Highly fluorescent semiconducting polymer dots for biology and medicine

Abstract

In recent years, semiconducting polymer nanoparticles have attracted considerable attention because of their outstanding characteristics as fluorescent probes. These nanoparticles, which primarily consist of π-conjugated polymers and are called polymer dots (Pdots) when they exhibit small particle size and high brightness, have demonstrated utility in a wide range of applications such as fluorescence imaging and biosensing. In this review, we summarize recent findings of the photophysical properties of Pdots which speak to the merits of these entities as fluorescent labels. This review also highlights the surface functionalization and biomolecular conjugation of Pdots, and their applications in cellular labeling, in vivo imaging, single-particle tracking, biosensing, and drug delivery. We discuss the relationship between the physical properties and performance, and evaluate the merits and limitations of the Pdot probes for certain imaging tasks and fluorescence assays. We also tackle the current challenges of Pdots and share our perspective on the future directions of the field.

Figure 1

Chemical structures of highly fluorescent semiconducting polymers.

Figure 2

Particle sizes of semiconducting polymer nanoparticles prepared by different methods. A) TEM image of nanoparticles of a ladder-type poly(p-phenylene) polymer prepared by the miniemulsion method. Reproduced from Ref. [36] with permission. B) TEM image of Pdots from a PFBT polymer prepared by the reprecipitation method. Scale bars in (A) and (B): 100 nm. Reproduced from Ref. [57] with permission.

Figure 3

Fluorescence spectra of Pdots. A) A photograph of aqueous Pdot suspensions under UV light illumination. Absorption (B) and emission (C) spectra. The spectra of PFO, PFPV, and PFBT Pdots were reproduced from Ref. [51] with permission. The spectra of CN-PPV Pdots were reproduced from Ref. [63] with permission. The spectra of polymer blend Pdots (PFBT+PF-DBT5) were reproduced from Ref. [58] with permission.

Figure 4

Two-photon excited fluorescence from Pdots. A) Photograph of aqueous Pdot dispersions excited with an λ= 800 nm mode-locked Ti:sapphire laser. B) Single-particle imaging of PFPV dots with two-photon excitation. C) Log-log plot of two-photon fluorescence intensities versus the excitation power. D) Semi-log plot of two-photon action cross sections (σ_2pϕ) versus the excitation wavelength. Reproduced from Ref. [48] with permission.

Figure 5

Comparison of single-particle fluorescence brightness. A) Single-particle fluorescence images of PFBT Pdots and particles of Qdot 565 obtained under identical λ = 488 nm excitation and detection conditions. Scale bar: 5 μm. The right panel shows the intensity distributions obtained from single-particle fluorescence data. Reproducedfrom Ref. [56] with permission. B) Single-particle fluorescence images of PBdot and Qdot 655. Under identical λ= 488 nm excitation, a neutral density filter with an optical density of 1 was used to obtain fluorescence images of PBdots but no attenuation was used for Qdot 655. Scale bar: 4 μm. The right panel shows the intensity distributions from single-particle fluorescence data. Reproduced from Ref. [58] with permission.

Figure 6

A) Fluorescence lifetimes of Pdots measured by the TCSPC technique. B) Fluorescence saturation of single PFBT Pdots with increasing excitation intensity. C) Photobleaching trajectories of single PFBT Pdots. D) Histogram of the photon numbers of individual PFBT Pdots of about a 10 nm diameter. Reproduced from Ref. [51] with permission.

Figure 7

Chemical stability of Pdots in the presence of biologically relevant ions, and ROS in aqueous solutions. Qdot 655 nanocrystals were a reference. The concentration for each metal ion was 500 μM. For the ROS stability test, the H₂O₂ and chlorine concentration was 0.1 wt% and 0.004 wt%, respectively.

Figure 8

Cellular toxicity studies of Pdots. A) Monitoring of the inflammatory markers in cells treated with Pdots. J774A.1 cells were incubated with 2 nM (4 ppm) PFBT Pdots for 4 h. RNA was extracted from each sample, the total RNA was analyzed for the expression of TNF-α, IL-1β, and actin by RT-PCR, and the amplified product was separated by agarose electrophoresis. Three independent observations are represented: C: vehicle control; NP: 2 nM (4 ppm) PFBT Pdots; and In: interferon-γ (60 ng mL⁻¹) + LPS (100 ng mL⁻¹) as a positive control. Reproduced from Ref. [68] with permission. B) Metabolic viability of NIH/3T3 fibroblast cells after incubation with large semiconducting polymer particles at different concentrations for 12, 24, and 48 h. Reproduced from Ref. [94] with permission.

Figure 9

Functionalized Pdots for biomolecular conjugation. A) Surface functionalization of PFBT Pdots with the amphiphilic polymer PS-PEG-COOH. B) TEM image of functionalized PFBT Pdots. C) Hydrodynamic diameter of functionalized PFBT Pdots measured by DLS. Reproduced from Ref. [56] with permission.

Figure 10

Clickable Pdots for bioorthogonal labeling. A) Pdot functionalization and subsequent bioorthogonal labeling through click chemistry. B) Gel electrophoresis of Pdots with different surface functional groups. C) A fluorescent assay using an alkyne-Alexa 594 dye to verify the presence of azide groups on the Pdot surface. Reproduced from Ref. [57] with permission.

Figure 11

Direct functionalization of Pdots with different side-chain carboxy groups. Bioconjugation was performed on the PFBT-C2 Pdots and the resulting Pdot-bioconjugates were specific for cellular labeling. Reproduced from Ref. [65] with permission.

Figure 12

Specific cellular targeting with Pdot-bioconjugates. A) Fluorescence imaging of the cell-surface marker EpCAM on MCF-7 cells incubated sequentially with the anti-EpCAM primary antibody and Pdot-IgG conjugates. Scale bar: 10 μm. B) Fluorescence imaging of the control sample in which the cells were incubated with Pdot-IgG alone (no primary antibody). C) Fluorescence intensity distributions for Pdot-streptavidin-labeled MCF-7 cells. D) Fluorescence intensity distributions for Qdot 565-streptavidin-labeled MCF-7 cells obtained under identical experimental conditions as those used in C). Reproduced from Ref. [56] with permission.

Figure 13

Specific subcellular imaging of Pdot-bioconjugates. A) Fluorescence imaging of microtubule structures in HeLa cells incubated sequentially with biotinylated anti-α-tubulin antibody and CN-PPV-streptavidin probes. Scale bar: 10 μm. B) Fluorescence imaging of the control sample in which the cells were incubated with Pdot-streptavidin alone (no primary antibody). Reproduced from Ref. [63] with permission.

Figure 14

Fluorescence imaging of newly synthesized proteins in the AHA-treated MCF-7 cells tagged with Pdot-alkyne probes. A) Positive Pdot labeling in the presence of copper(I). B) Negative control for Pdot labeling carried out under identical conditions as in A) but in the absence of the reducing agent, sodium ascorbate, which generates copper(I) from copper(II). The left four panels show fluorescence images; green fluorescence was from Pdots and blue fluorescence was from the nuclear stain Hoechst 34580. The right four panels show Nomarski (DIC) and combined DIC and fluorescence images. Scale bar: 20 μm. Reproduced from Ref. [57] with permission.

Figure 15

Semiconducting polymer nanoparticles for in vivo fluorescence imaging. A) Schematic diagram depicting colloidal synthesis of nanoparticles with a cyanovinylene backbone by a tetrabutylammonium hydroxide (TBAH) catalyzed Knoevenagel condensation in the hydrophobic core of solvent-free aqueous micelles. “C₈” stands for n-octyl chains. B) True-color photographs of water-dispersed polymer nanoparticles (left) and a nanoparticle-injected live mouse (right) under room light (top) and UV excitation at λ = 365 nm for fluorescence (bottom). Reproduced from Ref. [72] with permission.

Figure 16

Pdot-chlorotoxin bioconjugates for in vivo tumor targeting. A) Fluorescence imaging of healthy brains in wild-type mice (left) and medulloblastoma tumors in ND2:SmoA1 mice (right). Each mouse was injected through the tail vein with 50 μL of a1 μM solution of either the nontargeting PBdot-PEG (top), or targeting PBdot-CTX (middle). As a control, some mice did not receive an injection (bottom). B) Tumor-targeting efficiency by quantifying fluorescence signals in ND2:SmoA1 versus wild-type mice and cerebellum versus frontal lobe. C) Histological examination of the mouse brains in A). The dark purple regions in the H&E-stained cerebellum of ND2:SmoA1 mice confirmed the presence of tumor. Reproduced from Ref. [58] with permission.

Figure 17

Single-particle fluorescence tracking with highly fluorescent Pdots. A) Bright-field transmission image of a fixed cell. The color marks indicate the locations of the particles. Blue corresponds to a particle bound to the membrane, green corresponds to a particle outside the cell, and red corresponds to the cell interior. B) The trajectories for the three particles obtained from fluorescence tracking. C) Mean-square displacement (MSD) of the tracked PFBT Pdots inside a cell (red circles) and outside a cell (green circles), corresponding to diffusion constants of 3.3 × 10⁻³ μm²s⁻¹ and 3.6 × 10⁻³ μm²s⁻¹, respectively. D) Mean-square displacement of the Pdot adhered to the membrane. Reproduced from Ref. [53] with permission.

Figure 18

Energy-transfer-based Pdot sensors. A) Oxygen-dependent emission spectra of the PtOEP-doped PDHF Pdots. The inset shows UV illuminated photograph of the aqueous Pdot solutions saturated with nitrogen, air, and oxygen, respectively. Reproduced from Ref. [52] with permission. B) Fluorescence spectra of fluorescein-conjugated PPE Pdots at different pH values ranging from 5 to 8. Reproduced from Ref. [60] with permission. C) Fluorescence spectra of RhB-conjugated PFBT Pdots at different temperatures from 10 °C to 70 °C. Reproduced from Ref. [59] with permission. D) Fluorescence spectra of RhB-SL-doped PFBT Pdots in aqueous suspension as mercury ion concentration was increased from 0 to 0.57 μM. Reproduced from Ref. [77] with permission.

Figure 19

Semiconducting polymer nanoparticles for protease sensing. A) Protease-triggered “turn on” sensing scheme. The cross-linking effectively strains the nanoparticles into a more tightly aggregated and quenched state. The protease cleaves the peptide cross-linkers and releases the strain, resulting in a “turn-on” fluorescence response. B) Fluorescence spectra of shell cross-linked nanoparticle suspensions before (red) and after (blue) incubation with trypsin. Reproduced from Ref. [74] with permission.

Figure 20

Conjugated polymer nanoparticles for gene delivery. A) Lipid-modified cationic PFPL nanoparticles for delivery of pCX-EGFP plasmids encoding the GFP. Fluorescence imaging of A549 cells after incubation with PFPL/pCX-EGFP nanoparticles showed both blue fluorescence from PFPL and green fluorescence from GFP, thus indicating the PFPL nanoparticles successfully delivered plasmids into cells. Reproduced from Ref. [92] with permission. B) Loosely aggregated PPE nanoparticles for delivery of siRNA. The top shows a schematic illustration of loosely aggregated PPE particles complexed with siRNA. The bottom shows Western blots of actin B (target) and tubulin (control). Significant reduction in the target protein was observed from the CPN/siACT transfection. Actin B expression decreased about 94% under the transfection conditions. Reproduced from Ref. [85] with permission.