Self-Reporting Conjugated Polymer Nanoparticles for Superoxide Generation and Detection

Abstract

Conjugated polymer nanoparticles (CPNs or Pdots) have become increasingly popular fluorophores for multimodal applications that combine imaging with phototherapeutic effects. Reports of CPNs in photodynamic therapy applications typically focus on their ability to generate singlet oxygen. Alternatively, CPN excited states can interact with oxygen to form superoxide radical anion and a CPN-based hole polaron, both of which can have deleterious effects on fluorescence properties. Here, we demonstrate that CPNs prepared from the common conjugated polymer poly[(9,9-dioctylfluorenyl-2,7-diyl)-alt-co-(1,4-benzo-{2,1',3}-thiadiazole)] (PFBT, also known as F8BT) generate superoxide upon irradiation. We use the same CPNs to detect superoxide by doping them with a superoxide-responsive hydrocyanine dye developed by Murthy and co-workers. Superoxide induces off-to-on fluorescence switching by converting quenching hydrocyanine dyes to fluorescent cyanine dyes that act as fluorescence resonance energy transfer (FRET) acceptors for PFBT chromophores. Amplified FRET from the multichromophoric CPNs yields fluorescence signal intensities that are nearly 50 times greater than when the dye is excited directly or over 100 times greater when signal readout is from the CPN channel. The dye loading level governs the maximum amount of superoxide that induces a change in fluorescence properties and also influences the rate of superoxide generation by furnishing competitive excited state deactivation pathways. These results suggest that CPNs can be used to deliver superoxide in applications in which it is desirable and provide a caution for fluorescence-based CPN applications in which superoxide can damage fluorophores.

Keywords: conjugated polymer nanoparticles; electron transfer; energy transfer; fluorescence; semiconducting polymer dots; superoxide.

Scheme 1. Chemical Structures of Conjugated Polymer PFBT and the Reduced (HyCy5) and Oxidized (Cy5) Forms of Dye Dopants

Figure 1

(A) Absorbance of HyCy5 (tan), absorbance (black) and fluorescence (yellow-green) of PFBT CPNs, and absorbance (blue) and fluorescence (red) of Cy5 dyes with shading depicting donor–acceptor spectral overlap for FRET. (B) Size distribution of PFBT CPNs measured in aqueous suspension by dynamic light scattering. (C) Absorbance of PFBT CPNs before (black) and after (tan) doping with HyCy5 and after the doped sample was spun in a centrifugal filtration device (green).

Figure 2

(A) Fluorescence spectra of undoped (yellow-green) and HyCy5-doped (black) CPNs. (B) Stern–Volmer plot showing quenching of CPN fluorescence by HyCy5 dyes. Values are the average of 5 runs with error bars representing the standard deviation.

Figure 3

(A) Scheme for superoxide reporting by turn-on fluorescence in doped CPNs. (B) Jablonski diagram depicting key photophysical pathways in as-prepared HyCy5-doped CPNs. (C) Scheme for interaction of oxygen with CPN excited state. (D) Jablonski diagram depicting key photophysical pathways in doped CPNs after some HyCy5 dyes have been converted to Cy5. Internal conversion and other minor pathways are omitted for clarity in the Jablonski diagrams.

Figure 4

Fluorescence of 15 wt % HyCy5-doped CPNs before (black) and at 5 s intervals during irradiation (455 nm, 0.87 mW/cm²). Inset: normalized spectra recorded before irradiation and after 5 and 20 s of irradiation.

Figure 5

Fluorescence spectra of HyCy5-doped CPNs in aqueous suspension (A) under degassed conditions in the dark before (black) and after (blue) irradiation (40 s, 455 nm); (B) with methylene blue before (black) and after irradiation to produce singlet oxygen (red, 35 s, 625 nm) and after irradiation to produce a positive control (blue, 35 s, 455 nm). Fresh aliquots of the same sample were used for each irradiation exposure. (C) Difference in change of absorbance of Cyt C at 550 nm upon irradiation (2 min, 455 nm) in the presence and absence of SOD as a function of irradiation intensity. Results shown are the average of 3 measurements. Inset: representative traces of change of absorbance in presence and absence of SOD during irradiation (2 min, 455 nm, 2.6 mW/cm²).

Figure 6

(A) Evolution of fluorescence intensity of 15 wt % doped CPNs during irradiation (shaded area, 455 nm, 0.87 mW/cm²) in PFBT (537 nm, yellow-green) and Cy5 (671 nm, red) channels. (B) PFBT Fluorescence intensity as a function of time in undoped CPNs before, during (shaded area, 455 nm, 0.87 mW/cm²), and after irradiation. (C) Ratio of fluorescence intensities from panel A as a function of irradiation time.

Figure 7

(A) Fluorescence spectra of 15 wt % dye-doped CPNs after irradiation (40 s, 455 nm, 0.87 mW/cm²) under FRET conditions (450 nm excitation, blue) and upon direct excitation of Cy5 dyes (625 nm, red). Inset: scaled version of the direct excitation spectrum. (B) Cy5 fluorescence intensity as a function of irradiation time upon FRET (blue) and direct (red) excitation. (C) Antenna effect as a function of irradiation time.

Figure 8

(A) Evolution of CPN fluorescence signal in 15 wt % doped CPNs during irradiation (shaded area) as a function of irradiation intensity. (B) Derivatives of the trajectories in (A) with inset depicting lowest-intensity data. (C) Evolution of Cy5 fluorescence signal during irradiation at same irradiation intensities as (A).

Figure 9

(A) Evolution of CPN fluorescence signal in doped CPNs during irradiation (shaded area, 0.87 mW/cm²) as a function of dye loading (wt %). (B) Dynamic range of CPN fluorescence intensities accessed during the experiment in (A). (C) Ratio of fluorescence intensities as a function of irradiation time and dye loading. (D) Fluorescence spectra upon direct excitation of Cy5 dyes. (E) Evolution of Cy5 fluorescence in doped CPNs during irradiation (0.87 mW/cm²) after the first and fourth cycles of dye addition. Dye amounts equivalent to 2.5 wt % were added to the sample for each dope-irradiate cycle. Red dots depict the point at which half the signal growth is complete.