Rationally designed NIR-II excitable and endoplasmic reticulum-targeted molecular phototheranostics for imaging-guided enhanced photoimmunotherapy of triple-negative breast cancer

Abstract

Triple-negative breast cancer (TNBC) is a highly aggressive subtype of breast cancer characterized by an extremely poor prognosis. Photoimmunotherapy has emerged as a promising strategy for the treatment of TNBC. This approach works by selectively destroying tumor cells, releasing tumor-associated antigens, activating the immune system, and effectively inhibiting tumor proliferation and metastasis. However, the majority of current phototheranostic approaches are hindered by limited tissue penetration in the first near-infrared (NIR-I) and ultraviolet-visible (UV-Vis) regions. Additionally, due to the lack of specific subcellular targets, it may be difficult to effectively treat deep-seated lesions with ambiguous and extensive boundaries caused by TNBC metastases. Consequently, the development of effective, deep-penetrating, organelle-targeted phototheranostics is essential for enhancing treatment outcomes in TNBC. This work proposes a novel molecular design strategy of NIR-II phototheranostics to realize planar rigid conjugation and alkyl chain functionalization. The di-hexaalkyl chains in a vertical configuration on the donor (4H-cyclopenta[2,1-b:3,4-b'] dithiophene) and shielding units (fluorene) are introduced to construct a S-D-A-D-S type NIR-II phototheranostics (IR-FCD). The planar and rigid structure of IR-FCD exhibits a robust intramolecular charge transfer capability, a lower band gap, enhanced photon absorption properties, and significant steric hindrance from vertically arranged alkyl chains to minimize non-radiative energy loss. By incorporating N-(but-3-yn-1-yl)-4-methylbenzenesulfonamide at the terminus of an elongated alkyl chain, followed by self-assembly into DSPE-S-S-PEG2000, NIR-II excitable phototheranostics (IR-FCD-Ts NPs) with endoplasmic reticulum (ER) targeting capability were successfully synthesized for imaging-guided photoimmunotherapy of TNBC. The IR-FCD-Ts NPs demonstrate exceptional optical characteristics, with maximum absorption at 1068 nm (extending to 1300 nm) and emission at 1273 nm (extending to 1700 nm), along with a high molar absorption coefficient of 2.76*10⁴ L/mol·c at 1064 nm in aqueous solution. Under exposure to 1064 nm laser irradiation, IR-FCD-Ts NPs exhibit superior photothermal properties and have the potential for photodynamic therapy. By targeting ER, thereby inducing ER stress and significantly enhancing immunogenic cell death (ICD) in tumor cells, it triggers a strong antitumor immune response and inhibits the proliferation and metastasis of TNBC.

Keywords: Endoplasmic reticulum; Intermolecular π–π stacking interaction; NIR-II; Phototheranostics; Triple-negative breast cancer.

Scheme 1

NIR-II excitable molecular phototheranostics for NIR-II imaging-guided ER-targeted photoimmunotherapy for TNBC

Fig. 1

A Calculated HOMOs and LUMOs of IR-FTT and IR-FCD. B Optimized ground-state (S₀) and first singlet excited state (S₁) geometries of IR-FTT and IR-FCD. C Molecular dynamics simulations of IR-FTT and IR-FCD. D Interaction force of IR-FTT and IR-FCD. Diffusion coefficient of E IR-FTT and F IR-FCD

Fig. 2

A, B The absorption and C, D fluorescence emission spectra of molecular fluorophores IR-26, IR-FTT, IR-FCD, and IR-FCD-Ts in dichloromethane DCM and water solutions. E TEM images of IR-FCD-Ts NPs distribution analysis. F Hydrodynamic diameter size distribution of IR-FCD-Ts NPs. G Cumulative release of IR-FCD-Ts after incubating nanoparticles in GSH aqueous solutions of different concentrations for varying times. Error bars: mean ± SD (n = 3). H Heating curves of IR-FCD-Ts NPs at various concentrations upon 1064 nm laser irradiation. I Heating curve of IR-FCD-Ts NPs (30 µM) under 1064 nm laser irradiation of different power densities. J The photothermal conversion efficiency and K photothermal stability of IR-FCD-Ts NPs. L ROS production under 1064 nm laser irradiation with different times (I₀ and I represent the emission intensities of DCF at 525 nm before and after irradiation, respectively). Error bars: mean ± SD (n = 3)

Fig. 3

A CLSM images of 4T1 cells following incubation with IR-FCD-Ts/IR-FCD NPs and ER-Tracker. Scale bar: 20 µm. B Intensity profile of linear regions of interest between IR-FCD-Ts/IR-FCD NPs and ER-Tracker. C, D Cell viability after treatment with various concentrations of IR-FCD NPs and IR-FCD-Ts NPs without/with laser irradiation (1064 nm, 1.0 W/cm², 10 min). Error bars: mean ± SD (n = 3). E Calcein-AM/PI staining after various treatments. Scale bar:100 µm. F–J CLSM images of ROS, CHOP, Cleaved Caspase-3, CRT, and HMGB1 in 4T1 cells after different treatments. Scale bar:50 µm. K–O Flow cytometric analysis of ROS, CHOP, Cleaved Caspase-3, CRT, and HMGB1 in 4T1 cells following different treatments

Fig. 4

A Fluorescence images in real-time of 4T1 tumor-bearing mice following intravenous administration of IR-FCD-Ts NPs at various time intervals. B The fluorescence intensity of tumors was quantified at various time points following injection. C Schematic representation of in vivo antitumor therapy. D Photothermal images of 4T1 tumor-bearing mice after different treatments 24 h with 1064 nm laser irradiation (1.0 W/cm², 10 min). E Representative tumor photographs were collected from mice with different treatments (day 15). The relative tumor volumes in different treatment groups of the F primary and G distant tumor. H The schematic diagram of anti-lung metastasis treatment. I Representative images of the lung tissues after different treatments. J H&E-staining of lung tissues after different treatments. Scale bar: 500 µm. Error bars, mean ± SD (n = 3)

Fig. 5

A CHOP, Cleaved Caspase-3, CRT, HMGB1 staining of the primary tumor tissues after various treatments. The representative flow cytometric analyses of B DCs (CD80⁺ CD86⁺ CD11C⁺), C CTLs (CD3⁺ CD8⁺), and D Tregs (CD4⁺ CD25⁺ Foxp3⁺) in primary tumors from 4T1 tumor-bearing mice after different treatments. E–H TNF-α, IFN-γ, IL-6, and IL-10 levels of 4T1 tumor-bearing BALB/c mice after different treatments. Error bars: mean ± SD (n = 3)