Good Steel Used in the Blade: Well-Tailored Type-I Photosensitizers with Aggregation-Induced Emission Characteristics for Precise Nuclear Targeting Photodynamic Therapy

Abstract

Photodynamic therapy (PDT) has long been recognized to be a promising approach for cancer treatment. However, the high oxygen dependency of conventional PDT dramatically impairs its overall therapeutic efficacy, especially in hypoxic solid tumors. Exploration of distinctive PDT strategy involving both high-performance less-oxygen-dependent photosensitizers (PSs) and prominent drug delivery system is an appealing yet significantly challenging task. Herein, a precise nuclear targeting PDT protocol based on type-I PSs with aggregation-induced emission (AIE) characteristics is fabricated for the first time. Of the two synthesized AIE PSs, TTFMN is demonstrated to exhibit superior AIE property and stronger type-I reactive oxygen species (ROS) generation efficiency owing to the introduction of tetraphenylethylene and smaller singlet-triplet energy gap, respectively. With the aid of a lysosomal acid-activated TAT-peptide-modified amphiphilic polymer poly(lactic acid)12k-poly(ethylene glycol)5k-succinic anhydride-modified TAT, the corresponding TTFMN-loaded nanoparticles accompanied with acid-triggered nuclear targeting peculiarity can quickly accumulate in the tumor site, effectively generate type-I ROS in the nuclear region and significantly suppress the tumor growth under white light irradiation with minimized systematic toxicity. This delicate "Good Steel Used in the Blade" tactic significantly maximizes the PDT efficacy and offers a conceptual while practical paradigm for optimized cancer treatment in further translational medicine.

Keywords: aggregation-induced emission; nuclear targeting; photodynamic therapy; type-I photosensitizer.

Figure 1

Illustration of molecular design principle of TFMN and TTFMN, photophysical and photochemical mechanisms of type‐I and type‐II processes, construction of acid‐activated TTFMN NPs, and their applications in precise photodynamic nuclear targeting cancer therapy.

Figure 2

Optical properties of TFMN and TTFMN. A) Normalized absorption spectra of TFMN and TTFMN in ACN solution. B) PL spectra of TTFMN (10 × 10⁻⁶ m) in ACN/water mixtures with different water fractions (f _w). C) The plots of the relative emission intensity (I/I ₀) of TFMN and TTFMN versus water fraction, I ₀ and I are the peak PL intensities of AIEgens (10 × 10⁻⁶ m) in pure ACN and ACN/water mixtures, respectively. Inset: enlarged I/I ₀ plots of TFMN. D) Normalized PL spectra of TFMN and TTFMN in the solid state. E) Quantum yields of TFMN and TTFMN measured in ACN, ACN/water mixtures (f _w = 90%), and solid state. F) Time‐resolved decay profiles of TFMN and TTFMN.

Figure 3

ROS generation of TFMN and TTFMN upon white light irradiation. Relative changes in PL intensity of A) DCFH (for overall ROS detection) and B) HPF (for ^•OH detection) in the presence of TFMN or TTFMN (2 × 10⁻⁶ m) upon white light irradiation (22.1 mW cm⁻²) for different times. C) ESR signals of DMPO for type‐I ROS characterization in the presence of TFMN or TTFMN (1 × 10⁻³ m) before and after white light irradiation (200 mW cm⁻²). Relative changes in PL intensity of D) DHR123 (for ^• O₂ ⁻ detection), E) SOSG (for ¹O₂ detection), and F) decomposition rates of ABDA (for ¹O₂ detection) in the presence of TFMN or TTFMN (2 × 10⁻⁶ m) upon white light irradiation (22.1 mW cm⁻²) for different times.

Figure 4

Design and characterization of TTFMN‐NPs. A) DLS profile and TEM image (inset) of TTFMN‐NPs at pH 7.4. B) Zeta potential of TTFMN‐NPs characterized by DLS in aqueous solution at pH = 7.4. C) Stability analysis for size variations of TTFMN‐NPs with a concentration of 100 µg mL⁻¹ as a function of storage time at room temperature in H₂O, PBS, or PBS + 10% FBS measured by DLS. D) Normalized absorption and emission spectra of TTFMN‐NPs in aqueous solutions.

Figure 5

Cellular internalization and intracellular localization of TTFMN‐NPs on 4T1 tumor cells. A) TTFMN‐positive cells and MFI for quantitative cellular uptake after incubation with TTFMN‐NNPs, TTFMN‐NPs, TTFMN‐NPs (pretreated at pH 5.0 for 24 h), and TTFMN‐PNPs for 3 h at a unified TTFMN dose of 10 µg mL⁻¹ measured by flow cytometry. B) Intracellular tracking of TTFMN‐NPs (2 µg mL⁻¹ TTFMN) on 4T1 cells after incubation for 1 and 6 h imaged by CLSM. C) Nuclear targeting delivery of TTFMN‐NPs (2 µg mL⁻¹ TTFMN) on 4T1 cells after incubation for 1, 6, and 12 h determined by CLSM.

Figure 6

Photodynamic tumoricidal effect of TTFMN‐NPs on 4T1 tumor cell. A) Intracellular ROS level of 4T1 cells after various treatments indicated by DCFH‐DA. Laser irradiation (488 nm, 2% power, 3 min) was conducted after cells were incubated with TTFMN‐NPs (50 µg mL⁻¹ TTFMN) for 24 h. B) Cell viability of 4T1 cells incubated with TTFMN‐NPs at various concentrations in the dark and after white light irradiation (50 mW cm⁻²) determined by MTT assay (mean ± SD, n = 6). C) Apoptosis analysis using flow cytometry toward 4T1 cells after different treatments. White light irradiation (50 mW cm⁻²) was conducted after cells were incubated with TTFMN‐NPs (50 µg mL⁻¹ TTFMN).

Figure 7

In vivo fluorescence‐imaging‐guided photodynamic therapeutic efficiency of TTFMN‐NPs on 4T1‐tumor‐bearing BALB/c nude mice through systemic administration. A) Fluorescence images of tumor‐bearing mice at different monitoring times after administration of TTFMN‐NPs. B) Time‐dependent tumor growth curves of tumor‐bearing mice with various treatments (n = 5, *p < 0.001). Inset: photos of the tumors harvested at day 15 after different treatments. C) Weights of the tumors harvested at day 15 after different treatments (n = 5, *p < 0.001). D) H&E, TUNEL, Ki67, and CD31 staining analysis of tumor tissues after various treatments. TUNEL, Ki67, and CD31‐positive cells were stained red, green, and green, respectively.