Tuning the structure of thienoisoindigo (TIG) copolymers to afford bright near-infrared emission for bioimaging through aggregation-enhanced emission

Abstract

Near-infrared (NIR) emitting materials underpin emerging medical diagnostics and therapeutic bionanotechnologies. Conjugated polymer nanoparticles offer unique advantages due to their remarkable absorption cross-sections, photostability, synthetic tunability, and biocompatibility. Despite the vast library of NIR-absorbing conjugated polymers, relatively few narrow bandgap structures have been explored for NIR imaging. Herein, we investigate thienoisoindigo copolymers (PTIG-co-TT and PTIG-co-T), well-established semiconductors for organic bioelectronics and photovoltaics, as NIR emitters. Both polymers demonstrated weak emission in solution, which upon processing into the nanoparticle form factor displayed a remarkable, previously unexplored aggregation-enhanced emission (AEE) by multiple orders of magnitude. Careful matching of molecular weight and nanoparticle size between the two systems revealed the role of backbone flexibility on nanoparticle brightness; the AEE was more pronounced in the more planar PTIG-co-TT. Both formulations featured extraordinary brightness and photostability, outperforming clinical standard indocyanine green while displaying peak emission at ∼880 nm, making them strong alternatives that can be directly applied with current hardware for NIR cancer diagnostics and image-guided surgery. The unexpected NIR optical performance of otherwise well-established organic semiconductors suggests there may be untapped potential for this class of materials as imaging agents.

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1. Introduction

In vivo fluorescence imaging within the near-infrared (NIR) wavelength region (700–1700 nm), coined the biological transparency window,¹ fundamentally underpins emerging diagnostic and therapeutic bionanotechnologies. Chromophores operating within this region have been explored for guiding surgical resections of tumors in real-time, photoacoustic tomography, photothermal therapy, and complementing other imaging modalities.^2–7 The most clinically relevant NIR-emitting dye is small-molecule indocyanine green (ICG),¹ which has been utilized since the 1950s for medical diagnostics.^1,8 ICG has also been extensively investigated as an emitter for in vivo imaging technologies including cancer diagnostics, however, ICG displays a short half-life, poor photostability, hydrolytic instability, non-specific binding to proteins, and a lack of targeting modalities.^9–11 Challenges associated with using ICG have inspired a vast literature of various molecular and material-based emitters for NIR imaging including organic small-molecules, aggregation-induced emission agents (AIEgens),^12,13 engineered proteins, quantum dots (QDs),¹⁴ and carbon nanomaterials,⁴ among others.¹⁵

Of the materials that have been reported, conjugated polymers (CPs) are advantageous as they are (1) biocompatible,¹⁶ (2) easily processed into stable nanoparticles (CPNs) with cell/tissue labeling modalities, and (3) synthetically tunable to modulate the bandgap toward NIR emission.^17,18 Fluorescence quantum yields (QY) of NIR emitting CPs are generally low (<1%), which can be attributed to the energy gap law – nonradiative decay is accelerated by the vibrational overlap between the ground state and excited state because of the narrow polymer bandgap.^19,20 While currently reported CPNs have rather low QYs compared to alternative nanomaterials and some small molecules, the densely packed π-conjugated backbones of D–A CPs offers remarkably large absorption cross sections and therefore extraordinary brightness. Several CP/CPNs that have been reported for NIR imaging have demonstrated enhanced brightness and photostability in the NIR compared to well-established standards such as ICG, IR-1061, and IR-26.¹⁷

Of the limited examples that have been reported of relatively bright CP-derived NIR emitters, most rely on copolymers of specific heterocycles such as benzodithiophene (BDT) or triazole[4,5-g]-quinoxaline (TQ) derivatives; comparatively, a much larger library of heterocycles has been investigated for NIR absorption for photoacoustic, photothermal therapy, and organic photodetector applications.^{3,5–7,21,22} Here, we investigate the NIR emissive behavior of D–A copolymers consisting of thienoisoindigo (TIG) acceptors, and simple thiophene-based donors. A strong aggregation enhanced emission was observed upon processing the CPs into CPNs. Within the nanoparticle state, both materials displayed significant photostability and brightness compared to widely adopted small molecule standards. To our knowledge, this is the first investigation into the emissive properties of TIG-derived copolymers, despite their known absorption throughout the infrared.^23–28 The relatively bright NIR emission of these well-established organic electronic materials suggests that many other unexplored CPs may prove efficacious for NIR imaging technologies as well.

2. Results and discussion

2.1. Molecular design of TIG-derived near infrared emitters

Conjugated polymers featuring isoindigo (IG) acceptors are well-established within the organic electronic community and are typically utilized for their charge-transporting properties. Furthermore, π-extension of the IG unit with fused thiophenes affords a thienoisoindigo (TIG) acceptor with enhanced structural planarity (S⋯O coulombic/chalcogen interactions),²⁹ increased electron affinity, and redshifted absorption for improved light-harvesting. While D–A copolymers featuring TIG acceptors (A) have been reported for organic thin-film transistors (OTFTs),^24–28 organic electrochemical transistors (OECTs),^30,31 and organic photovoltaics (OPVs),^24,32–34 their emissive properties have yet to be explored. To investigate the potential of TIG copolymers for applications requiring NIR emission such as bioimaging, two D–A copolymers were synthesized featuring either the thieno[3,2-b]thiophene (TT) or thiophene (T) donor, both of which have been previously reported for organic electronic applications.^25,34

The investigated polymers were synthesized using established synthetic methodologies by first constructing TIG cores with aliphatic side chains (Fig. 1).³¹ The reactive monomers were subjected to Stille polycondensation with either the 2,5-bis(trimethylstannyl)thiophene or 2,5-bis(trimethylstannyl)thieno[3,2-b]thiophene donors to afford alternating copolymers PTIG-co-T and PTIG-co-TT, respectively (abbreviated as PTIG-T and PTIG-TT). The crude polymers were purified by Soxhlet extractions in methanol, acetone, and hexanes/THF, after which the polymers were obtained by precipitation from chloroform. The polymers (dark green solids) are soluble in chloroform, 1,2,4-trichlorobenzene, and THF, the latter of which is required for nanoprecipitation (See section 2.2). The weight-average molecular weights (M_w) were measured via high-temperature gel-permeation chromatography (See Experimental Section) to be 24.9 and 23.3 kDa for PTIG-T and PTIG-TT, respectively. It is important that both CP batches feature similar molecular weight distributions, as chain length directly impacts molar absorptivity and quantum yield, which are proportional to fluorophore brightness, and therefore performance.^35–37

Fig. 1 Synthesis of (top) functional thienoisoindigo (TIG) monomer and (bottom) corresponding polymerization of PTIG-TT and PTIG-T copolymers via Stille polycondensation.

The UV-vis-NIR absorption spectra of PTIG-TT and PTIG-T in THF solution display dual absorption band characteristics (Fig. 2a), corresponding with prior literature reports of the same materials.^25,34 The short-wavelength absorption bands from 350–500 nm are assigned to the delocalized excitonic π–π* transition of the TIG moieties, whereas the long-wavelength absorption bands are due to intramolecular charge transfer (ICT) interactions from the donors (T or TT) to the TIG acceptor unit of the polymeric backbone. The absorption spectra of both polymers span the NIR spectral window, and can conveniently be excited by an 808 laser, which is typical for NIR imaging applications. The maximum emission peak (λ_em, max) for both materials is at 898 nm, slightly farther into the infrared than the current standard ICG (λ_em, max = 810–840), but directly compatible with current NIR-imaging hardware which is designed for ICG. Both polymers feature nearly identical emission profiles (Fig. 2b), suggesting that the electronic structure is primarily governed by the TIG units, rather than the thiophene based donors. This is supported by the nearly identical optical band gap (E^opt_g) between the PTIG-TT and PTIG-T which were both 0.97 eV (See SI), which also correspond with prior reports of these materials.^25,34 Furthermore, the similar experimental highest occupied molecular orbital energy levels (HOMOs) and calculated emission energy and bandgap via DFT (Tables S2 and S4) suggest the two investigated donors were similarly weak.

Fig. 2 (a) Absorption spectra of TIG copolymers in THF solutions (20 μM). (b) Fluorescence emission spectra of the polymers in THF (OD₄₄₄ = 0.02). (c) Integrated fluorescence intensity plotted as a function of OD at 444 nm for the polymers in THF, and IR-1061 in dichloromethane. (d) Photostability of the polymers in THF, and ICG in H₂O with the same molar concentration under continuous radiation at 444 nm and collection at their respective maximum emission peaks (λ_em, max).

The fluorescence QYs of both polymers in THF were determined by using IR-1061 as a reference (Fig. 2c, See Experimental Section) – a typical standard for NIR emitting organic materials.¹⁷ The optical profiles were strikingly similar with the QYs of the two polymers at 0.03 and 0.01% for PTIG-T and PTIG-TT, respectively. DFT calculations of relative lifetime (ns) and oscillator strength obtained at the M06-2X/6-31G* level of theory also supports a similar radiative decay for both polymers, suggesting comparable QY (Table S3). However, despite the lower QY of both polymers, the relatively high molar absorptivity of the π-conjugated D–A polymer structure leads to enhanced brightness of the polymers compared to small molecules ICG and IR-1061 at the same concentration (Fig. S2). Furthermore, the photostability of both polymers were examined under continuous irradiation, demonstrating superior photostability compared to ICG (Fig. 2d).

2.2. Processing and performance of PTIG CPNs

A significant advantage of CP derived emitters for bioimaging compared to organic small molecules is the ease at which nanoparticles/microparticles (10–500 nm) can be processed with colloidal stability, and orthogonal surface functionalities for cell/tissue labeling.¹⁷ Through techniques such as nanoprecipitation, dilute CP solutions in organic solvent can be introduced to an excess, miscible nonsolvent, usually water – where the rapid decrease in solvent quality induces aggregation/precipitation of the conjugated polymer nanoparticle (CPN), stabilized by a surfactant shell. However, the already low QY of the CPs typically decreases further due to aggregation caused quenching (ACQ) – prior reports display an order of magnitude reduction in QY from the CP to CPN form factor for NIR emitting D–A systems.^38,39 Alternatively, chromophores that display enhanced brightness in the aggregated state, also known as solid-state luminescent enhancement (SLE) or aggregation-induced emission (AIE), can be incorporated within the D–A polymer structure – these work by restricting rotations that otherwise allow for nonradiative dissipation.¹⁵ While several examples have reported the incorporation of twisted acceptors within NIR emitting D–A CPs to enable AIE,^40–42 there is a tradeoff between enhanced QY and reduced absorptivity from disrupted conjugation.^17,43 Recently, thienoisoindigo (TIG) has been demonstrated as a strong planar acceptor that can act as an AIE block within small molecule D–A–D NIR emitters,^44,45 but it has yet to be leveraged within D–A CP NIR systems. The AIE behavior of PTIG-TT and PTIG-T were assessed by measuring their fluorescent spectra in THF solution containing different volume fractions of H₂O (f_w) at room temperature (Fig. 3). For both polymers, there was a significant enhancement in relative fluorescence intensity (I/I₀) at approximately f_w = 80%; emission then dramatically decreased at around f_w = 88% which can be attributed to uncontrolled polymer precipitation.⁴⁶ Upon aggregation at f_w = 88%, 50-fold and 30-fold enhancements in emission at λ_em, max (∼882 nm) were observed for PTIG-TT and PTIG-T respectively. The remarkable enhancement in emission intensity suggests that PTIG derivatives could be strong emitters for NIR imaging within the aggregated nanoparticle state.

Fig. 3 Fluorescent spectra of (a) PTIG-TT and (b) PTIG-T in solutions of THF with different H₂O fractions (%) – all solutions were set to 1.2 μM. The insets display the normalized maximum peak fluorescent intensities (λ_em, max = 882 nm) of each polymer in solution of THF containing different H₂O fractions.

Both PTIG copolymers were then independently processed via nanoprecipitation to afford surfactant stabilized CPNs. Traditional batch methods introduce the CP organic solution to the aqueous bath manually, precluding precision control of nanoparticle size, a factor that has been directly correlated to brightness of CPNs in recent reports.^17,47 To ensure similarly sized CPNs for direct comparisons between the two systems, a syringe pump was used to control CP flow rate (0.5 mL min⁻¹) during nanoprecipitation with a DSPE-PEG₂₀₀₀ surfactant (Fig. 4a). The hydrodynamic diameters of the resulting CPNs were measured by dynamic light scattering to be 75.9 nm and 77.7 nm for PTIG-T and PTIG-TT, respectively (Fig. 4b). As CPNs are self-assembled in aqueous solution and purified via centrifugation (see Experimental section), it is challenging to assess final CPN concentration accurately. As absorptivity of CPs is conformation dependent, calibration curves from CP absorptivity in solution cannot accurately inform CPN concentration on a molar basis. The current standard in the field is to lyophilize the colloidal suspension and use the afforded microgram quantities to retroactively calculate mass extinction coefficients rather than molar extinction coefficients;^36,48,49 such calculations are challenging due to the hygroscopic nature of the DSPE-PEG surfactant, and small quantity of final material. Instead, nanoparticle tracking analysis (NTA) was used as a nondestructive technique to directly measure particle concentration in aqueous solution (Fig. 4c).⁵⁰ Using NTA, accurate CPN concentrations on a molar basis were measured to enable accurate comparisons of relative brightness between the two CPN systems, and with small molecule NIR standards.

Fig. 4 (a) Diagram demonstrating the use of a syringe pump to finely control CPN size during nanoprecipitation. (b) Hydrodynamic diameter distribution of both CPNs measured by dynamic light scattering in H₂O (∼ 0.2 × 10⁻¹⁰ M). The inset displays the zeta (ξ) potential analysis of PTIG-TT (−35.5) and PTIG-T (−33.3). (c) Size distribution and particle count measured by nanoparticle tracking analysis (NTA). The inset displays a snapshot of the real time reading from NTA with the corresponding concentration that was measured.

As with the CPs, both CPNs displayed near identical optical profiles (Fig. 5a and b). As anticipated, the AIE phenomena of the CPs translated to the CPNs – displaying remarkable enhancements in emission between the polymers (Fig. 2b) and particles (Fig. 5b) under similar conditions. The fluorescence QYs of both CPNs in H₂O were determined by using IR-1061 as a reference (see Experimental section); both displayed significant enhancements in QY of 1.4 and 1.3% for PTIG-T and PTIG-TT CPNs, respectively. Such enhancements are rare for D–A CP derived CPNs, which typically feature reductions in emission from the CP to CPN form factor.^38,39 While the QY is slightly higher for the PTIG-T CPN, relatively larger molar absorptivity of the more planar PTIG-TT (Fig. S2) afforded a slightly elevated brightness; both materials display significantly enhanced emission compared to ICG and IR-1061 small molecule standards when measured at the same concentration (Fig. 5d). The brightness of CPNs can be further tuned through the manipulation of CP molecular weight before nanoprecipitation. Extending the chain length of a CP has two inversely related impacts on performance; (1) an enhancement in molar absorptivity, and (2) a decrease in QY. For example, multiple reports have demonstrated over an order of magnitude difference in QY of similar CPs based on only small molecular weight differences.^35,36 To further evaluate the potential of PTIG derived CPNs, a lower molecular weight (9.7 kDa) PTIG-TT derivative was synthesized and processed into nanoparticles of comparable size (d = 81.54 nm) to the other two batches (Fig. S3). The lower molecular weight derived PTIG-TT CPNs featured significantly enhanced experimental relative brightness compared to the other two CPN formulations, primarily due to a remarkably high QY of 2.9%. Compared to other NIR emitting CPNs with peak emission between 800–900 nm, including commercial formulations, the QY is remarkable (Table S5). The performance of the PTIG derived CPNs can potentially be tuned further via molecular weight adjustments, and modulation of nanoparticle size.

Fig. 5 (a) Absorption spectra of PTIG CPNs in H₂O (0.2 nM). (b) Fluorescence emission spectra of the CPNs in H₂O (0.2 nM). (c) Integrated fluorescence intensity plotted as a function of OD at 444 nm for the CPNs in H₂O, and IR-1061 in dichloromethane. (d) The mean fluorescence intensity of ICG in H₂O, IR-1061 in dichloromethane, and CPNs in H₂O with the same molar concentration (0.2 nM, λ_ex = 444 nm, emission at λ_em, max). The inset displays corresponding NIR fluorescent images from the IR VIVO animal imager. (e) Photostability of the CPNs and ICG in H₂O with the same molar concentration (0.2 nM) under continuous radiation at 808 nm. (f) Size stability as measured by fluctuation in the hydrodynamic diameter (D_h) over time (168 h) for (top) PTIG-T, and (bottom) PTIG-TT in buffers of varying pH. (g) In vivo NIR fluorescence imaging of CD-1 mice after injection of 50 μL of PTIG-TT CPNs with corresponding (h) resolution of a vein. (i) Ex vivo fluorescence imaging of various viscera of a mouse: (i) heart, (ii) lungs, (iii) liver, (iv) stomach, (v) spleen, (vi) kidneys, (vii) intestines.

Beyond enhanced brightness, the CPNs require long-term photostability and structural stability to be efficacious for in vivo imaging. The photostability of both CPNs were high under continuous laser excitation (Fig. 5e), as observed with the corresponding CPs (Fig. 2d). CPNs typically display long-term photostability,¹⁷ compared to ICG which demonstrates significant emission decay within 30 minutes (I/I₀, t = 30 min = 0.5, Fig. 5e). To assess colloidal stability under biologically relevant conditions, the size of both CPNs was monitored by dynamic light scattering in H₂O and under acidic (acetate), physiological (PBS), and basic (borate) buffers, which span the pH ranges relevant to various bioimaging applications. Both systems displayed stability over the span of seven days (Fig. 5f). An MTT assay was used to confirm the cytocompatibility of the CPNs (Fig. S6); CP and CPNs are widely considered to be broadly biocompatible.^16,17 To demonstrate the efficacy of the PTIG CPNs in vivo, high performing PTIG-TT derived CPN (9.7 kDa CP batch) was investigated in mice (see Experimental section) using an IR VIVO animal imager. The CPN displayed strong emission within the mouse (Fig. 5g), and line analysis of a vein demonstrates the resolution of vasculatures (Fig. 5h). Consistent with other reports of CPNs, emission intensity suggests elevated biodistribution into the liver and spleen (Fig. 5i). Future work can use surfactant labeling or other coating strategies to provide tumor targeting moieties for engineering formulations for cancer diagnostics and image guided surgery, as well as other targeted bioimaging technologies.¹⁷

3. Conclusions

Herein, we have offered a strategy to develop PTIG-derived copolymers into bright NIR emitters for bioimaging. The controlled precipitation of both the PTIG-TT and PTIG-T into CPNs displayed remarkable aggregation-induced enhancements in peak emission by a factor of ∼40 between each CPN and their corresponding CP. Potential confounding factors such as molecular weight, CPN particle size, and CPN concentration were carefully matched to enable an accurate comparison between the two systems, and standard small-molecule NIR chromophores. The CPNs displayed (1) significantly enhanced brightness and photostability compared to current standard ICG, (2) long term structural stability, and (3) were efficacious for in vivo NIR imaging. The high performance of two well-established organic electronic materials for NIR imaging, that previously were widely explored for OPVs and OTFTs, suggests there may be significant untapped potential to further develop CP-derived NIR emitters for bioimaging and diagnostics.

4. Experimental section

4.1. Materials

PTIG copolymers were synthesized according to previously reported procedures.^25,31,34 Monomers 2,5-bis(trimethylstannyl)thiophene and 2,5-bis(trimethylstannyl)thieno[3,2-b]thiophene and other reagents were purchased from Sigma-Aldrich and used without further purification, unless otherwise specified. Xylenes was degassed and dried over 4 Å molecular sieves. Tetrakis(triphenylphosphine)palladium(0) (Pd(PPh₃)₄) was purchased from Strem Chemicals and used without further purification. All manipulations of air and/or moisture sensitive compounds were performed under an inert atmosphere using standard glove box and Schlenk techniques.

4.2. Molecular weight characterization

The weight average molecular weight (M_w) weight and dispersity (Đ) were determined by high-temperature gel permeation chromatography (HT GPC) at 130 °C in 1,2,4-trichlorobenzene (1 mg mL⁻¹; TCB, stabilized with 125 ppm of BHT) in a Tosoh EcoSEC High Temperature GPC system using a TSKgel G2000Hhr (20) HT2 column (1 mL min⁻¹).

4.3. Nanoprecipitation

Conjugated polymer nanoparticles were prepared according to previous reports via nanoprecipitation.⁵¹ Briefly, conjugated polymer (0.5–1.0 mg) and DSPE-PEG₂₀₀₀ (9.0 mg) were dissolved in THF (2 mL), and the solution was then injected into 7 mL DI H₂O under continuous sonication for 3 min (70% amplitude, phase on for 2 s and phase off for 1 s) using a syringe pump (CHEMYX Fusion 4000X) at 0.5 mL min⁻¹. Subsequently the solution was stirred overnight. The aqueous solution was then washed three times using centrifugal filter (Vivaspin 15R, 30 000 MWCO) under centrifugation at 3700 rpm for 10 min, and resuspended in DI H₂O or buffer. The washed solution was then filtered through PVDF syringe filter (13 mm, 0.22 μm) to remove impurities and large particles. The CPN solutions were stored in a cool and dark environment.

4.4. NIR fluorescence of polymers and nanoparticles

Optical characterization was performed using a dual absorbance and fluorescence spectrophotometer (Olis DSM 142 UV/Vis/NIR) featuring extended InGaAs detectors to assess the NIR absorption and emission of each CP/CPN. Fluorescence quantum yield (QY) was measured using IR-1061 dye (QY = 0.32%) as the reference.⁵² The IR-1061 dye was diluted with dichloromethane (DCM) solution to different optical densities (OD) at 444 nm. Five different concentrations around or less than an OD of 0.1 were measured and analyzed at room temperature. The absorption and emission of polymer (THF) or CPNs (aqueous) were measured. The integrated fluorescence intensity was plotted against the absorbance at the excitation wavelength of 444 nm and fitted into a linear function as previously reported. And the QY was calculated using eqn (1).⁵³

QY_s = QY_r × (K_s/K_r) × (n_s/n_r)² (1)

Where subscripts s and r denote sample and reference, respectively. K is the slope of the integrated fluorescence intensity against the absorbance plot (linear fitting). n_s = 1.333 is the refractive index of water, n_s = 1.407 is the refractive index of THF, and n_r = 1.424 is the refractive index of DCM. For aqueous nanoparticle solutions, solution concentrations were measured using nanoparticle tracking analysis (NTA),⁵³ which utilizes the properties of both light scattering and Brownian movement to acquire a nanoparticle size distribution of samples in liquid dispersion (ZetaView Quatt, Particle Metrix, λ_ex = 488 nm).

4.5. In vivo imaging

All experiments involving animals were carried out according with the guidelines stipulated by the Animal Care and Use Committee (IACUC), Texas Tech University Health Sciences Center (TTUHSC). A CD-1 mouse was obtained from Charles River Laboratories and used at ten-weeks-old. The mouse was injected with 50 μL of PTIG-TT CPN through the jugular vein for the imaging study. NIR fluorescence imaging was performed using an IR VIVO whole animal imaging system (Photon etc., Canada) with excitation at 808 nm. The ex vivo biodistribution studies were further performed after 12 h post-injection of the nanoprobe to evaluate the distribution of the CPN in major organs.

Conflicts of interest

J. T., R. P., and N. G. have a pending patent application related to the disclosed materials.

Data availability

The data supporting this article have been included as part of the SI. See DOI: https://doi.org/10.1039/d5tb01093d

Any data not provided as SI will be made available on request from the corresponding author.

Acknowledgements

J. T. acknowledges Texas Tech University for financial support. These studies were funded, in part, by the Core Facility Support Award to U. B. (grant number RP200572) from the Cancer Prevention and Research Institute of Texas (CPRIT) to the Imaging Core, Texas Tech University Health Sciences Center at Amarillo.

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Footnote

† R. P. and N. G. contributed equally to this work.

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