1. Introduction

Poly (3, 4-ethylenedioxythiophene) (PEDOT) is a particularly promising conductive polymer, showing high transparency and conductivity, narrow band gap, excellent environmental stability, favorable biocompatibility, and broad visible and near infrared (NIR) absorption and scattering properties [1]. Because of its outstanding electrical, physical and optical properties, PEDOT has been widely applied in electrochromic devices [2], antistatic, microwave sorption [3], photocatalytic [4], drug release [5], etc. However, to the best of our knowledge, the application of PEDOT in biological imaging has never been reported.

Diffusion reflection (DR) spectroscopy is an optical diagnostic method that is simple, safe, and inexpensive. It is capable of revealing morphological information of tissues, using low radiation with high penetration depths [6, 7]. In DR, the reflected light intensity profile of a tissue (Γ) is measured across a pre-specified range of light source-detector distances (ρ) [7, 8]. In previous publications, we demonstrated the high sensitivity of DR for tumor [9–11] and atherosclerosis [12] detection, using gold nanoparticles (GNPs). GNPs were used as contrast agents due to their nontoxicity and biocompatibility [13, 14], combined with their advantageous optical properties, which include a large absorption cross-section [15] and tunable scattering properties [16] in the visible spectrum. Gold nanorods (GNRs) are especially appealing, due to their extreme absorption and scattering properties in the visible and NIR regions of the spectrum, which are enhanced by surface plasmon resonances (SPR) [17]. These distinct properties of GNRs suggest that their use as optical contrast agents can add specificity and sensitivity to the imaging method. However, GNPs, as well as GNRs, are unstable due to their high surface energy, and therefore require suitable surface modifications for their stabilization, which will prevent aggregation. Various functional groups, such as cyano (-CN), mercapto (-SH), and amino (-NH₂) groups have high affinity to gold. Hence, using protective polymers with such functional groups can inhibit the aggregation of GNPs [18]. Moreover, the synergy effect between the two components is likely to improve the SPR of GNPs. However, it is still a great challenge to prepare suitable surface modified GNRs-organic or GNRs-inorganic composites as optical contrast agents with high specificity and sensitivity for biomedical applications.

With its broad visible, NIR to IR absorption properties [19] and favorable biocompatibility, PEDOT has great potential in the biological field. The PEDOT polymer backbone could connect with GNPs through Au–sulphur (thiophene) interactions [20]. Thus, a composite consisting of GNRs and PEDOT, which exhibits the unique properties of both PEDOT and GNPs, is an attractive prospect. First, the PEDOT coating can decrease the aggregation of GNRs. In addition, the NIR, and especially the IR, absorption of PEDOT is expected to increase the SPR of GNRs by the synergistic effect between GNRs and PEDOT.

Fabrication of GNP-PEDOT nanomaterials has been reported in literature, employing several techniques. An Au-incorporated PEDOT composite was fabricated using 3, 4-ethylenedioxythiophene (EDOT) as a reductant, and polystyrene sulfonate (PSS^-) as a particle stabilizer and dopant for PEDOT. However, the morphology of GNPs was difficult to control, and the outer GNPs easily aggregated [21, 22]. Li et al. reported on the self-assembly of GNPs prepared using EDOT as a reductant, by the reduction of HAuCl₄ in tetrahydrofuran solutions with alkylamines as stabilizers. They concluded that the as-formed PEDOT coated on the surface of GNPs, and the PEDOT-GNP composite, showed a red-shift of the surface plasmon absorption. However, the formed PEDOT was insoluble in water, forcing GNPs to aggregate and sediment [23]. Thus, although different chemical methods have been developed to prepare GNP-PEDOT composites, the preparation of highly regulated core-shell GNP-PEDOT composites with superb dispersion remains a challenge that has rarely been reported, and the potential applications of this kind of material need to be studied as well.

In this report, we introduce a facile method for preparing GNR-PEDOT composite nanoparticles (NPs). We suggest that the obtained GNR-PEDOT composite could perfectly achieve the isolation of GNRs and tailor the properties of GNRs, due to its underlying synergistic effect. We performed Monte Carlo (MC) simulations of light path in tissues with different GNR-PEDOT concentrations. In addition, DR measurements of GNR-PEDOT composites showed enhanced SPR optical properties after the PEDOT coating. The porous structure of the new GNR-PEDOT composite, which originates from the crosslinking of polymer chains and the modified –OH group, suggests that the new NPs can be applied as drug carriers for drug delivery. Thus, using this composite, real-time biological imaging and drug release and can be concurrently achieved.

2. Materials and methods

2.1 Monte Carlo simulation of reflected light intensity from irradiated tissues

In order to substantiate and extend the experimental results, an MC simulation of photon migration within irradiated tissues was built. The simulated tissues presented optical properties that were chosen according to skin optical properties [9]. A constant reduced scattering coefficient μ_s' = 1.6 mm⁻¹ and varying concentration of GNR-PEDOT that changes the absorption coefficients to µ_a = 0.0115, 0.0126, 0.0182 and 0.0227 mm⁻¹ were used. The absorption coefficients just slightly differed from each other, in order to test the sensitivity of the reflected light profiles to different absorption properties of the tissue. The main assumptions of the simulations were described before [9].

The simulation displayed the radial distribution of reflected photons around the injection point to perform simulated $\ln ({\rho }^2\Gamma (\rho ))$ graphs for the different absorption coefficients.

2.2 Nanoparticle fabrication and coating

2.2.1 Materials

All reagents were analytical grade and purchased from Sinopharm Chemical Reagent Beijing Co. Ltd., and used without further purification unless otherwise mentioned.

2.2.2 Preparation of GNPs

For the purposes of this study we created two shapes of GNPs, namely, gold nanospheres (GNSs) and GNRs, as previously described [9]. Briefly, the method of Enüstün and Turkevich was used to assemble GNSs with a diameter of about 20 nm. For this process, 414 µL of 50% HAuCl₄ was mixed into 200 mL distilled water, and then boiled. Upon boiling, 4.04 mL 10% sodium citrate was added, and the solution was stirred with heat for 5 minutes. The mixture was then left at room temperature until cooled. The nanoparticles were then gathered through repeated centrifugation.

GNRs were constructed using a modified version of the Seed-Mediated Growth Method [24]. Gold seeds were created by mixing 250 µL HAuCl₄ (0.01 M) with 9.75 mL CTAB (0.1 M), and left to stir. Then 600 µL NaBH₄ (0.01 M) was added, and the solution was left to stir for 10 minutes. After 10 minutes the mixture was removed from stirring, and allowed to sit for at least 1 hour. In a flask, 95 mL CTAB (0.1 M) was mixed with 5 mL HAuCl₄ (0.01 M). Silver nitrate (0.01 M) was added, with 0.6 mL to create shorter rods, and 1.2 mL for longer rods. Afterwards, 550 µL of ascorbic acid (0.1 M) was added, turning the solution clear. From the seed created previously, 120 µL was added to the flask, and the solution was allowed to sit overnight. The following day, the particles were concentrated through centrifugation until reaching clear suspensions.

2.2.3 Preparation of GNR-PEDOT NPs

GNR-PEDOT NPs were prepared by in situ polymerization of EDOT on the surface of GNRs in the presence of sodium dodecyl sulfonate (SDS). In a typical experimental procedure, first, excess CTAB was removed from the as-prepared GNRs (1 mL) by centrifuging for 15 min at 10000 rpm. Next, transferring the clean GNRs into a 2 mL EDOT (0.28 mmol) and SDS (0.15 mmol) mixture aqueous solution, the mixture was stirred for 5 min and 0.75 mmol H₂O₂ was added as an oxidant. After being stirred for 16 h at 60°C, the resulting solution was isolated by centrifugation and washed several times with a solvent of deionized water/ethanol (1/1, v/v). The precipitate was dried under vacuum at 50°C for 24 hours.

2.2.4 Preparation of PEDOT NPs

A 2 mL EDOT (0.28 mmol) and SDS (0.15 mmol) mixture aqueous solution was stirred for 5 min and 0.75 mmol H₂O₂ was added as an oxidant. After being stirred for 16 h at 60°C, the resulting solution was isolated by centrifugation and washed several times with a solvent of deionized water/ethanol (1/1, v/v). The precipitate was dried under vacuum at 50°C for 24 hours.

2.3 Apparatus

Transmission electron microscope (TEM) measurements were made using JEM-2100F to characterize the morphology of the products. Powder X-ray diffraction (XRD) measurements were carried out on a D8 Focus Diffractometer (Germany) using Cu K_α radiation (λ = 0.15406 nm). Fourier transform infrared (FT-IR) spectra were measured on sample pellets with KBr by means of an infrared spectrophotometer (Excalibur 3100, America, Varian).

2.4 Solid phantom construction

Solid phantoms were created by mixing 10% Intralipid (Lipfundin MCT/LCT 20%, B. Braun Melsungen AG, Germany) for scattering, 3% India ink (0.1%) for absorption, and the other 87% of the volume completed either with distilled water or NPs solution. Agarose powder (SeaKem LE Agarose, Lonza, USA) was added at 1.2% in order to solidify the solution. The components were stirred together while heated until even mixing was achieved, and then poured into wells capable of holding the volume. The wells were placed into vacuum to help cool and solidify over a few hours.

2.5 Diffusion reflection measurements

DR measurements were conducted on a device designed for the noninvasive optical imaging technique (NEGOH-OP TECHNOLOGIES, Israel), and the method has been previously described [9, 10]. Two laser diodes, of wavelengths 650nm and 780nm, served as excitation sources, and a 125μm diameter optical fiber was used for irradiation. A photodiode was placed in contact with the phantom’s surface to detect scattered light while minimizing ambient light and potential losses due to the phantom’s edges. The light source was moved on a micrometer plate in steps of 250μm so that the light intensity reaching the photodiode could be measured at varying distances between the source and detector (ρ), with an initial distance of approximately 1mm, and a final distance of about 5 or 6 mm. The reflected light intensity at the photodiode, Γ(ρ), was measured using a digital scope (Agilent Technologies, Mso7034a, Santa Clara, CA, USA), and data was processed using Lab View.

3. Results and discussion

Simulations of the reflected light intensity from tissues with different optical properties were performed according to the description in Materials and Methods (section 2.1). Photons penetrated and advanced randomly in the tissue. Several absorption coefficients describing the GNR-PEDOT NPs were considered, and the resultant logarithmic graphs of the reflected light intensity are shown in Fig. 1.

 

Fig. 1 MC simulation results of tissues presenting different absorption coefficients, but a constant µ_s'. Simulated $\ln ({\rho }^2\Gamma (\rho ))$profiles of homogeneous tissues with GNR-PEDOT presenting four absorption coefficients of 0.0115 (diamonds), 0.0126 (squares), 0.018 (triangles) and 0.0227 (circles) mm⁻¹.

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The MC simulation results in Fig. 1 present the predicted dependence of the reflected light intensity profile on the lattice and the GNR-PEDOT absorption coefficient: the higher the absorption coefficient, the sharper the decay of the reflected light intensity profile. This is in agreement with the following equation:

GNR-PEDOT composite NPs were prepared by a simple emulsion polymerization method. PEDOT coats the surface of GNRs by employing the interactions between sulphonate of surfactant SDS and PEDOT, and Au–Sulphur (thiophene) of the PEDOT polymer backbone. Moreover, using H₂O₂ as a weak oxidant ensured that the EDOT gradually polymerized on the surface of GNRs, rather than dissociated in the reaction system. The new GNR-PEDOT NPs, GNSs, PEDOT NPs, and GNRs were characterized via TEM (Figs. 2(a)–2(d)) and absorbance spectroscopy (Fig. 2(e)). The GNSs with a diameter of about 20 nm had an absorption peak at 530 nm. The PEDOT NPs with a diameter of about 150 nm had broad absorption spectra from 400 to 900 nm. GNRs with average dimensions of 45 ± 5 nm by 15 nm as width had a peak resonance at 510 nm and 690 nm. After coated with PEDOT, the GNRs were covered by the PEDOT shell, and uniform spherical GNR-PEDOT NPs were obtained with diameters of about 80 nm.

 

Fig. 2 TEM images of (a) GNSs; (b) PEDOT; (c) GNRs; (d) GNR-PEDOT NPs; (e) UV-vis spectra of GNSs (green), PEDOT (black), GNRs (blue) and GNR-PEDOT NPs (red).

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The protection provided by the outer PEDOT layers prevented aggregation of the original GNRs. The UV-Vis spectra (Fig. 2(e)) showed that as compared to the GNRs, GNR-PEDOT NPs had a broad absorption at 530 nm and a significant peak at 790 nm, which is a 100nm red-shift compared with that of GNRs. Because organic micro-molecules have higher refractive indexes than solvents, the surrounding PEDOT produced the red-shift effect for the SPR of GNRs [25]. This result indicates the dual benefit of GNR-PEDOT NPs, namely, protection of GNRs and enhancement of the GNRs' SPR.

Figure 3 shows the FT-IR spectra of the purified GNR-PEDOT composite. It is clear that the peaks at 1538, 1487, 1437,1388 cm⁻¹ are attributed to the C = C stretching of the quinoid structure of thiophene ring; the peak at 1359cm⁻¹ is assigned to C - C stretching of the quinoidal structure; vibrations at 991, 842cm⁻¹ are associated with the C - S bond in the thiophene ring; the bands at 1068, 1245cm⁻¹ are due to the ethylenedioxy group; the peak around 933cm⁻¹ originates from the ethylenedioxy ring deformation mode, and the band at 891 is ascribed to the C - H bending mode, demonstrating the formation of PEDOT chains with α, α'- coupling. All the peaks confirm the polymerization of PEDOT [3, 20]. The peak near 3435cm⁻¹ corresponds to the hydrogen-bonded O-H stretching vibration, which may stem from the use of oxidant H₂O₂. This finding emphasizes that the GNR-PEDOT composite is modified by a functional group that can be highly important for further functionalization and application of this composite, such as attachment to drug molecules for drug delivery.

 

Fig. 3 Left: FT-IR spectra of the GNR-PEDOT NPs; Right: enlarged image of the 600-1600 nm section.

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The XRD pattern of GNR-PEDOT NPs is presented in Fig. 4. The broad peak at 2θ = 25.6° can be ascribed to PEDOT, based on the literature [26]. Besides the PEDOT peak, four additional peaks found at 2θ = 37.91, 44.06, 64.39 and 77.44° corresponded to the (111), (200), (220) and (311) planes of Au, which indicate the existence of Au in the GNR-PEDOT core–shell composite. Based on these results, we conclude that by using this simple emulsion polymerization method, GNR-PEDOT core-shell nanoparticles with excellent dispersion and SPR properties can be successfully prepared.

 

Fig. 4 XRD pattern of GNR-PEDOT NPs.

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DR measurements were used to detect the presence of GNP-PEDOT NPs in solid phantoms. By measuring the intensity of reflected light from the phantom (denoted by Γ(ρ)) over varying distances between the 780nm light source and the detector, the slope of ln(ρ²Γ(ρ)) versus ρ was calculated for phantoms containing either water, GNSs, or GNRs with or without coating of PEDOT. Figure 5 shows results for each of these types of phantoms. In such a DR figure, a more pronounced slope indicates greater particle absorption. As expected, the GNR-PEDOT with a peak at 790 nm absorbed the source light most efficiently (as indicated in Fig. 5 by the thick black line), and the GNRs without the PEDOT coating absorbed less efficiently at 780 nm (Fig. 5, dotted red line). The GNSs and water phantoms (Fig. 5, dashed blue line and thin purple line, respectively) performed in a manner similar to the GNRs without coating, due to their low absorption at 780 nm. These results indicate that tuning of the probing light used by the DR system enables efficient testing for presence of corresponding particles within the volume of a sample, and that the PEDOT coating can efficiently improve the optical properties of GNRs. As an additional control measurement, we prepared the same phantoms, containing PEDOT only (without GNRs). We found that the slope of the DR was the same at the 650nm and 780nm excitation wavelengths (as expected from Fig. 2). This indicates that PEDOT alone cannot be used as the contrast agent in DR experiments, as it has no SPR effect, and has the same spectra in the relevant SPR wavelengths. However, PEDOT-coated GNRs generate a change in reflected light intensity, meaning that this composite has great potential as a contrast agent for molecular imaging. Moreover, the promising new combination with the DR imaging technique enables efficient and sensitive molecular imaging.

 

Fig. 5 DR results from four different phantoms, each denoted by a different color: a water-only base (thin purple line), GNRs with absorption peak at 690nm (red dotted line), GNRs with PEDOT (black line), and GNSs (dashed blue line). Each phantom was measured twice. The light source had a 780nm wavelength.

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4. Conclusions

In the present study, we demonstrated a new, facile in situ polymerization method for PEDOT-based coating of GNRs. The GNR-PEDOT NPs exhibited the standard optical properties of GNRs, together with a unique SPR. Thus, our efficient GNP-PEDOT constructs enabled production of enhanced images and sensitive detection of GNP presence using DR measurements. By detecting changes in optical properties of tissue-like phantoms, we were able to gain insight into tissue behavior during similar imaging conditions.

In summary, the combination of GNR-PEDOT with DR detection − a non-invasive, simple, and highly sensitive method − offers a promising imaging tool for medical diagnostics.