Abstract
Ring-functionalized semiconducting polythiophene dots (Pdots) were synthesized rapidly and in one step by the hydrazine hydrate reduction of doped parent polythiophene, obtained by conventional chemical oxidation of thiophene monomer by FeCl3 in anhydrous acetonitrile. Dispersions of these Pdots display robust (pseudo) solvatochromism and solvatofluorism. Polythiophene Pdots exhibit significant cytotoxicity towards prostate cancer cells (expected) although when injected subcutaneously in vivo in live mouse, no toxicity is observed for 24 days when monitored in real time using fluorescence imaging.
1. Introduction
In contrast to the wide technological use of conducting polymers in electronic and optoelectronic devices, their application in biomedicine (particularly in vivo) has been limited due to dopant leaching and attendant conductivity issues at physiological pH. It is only fairly recently that conducting polymers have made a genuine transition into biomedicine as semiconducting polymer dots (Pdots), where the strong fluorescence exhibited in their undoped electrically insulating state is used to advantage [1–4]. A combination of high intrinsic absorptivity of conjugated polymers and densely packed nanoscale morphology confers Pdots with high chromophore density and very high brightness [5]. Their all-organic nature also often leads to biocompatibility and low toxicity [6].
In this study, we show that semiconducting parent polythiophene Pdots are readily synthesized and can be used as versatile bioimaging agents. Unlike ring-substituted polythiophene, the parent system stands somewhat alone among conducting polymers where its insolubility, intractability, and infusibility severely limits its use [7]. We have described a workaround where thin films of nanofibrillar doped parent polythiophene deposit directly on inert substrates during a chemical oxidative polymerization of thiophene by in situ adsorption polymerization, thereby bypassing all postsynthesis processing steps [8]. We now show that the doped polythiophene powder obtained during the synthesis, when treated with hydrazine hydrate, yields undoped ring-functionalized (Cl, OH) polythiophene powder that disperses in a variety of organic solvents as 10–25 nm diameter Pdots that are highly fluorescent.
2. Experimental Section
In a typical synthesis of polythiophene Pdots (Scheme 1), a solution of 10 ml of 1.0 M anhydrous FeCl3 in anhydrous acetonitrile is added via syringe to a magnetically stirred solution of thiophene monomer (1 ml, 12.5 mmol) in anhydrous acetonitrile (60 ml) in a flask that is continuously purged with nitrogen. The mixture turns dark immediately upon addition of FeCl3, and after 2 h, the resulting black precipitate is suction filtered and washed with copious amounts of acetonitrile. Vacuum drying in an oven at 80°C for 12 h yields ~250 mg of doped polythiophene exhibiting a room temperature conductivity of ~3 S/cm (4-probe, pressed pellet). About 100 mg of freshly synthesized doped polythiophene is then dispersed (magnetically stirred) in a beaker containing ethanol (100 ml), and hydrazine hydrate (2 ml, 40 mmol) is added all at once. Over a period of 12 h, the precipitate changes color from black to red that is consistent with a conversion to undoped polythiophene.
3. Results and Discussion
3.1. Characterization of Polythiophene Pdots
Elemental analysis of the red-brown powder obtained after hydrazine hydrate treatment, C: 39.82, H: 1.68, N: 2.11, S: 25.42, O: 11.84, and Cl: 6.62, total: 87.49 (ash residue), is consistent with a polythiophene backbone structure with a C/S ratio of 1.57 (theoretical C/S = 1.50) containing a significant amount of chlorine and oxygen functional groups. X-ray photoelectron spectroscopy indicates that oxygen is present predominantly as OH (along with some C=O) (SI).
This hydrazine hydrate-reduced polythiophene powder disperses readily in water containing 3% polysorbate surfactant (Tween-80) as Pdots with average diameter of ~45 nm. The particle size can be lowered further (to ~14 nm) when it is precipitated from a solution in N-methyl-2-pyrrolidone (NMP). Transmission electron microscopy (TEM) images of Pdots obtained from aqueous dispersions show that while there are some larger-size particles (~45 nm), most of the Pdots cluster around ~14 nm (Figure 1, SI) [9, 10]. Dynamic light-scattering data also show an average particle size of 14 nm. Analytical, spectroscopic, and particle size data are consistent across multiple synthetic batches showing that while the product composition might be complex as one might expect from an as-synthesized product, the simple two-step oxidation-reduction synthesis route yields polythiophene Pdots having reliable and reproducible physical and chemical properties.
Freshly synthesized undoped polythiophene powder is also sparingly soluble in various organic solvents, such as diethyl ether, methanol, tetrahydrofuran (THF), dimethylsulfoxide (DMSO), dimethylformamide (DMF), and NMP. For example, ~23% of the polymer dissolves in NMP while ~5% dissolves in tetrahydrofuran (THF). Solutions of Pdots in organic solvents display unusual properties. For instance, there is no “streaking” in a thin-layer chromatography (TLC) plate as one would expect from a complex polymeric mixture. The polymer spotted on a TLC plate moves as a single spot in solvent mixtures of DMSO and hexane very much like a pure compound. In contrast, MALDI/TOF indicates a complex mixture (SI). Importantly, 1H NMR in DMSO d6 shows large anomalous upfield peaks (~0 ppm) that are inconsistent with the elemental composition. It is tempting to suggest that these are highly solvated dispersions that happen to display typical solution properties.
For example, solutions of Pdot in organic solvents display marked solvatochromic shifts that are similar in magnitude to what is observed in substituted polythiophenes. The electronic absorption peak (λmax) shifts from 350 nm in diethyl ether to 460 nm in NMP (Figure 2(a)). Typically, solvatochromic shifts in polythiophene derivatives have been attributed to charge transfer between the polymer and various solvents [11] and/or to a different polymer conformation in solution [12, 13]. In contrast, in our Pdot solutions where chains are very tightly packed (almost dispersions), one cannot readily extend these rationalizations to account for what appear to be pseudosolvatochromic shifts. Still, it is possible to look for empirical correlations between changes in λmax in different solvents and known solvent parameters, such as dipole moment and dielectric constant. The solvatochromic shift in λmax of Pdot solutions show a relatively solid correlation with the ET(30) scale, which is a polarity scale derived from changes in the λmax of the lowest energy charge-transfer peak in the electronic spectrum of a highly aromatic zwitterionic betaine dye, pyridinium-N-phenolate betaine, in a given solvent [14, 15].
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Importantly, our Pdot dispersions are highly fluorescent and show strong solvatofluoric phenomena (Figure 2(b)). The emission maximum (λmax) shifts from 497 nm in diethyl ether to 555 nm in NMP. Once again, there is a definite but somewhat weaker correlation between emission maxima (λmax) in different solvents with the ET(30) polarity scale that is to be contrasted with the very weak correlation with other solvent polarity scales. Again, it is important to point out that only a small fraction of the polythiophene powder is “soluble” in organic solvents. Since different solvents have different amounts of Pdot, one cannot readily rationalize solvatochromic and solvatofluoric shifts in λmax with the ET(30) scale. The intense fluorescence observed, however, opens new opportunities for use as bioimaging agents [3].
The relative quantum yield (QY) versus fluorescein was calculated using the equation [16]where QS, IS, As, and ηs are the QY, area under the curve, absorbance intensity, and refractive index values, respectively, for Pdots in NMP and Qr, Ir, Ar, and ηr are the corresponding values for fluorescein in water. A relative QY value of 72.7% was obtained for Pdots at an excitation wavelength of 460 nm (λmax of Pdot in NMP) using values of 0.91, 1.33, and 1.47 for Qr, ηr, and ηs, respectively (SI).
The aqueous Tween-80-supported Pdot dispersion is also highly fluorescent with an emission peak at 485 nm (excitation at 365 nm) with a full width at half maxima (FWHM) value of 110.9 nm. For instance, luminescent organic dyes and QDs display FWHM values typically in the 50–100 nm range.
3.2. Bioimaging
Fluorescent materials for biosensing and bioimaging include metal ions [17], conjugated polyelectrolytes [18], functionalized nanoparticles (NPs) involving quantum dots (QDs) [19–22], magnetic particles (MPs) [23, 24], noble metal NPs [25–28], functionalized proteins [29–31], carbon dots [32, 33], and, more recently, conjugated polymers. The effect of polythiophene Pdots on prostate cancer cell PC3 viability was determined by crystal violet assay. Pdots of different concentrations (1, 5, 25, 50, and 100 μg/ml) were tested on human prostate cancer cells by diluting solutions in DMSO or DMF. The final concentration of DMSO was <0.5%. Expectedly, the plot of Pdot concentration versus relative viable cells (Figure 3) shows that it is very cytotoxic to human prostate cancer cells (PC3) with cytotoxicity increasing after 24 h.
For in vivo studies, this concentration was halved, that is, diluted further to 12.5 μg/ml. Based on the cytotoxicity data above and looking at the chemical composition with a significant amount of Cl groups, we anticipated polythiophene Pdots to display even greater toxicity in vivo.
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Surprisingly, polythiophene Pdots show very low cytotoxicity in vivo (Figure 4). For example, Pdots were injected subcutaneously in vivo in a live mouse and its photostability [34, 35] and cytotoxicity [36] were probed relative to classically applied NPs, MPs, and QDs. For in vivo fluorescent imaging experiments, 20 μl of a Pdot dispersion in water (12.5 μg/ml) was injected subcutaneously into two spots of the flank of C57BL/6mice. Mice were anesthetized using a mixture of ketamine, xylazine, and acepromazine, and then fluorescent images were acquired in vivo by in situ monitoring at 10 min, 30 min, 60 min, and 24 h after injection. For clearance studies, fluorescent images were acquired with several day intervals during a 24-day period. To further understand the temporal effects of cytotoxicity, Pdots with fluorescence emission peaks in the blue range were used and imaged using a multispectral CRi Maestro fluorescent imaging system with blue filter and green filter (Figures 4(a) and 4(b)). The fluorescence intensity changes over a 24-day period indicating excellent fluorescence stability [37], slow degradation [38], and low toxicity.
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Polythiophene Pdots appear to be toxic to cells but not to live mice. This apparent contradiction between in vitro and in vivo toxicities can be rationalized on the basis of differences in dosage. A similar discrepancy has been observed in the toxicity of CdSe QDs in vivo and in vitro studies. Under cell culture conditions, the concentration of Pdots remains high for an extended period of time which induces cytotoxicity, whereas when injected into a live mouse, the concentration is continuously in flux; that is, the site-specific (subcutaneous) dose is not elevated enough to induce toxicity. Compared to QDs, our Pdots are expected to be safer because they are completely excreted over 24 days whereas even QDs that are found to be nontoxic are retained in the liver or spleen [39]. It is important to note, however, that these results are for subcutaneous injection and not intravenous injection, so one has to be careful while making comparisons with existing organic fluorescent imaging probes. Importantly, the emission range is still short of the near-IR range that is typical of developmental bioimaging probes. However, the direct one-step access to ring-functionalized undoped polythiophene opens new opportunities to modify the synthesis to tailor Pdots having these desirable properties including how to target specific organs and/or to reduce clearance time in the body. For example, pegylation of the polymer backbone during the reduction step (or postsynthesis) is expected to increase hydrophilicity and reduce (renal) clearance times significantly [3]. All this is enabled by facile access to a hydroxyl-functionalized polymer backbone in essentially one step, for example, simultaneous reduction and ring substitution of the polythiophene backbone during the reaction with hydrazine hydrate. By leveraging the reduction reaction, it might be possible to gain access to a large family of luminescent polythiophenes with preselected properties. In this sense, the current study is more about a simple and rapid new synthetic method to Pdots with the imaging data providing early validation for surprisingly low in vivo toxicity and overall potential for bioimaging.
4. Conclusions
In summary, this is the first account of solvatochromism and solvatofluorism in polythiophene obtained directly from thiophene monomer and of corresponding Pdots that can be used for in vivo imaging. This rediscovery of the technological uses of the parent polythiophene system could lead to new opportunities in biomedicine, such as fluorescent probes for peptides and nanotherapeutics and in other areas such as related to photovoltaics. Importantly, our synthetic route using hydrazine hydrate is simple, inexpensive, and rapid and can be readily extended to other conducting polymers, such as PEDOT and polypyrrole.
Conflicts of Interest
The authors declare that they have no conflicts of interest.
Authors’ Contributions
Zhen Liu and Alaa Nahhas contributed equally.
Acknowledgments
The authors gratefully acknowledge financial support from Auburn University, the Robert and Gail Ward Endowed Professorship at the University of Massachusetts Lowell (UML), and the University of Texas Southwestern Medical Center.
Supplementary Materials
Additional information can be found in the Supporting Information section on (i) synthesis of doped and undoped polythiophene; (ii) X-ray photoelectron spectroscopy (XPS) of undoped polythiophene; (iii) transmission electron microscopy (TEM) images of soluble, filtered fractions of undoped polythiophene in DMSO and THF; (iv) Fourier transform infrared spectroscopy of doped polythiophene, as-synthesized undoped polythiophene, and DMSO-insoluble fraction of undoped polythiophene; (v) dynamic light-scattering (DLS) data on aqueous dispersions of undoped polythiophene in water containing 2 wt% Tween-80; (vi) matrix-assisted laser desorption ionization mass spectrometry (MALDI) of the DMSO soluble fraction of undoped polythiophene; (vii) thermogravimetric analysis (TGA) of doped and undoped polythiophene; (viii) details of the cytotoxicity assay; and (ix) details on fluorescence imaging studies on undoped polythiophene in DMSO and DMF. Also included is Table 1 that summarizes the optical properties of dispersions of undoped polythiophene in different solvents. (Supplementary Materials)