Molecular Design of Conjugated Small Molecule Nanoparticles for Synergistically Enhanced PTT/PDT

Abstract

Simultaneous photothermal therapy (PTT) and photodynamic therapy (PDT) is beneficial for enhanced cancer therapy due to the synergistic effect. Conventional materials developed for synergistic PTT/PDT are generally multicomponent agents that need complicated preparation procedures and be activated by multiple laser sources. The emerging monocomponent diketopyrrolopyrrole (DPP)-based conjugated small molecular agents enable dual PTT/PDT under a single laser irradiation, but suffer from low singlet oxygen quantum yield, which severely restricts the therapeutic efficacy. Herein, we report acceptor-oriented molecular design of a donor-acceptor-donor (D-A-D) conjugated small molecule (IID-ThTPA)-based phototheranostic agent, with isoindigo (IID) as selective acceptor and triphenylamine (TPA) as donor. The strong D-A strength and narrow singlet-triplet energy gap endow IID-ThTPA nanoparticles (IID-ThTPA NPs) high mass extinction coefficient (18.2 L g^-1 cm^-1), competitive photothermal conversion efficiency (35.4%), and a dramatically enhanced singlet oxygen quantum yield (84.0%) comparing with previously reported monocomponent PTT/PDT agents. Such a high PTT/PDT performance of IID-ThTPA NPs achieved superior tumor cooperative eradicating capability in vitro and in vivo.

Keywords: Conjugated small molecule nanoparticles; Isoindigo; Molecular design; Singlet–triplet energy gap; Synergistic PTT/PDT.

Scheme 1

Illustration of molecular design of IID-ThTPA, preparation of IID-ThTPA NPs, and biomedical application of IID-ThTPA NPs. a Acceptor-oriented molecular design of IID-ThTPA with narrow singlet–triplet energy gap. b IID-ThTPA NPs prepared by a nanoprecipitation method. c PAI-guided synergistically enhanced PTT/PDT under a single NIR laser irradiation enabled by IID-ThTPA NPs

Fig. 1

Basic properties of IID-ThTPA and IID-ThTPA NPs. a UV–Vis–NIR absorption spectra of IID-ThTPA in THF and IID-ThTPA NPs in water (20 μg mL⁻¹) (insets are the digital photographs of IID-ThTPA in THF and IID-ThTPA NPs in water). b UV–Vis–NIR absorption spectra of IID-ThTPA NPs with different concentrations. c Mass extinction coefficient of IID-ThTPA NPs at 671 nm. Normalized absorbance intensity at 671 nm divided by the characteristic length of the cell (A/L) at different concentrations (mass extinction coefficient is calculated by the Lambert–Beer law: A/L = εc (L = 1 cm)). d Fluorescence spectra of IID-ThTPA in THF and IID-ThTPA NPs in water (20 μg mL⁻¹) (excitation wavelength: 550 nm). e DLS profile of freshly prepared IID-ThTPA NPs (inset shows the TEM image of IID-ThTPA NPs). f DLS profile of IID-ThTPA NPs after one week’s storage at room temperature and the size change of IID-ThTPA NPs during one week’s storage at room temperature (inset is the autocorrelation functions of IID-ThTPA NPs at 0 and 7 days, respectively)

Fig. 2

Photothermal effect of IID-ThTPA NPs. a Photothermal heating curves of IID-ThTPA NPs with different concentrations (671 nm, 1.00 W cm⁻²). b Plot of the temperature changes of IID-ThTPA NPs versus the absorbance at different concentrations with a fitted curve derived from Lambert–Beer law. c Infrared thermographs of IID-ThTPA NPs with different concentrations under laser irradiation at various time points (671 nm, 1.00 W cm⁻²). d Photothermal heating curves of IID-ThTPA NPs (80 μg mL⁻¹) under 671-nm laser irradiation with different power densities. e Plot of the temperature changes of IID-ThTPA NPs versus the laser power densities with a linearly fitted line. f Infrared thermographs of IID-ThTPA NPs (80 μg mL⁻¹) under 671-nm laser irradiation with different power densities at various time points. g Photothermal heating curve to calculate the photothermal conversion efficiency. IID-ThTPA NPs (80 μg mL⁻¹) were irradiated by a 671-nm laser with a power density of 1.00 W cm⁻² to reach a temperature plateau; then, the laser was shut off and the temperature was allowed to cool to room temperature naturally. h Plot of linear time data from the cooling period of IID-ThTPA NPs versus negative natural logarithm of the driving force temperature (θ). i Photothermal heating curve of IID-ThTPA NPs (40 μg mL⁻¹) for five laser on/off cycles under irradiation (671 nm, 1.00 W cm⁻²) (insets are the infrared thermographs of IID-ThTPA NPs at different time points during the laser on/off cycles)

Fig. 3

Singlet oxygen-generating capability of IID-ThTPA NPs. a UV–Vis–NIR absorption spectra of IID-ThTPA NPs and a standard reference MB in water. Degradation of DPBF in the presence of b IID-ThTPA NPs and c MB under laser irradiation. Degradation kinetics of DPBF in the presence of IID-ThTPA NPs and MB under laser irradiation, where d A₀ and A_t are the characteristic absorbance of DPBF at 410 nm before and after laser irradiation, respectively; e A₀ and A_t are the absorbance at 580 nm (IID-ThTPA NPs) or 665 nm (MB) before and after laser irradiation, respectively. f DFT-calculated optimized geometries, HOMOs, LUMOs, S₁, and T₁ energy levels of IID-ThTPA and a previously reported conjugated small molecule DPP-TPA to depict the ultrahigh singlet oxygen quantum yield of IID-ThTPA NPs

Fig. 4

In vitro phototherapy of IID-ThTPA NPs. a CLSM images of 4T1 cells after incubating with IID-ThTPA-RITC NPs for different times (1, 2, 4, and 6 h). b Quantitative analysis of RITC fluorescence intensity in a. c Cell viability of 4T1 cells incubating with IID-ThTPA NPs (0, 5, 10, 20, 40, and 80 μg mL⁻¹) under different experimental conditions (dark, PTT, PDT, and PTT + PDT) (the results are presented as mean ± SD, n = 6). d Intracellular ROS level of 4T1 cells under different treatments (control, IID-ThTPA NPs, laser, and IID-ThTPA NPs + laser). e Quantitative analysis of DCF fluorescence intensity in d

Fig. 5

In vivo PAI-guided phototherapy enabled by IID-ThTPA NPs. a PA images of the tumor sites at 680 nm at different time points post-injection (0, 2, 4, 6, 12, and 24 h). b Infrared thermographs of the mice under laser irradiation (671 nm, 1.00 W cm⁻²) in laser and IID-ThTPA NPs + laser groups. c Tumor temperature elevation curves of the mice under laser irradiation (671 nm, 1.00 W cm⁻²) in laser and IID-ThTPA NPs + laser groups. d Tumor volume variation curves of the mice in control, IID-ThTPA NPs, laser, IID-ThTPA NPs + Vc + laser (PTT), and IID-ThTPA NPs + laser (PTT + PDT) groups during the treatment period (the results are presented as mean ± SD, **P < 0.01 by two-tailed unpaired Student’s t tests, n = 5). e Digital photographs of the tumors dissected from the mice in control, IID-ThTPA NPs, laser, IID-ThTPA NPs + Vc + laser (PTT), and IID-ThTPA NPs + laser (PTT + PDT) groups at the end of treatment. f Tumor weights of the mice in control, IID-ThTPA NPs, laser, IID-ThTPA NPs + Vc + laser (PTT), and IID-ThTPA NPs + laser (PTT + PDT) groups at the end of treatment (the results are presented as mean ± SD, **P < 0.01 by two-tailed unpaired Student’s t tests, n = 5). g H&E, Ki-67, and TUNEL staining of tumors of the mice after different treatments. h H&E staining of the major organs (heart, liver, spleen, lung, and kidney) of the mice after different treatments

Fig. 6

In vivo toxicity evaluation. a Hematological index (WBC, RBC, HGB, HCT, MCH, MCHC, MCV, and PLT) of the mice in control, 7 d post-injection, and 14 d post-injection groups. b Biochemical blood analysis (ALT, AST, BUN, CREA, and TBIL) of the mice in control, 7 d post-injection, and 14 d post-injection groups (the results are presented as mean ± SD, n = 3)