-27.6 ± 0.6

-30.8 ± 0.3

-30.4 ± 2.0

-33.3 ± 4.0

Hydrodynamic Diameter (nm)

75 ± 6

134 ± 7

77 ± 3

84 ± 2

82 ± 7

their corresponding absorption spectra. All the low-bandgap CPdots in this study have substantial spectral intensity in the NIR region. The λmax values of fluorescence of the lowbandgap CPdots are listed in Table 1. All the low-bandgap CPdots show a fluorescence maximum either in the red (PCDTBT, PBDTTPD) or NIR (PCPDTBT, PSBTBT and PTB7) spectral region. Most notably, PSBTBT CPdots exhibit a fluorescence maximum at 980 nm and a spectrum nearly entirely in the NIR spectral region. The fluorescence spectra of low-bandgap CPdots and their corresponding conjugated polymers as isolated chains in THF are shown in Figure S2 in the Supporting Information. The spectra of the CPdots exhibit a red shift compared to those of the low-bandgap conjugated polymers. The red shift has been attributed to aggregation, in which the emitting chromophoric units in the CPdots have a longer conjugation length than those in isolated chains. In our previous study, we showed that the chromophores with a long conjugation length are found in the interior of the CPdot, where effective π-stacking of chromophores occurs. 57 The presence of an emission site away from the CPdot surface, where dissolved oxygen can be found, has significant implication on the use of CPdots as emitters, particularly in applications in the aqueous environment. The photo-stability of CPdots is a particular advantage for their applications in cell imaging, 30,32,33,36,37 as shown below. The φf values of the low-bandgap CPdots are reported in Table 1, with values ranging

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from 0.57% (PTB7) to 7.18% (PCDTBT). In contrast, low-bandgap conjugated polymers in solution exhibit higher φf values, with PCPDTBT, PSBTBT, PTB7, PCDTBT and PBDTTPD having φf of 6%, 58 20%, 1.8%, 31%, 59 and 61%, respectively, according to either the cited references or using the data including those presented in Figure S1. In comparison to the CPdots prepared using conjugated polymers that emit in the visible range, including PDOF and F8BT, 37,49 the φf values of low-bandgap CPdots are modest. It should be noted that φf for NIR emitters are inherently lower than those of visible emitters as a result of a smaller energy gap between the ground state and emitting state, often making non-radiative relaxation pathways competitive with radiative relaxation. In contrast, relative to common NIR emitters including IR-125 and IR-140, the φf values of these low-bandgap CPdots are comparable, 60 paving the way for their applications as attractive NIR emitters.

Colloidal Properties and Sizes of Low-bandgap CPdots The low-bandgap CPdots exhibit excellent colloidal stability in water, showing no sign of particle aggregation over the course of our studies. This high degree of colloidal stability has been observed previously in other CPdots, including those composed of MEH-PPV, PDOF and F8BT. 34,48,49 It is important to stress that preparation of CPdots using nanoprecipitation is a surfactant-free approach and the colloidal stability arises from inherent surface characteristics of the CPdots. The resistance of low-bandgap CPdots to undergoing particle aggregation in water is attributable to a substantial and negative zeta potential, as shown in Table 1, ranging from −27.6 to −33.3 mV. It is well known that colloidal systems with a zeta potential value of −25 mV or lower (i.e., more negative) exhibit a long-term colloidal stability. 61 The origin of a negative zeta potential of CPdots was addressed in our previous study, 48 where evidence for oxidative defects on the surface of CPdots was presented. In particular, the presence of electron-rich carboxylate groups on the CPdot surface is consistent with a negative zeta potential. 48 It should be noted that even in the absence of oxidative defects, organic nanoparticles, with an expected degree of hydrophobicity, would associate 14


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hydroxyl anions in solution at their surface, providing another source of negative charge. 62,63 However, it is likely that such a charge would not be sufficient to engender the degree of colloidal stability observed for the CPdots, as any hydrophobic particle suspension would be subject to attractive van der Waals forces and hydrophobic interactions, with the former enhanced by gas nucleation on the particles. 64–66

Figure 3: AFM images and particle height distributions of (a) PCPDTBT, (b) PSBTBT, (c) PTB7, (d) PCDTBT, (e) PBDTTPD CPdots. The scale bar in the images is 400 nm.

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Two techniques were used to determine the size of the low-bandgap CPdots. First, DLS studies reveal that the low-bandgap CPdots have hydrodynamic diameters ranging from 75 to 134 nm. The distribution of hydrodynamic diameters of the low-bandgap CPdots are shown in the Supporting Information. Second, AFM was used to determine the height of the lowbandgap CPdots. As evident from the AFM images in Figure 3 a–e, the height of the isolated CPdots, which correlates to the particle diameter and is a parameter not affected by the artefacts related to the AFM tip size, is significantly smaller than the hydrodynamic diameter measured by dynamic light scattering and listed in Table 1. This discrepancy is most likely due to the relatively high concentration of CPdots used for the DLS experiment, as well as the hydrophobic surface of CPdots. The discrepancy also indicates that some aggregation does occur under certain solution conditions, even in the presence of the oxidative defects (most clearly shown in the DLS result of the PBDTTPD CPdots), and this point should be borne in mind during potential deployment of these nanoparticles for imaging applications. Both factors facilitate some aggregation of the particles, however hydrophobicity (and potential gas nucleation at the CPdots’ surface leading to bridging of multiple CPdots to form aggregates) may be the main driving force. The particle heights measured from AFM images of all CPdots range between 10 and 64 nm, with PCDTBT and PBDTTPD having the largest fraction of the smallest particles (height below 20 nm) and PSBTBT not having this fraction at all. The smallest height measured for PSBTBT was 20 nm, and the largest 49 nm. The fraction with the height above 44 nm is absent for PTB7 and PCDTBT particles, while the largest height measured for PBDTTPD was only 39 nm. The largest particles, with a height of up to 64 nm, were measured for PCPDTBT using AFM. The heights of the low-bandgap CPdots are similar to other CPdots previously studied. 48,49 In addition to the heights, the widths of the low-bandgap CPdots were also analyzed using the AFM images. The distributions of particle width are shown in the Supporting Information, with an average width ranging from 50 to 150 nm. In short, the results from DLS and AFM show that the low-bandgap CPdots have a size of approximately 100 nm. The size of these CPdots is

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sufficiently small to be internalized by cells, 32,34 highlighting the potential application of these CPdots in NIR fluorescence cell imaging.

Cell Imaging using Low-bandgap CPdots Figure 4 shows confocal fluorescence and bright-field images of macrophages treated with PBS and low-bandgap CPdots. The presence of CPdots in the macrophages after treatment of CPdot solutions with a concentration of 3 – 8 ppm for 18 h indicates efficient uptake of CPdots by the macrophages. First, Figure 4a shows that with an excitation wavelength of 561 nm, the macrophages treated with PBS only (i.e., no CPdots) exhibit negligible fluorescence. Second, the macrophages treated with low-bandgap CPdots (Figure 4b – 4f) exhibit a substantial level of fluorescence in the far-red and NIR spectral regions. A comparison of the confocal fluorescence images with the bright-field images reveals that the CPdots are present in the macrophages. It is interesting that the CPdots are found in the macrophages after treatment because previous studies have shown that CPdots are unstable as a colloidal system in a medium with a high ionic strength such as PBS. 34,37 Under this condition, CPdots tend to agglomerate to form sizeable aggregates unless the CPdot surface is functionalized with polar groups to inhibit aggregation. 34,43 In this case, it appears that the low-bandgap CPdots are able to be internalized by the macrophages prior to significant aggregation. As a consequence of labeling the macrophage nuclei with DAPI (blue areas in Figure 4) and a lack of colocalization of the CPdot and DAPI fluorescence (Figure S5 in the Supporting Information), it appears that the low-bandgap CPdots are excluded from the macrophage nuclei. This result is expected because it is well known that positively charged molecular species or colloidal systems have the ability to penetrate the nuclear membrane. 67,68 In contrast, the low-bandgap CPdots have a negatively charged surface, which limits their likelihood to be found in cell nuclei. Low-bandgap CPdots are attractive light-emitting materials for cell imaging. First, owing to a low bandgap, light absorption of these CPdots occurs in the red or far-red spectral region. 17



Figure 4: Fluorescence (top) and bright-field (bottom) images of macrophages treated with (a) phosphate buffer saline (control), (b) PCPDTBT, (c) PSBTBT, (d) PTB7, (e) PCDTBT, (f) PBDTTPD CPdots. The cell nuclei (blue) are stained with DAPI. The scale bar is 10 µm.

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Excitation of low-bandgap CPdots using this low-energy light results in negligible emission of intrinsic fluorophores within the cell (Figure 4a), thereby enabling a high sensitivity for cell imaging. In contrast, some of the visible-light absorbing CPdots have overlapping absorption bands with intrinsic fluorophores in cells, limiting their sensitivity. Second, the red and far-red absorption band of the low-bandgap CPdots can result in the potential application of these emissive nanoparticles in fluorescence imaging in a medium with a high Rayleigh scattering cross section (e.g., in biological tissues). Owing to I ∝ 1/λ4 , where I is the scattering intensity and λ is the wavelength of light, red or far-red light is significantly less affected by Rayleigh scattering than light of shorter wavelengths, resulting in substantial penetration depth in a medium that resembles biological tissues. Third, the NIR emission of the low-bandgap CPdots results in significant penetration in a turbid medium, facilitating a sensitive detection of these emissive nanoparticles in biological tissues. Moreover, another advantage of CPdots in cell imaging is that they exhibit low cytotoxicity, 27,28,38,40,42,43 with a 90% cell viability at 10 µg mL−1 for PSBTBT. 42 In short, the red or far-red absorption and NIR emission are attractive aspects of the low-bandgap CPdots, highlighting their significant potential to be employed in advanced fluorescence imaging of biological tissues.

Conclusions In this paper, we report the spectral and colloidal properties of CPdots prepared by nanoprecipitation using a series of low-bandgap conjugated polymers, namely PCPDTBT, PSBTBT, PTB7, PCDTBT and PBDTTPD. The low-bandgap CPdots exhibit absorption in the red or far-red and emission in the NIR spectral region. These CPdots exhibit colloidal stability owing to a negatively charged surface, with a zeta potential of ∼−30 mV. The average size of the CPdots is ≤100 nm, as determined using DLS and AFM. We also report the applications of the low-bandgap CPdots in cell imaging. Confocal fluorescence images show that low-bandgap CPdots are internalized by macrophages.

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Acknowledgement Part of this work was performed at the South Australian node of the Australian National Fabrication Facility, a company established under the National Collaborative Research Infrastructure Strategy to provide nano- and microfabrication facilities for Australia’s researchers.

Supporting Information Available UV-visible absorption and fluorescence spectra of low-bandgap conjugated polymers and CPdots, hydrodynamic diameter distributions, zeta potential and AFM measurement of titania surfaces and nanoparticle width distributions, colocalization of CPdots and cell nuclei. This material is available free of charge via the Internet at http://pubs.acs.org/.

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Low-Bandgap Conjugated Polymer Dots for Near-Infrared

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