Peptide-based semiconducting polymer nanoparticles for osteosarcoma-targeted NIR-II fluorescence/NIR-I photoacoustic dual-model imaging and photothermal/photodynamic therapies

Abstract

Background: The overall survival rate of osteosarcoma (OS) patients has not been improved for 30 years, and the diagnosis and treatment of OS is still a critical issue. To improve OS treatment and prognosis, novel kinds of theranostic modalities are required. Molecular optical imaging and phototherapy, including photothermal therapy (PTT) and photodynamic therapy (PDT), are promising strategies for cancer theranostics that exhibit high imaging sensitivity as well as favorable therapeutic efficacy with minimal side effect. In this study, semiconducting polymer nanoparticles (SPN-PT) for OS-targeted PTT/PDT are designed and prepared, using a semiconducting polymer (PCPDTBT), providing fluorescent emission in the second near-infrared window (NIR-II, 1000 - 1700 nm) and photoacoustic (PA) signal in the first near-infrared window (NIR-I, 650 - 900 nm), served as the photosensitizer, and a polyethylene glycolylated (PEGylated) peptide PT, providing targeting ability to OS.

Results: The results showed that SPN-PT nanoparticles significantly accelerated OS-specific cellular uptake and enhanced therapeutic efficiency of PTT and PDT effects in OS cell lines and xenograft mouse models. SPN-PT carried out significant anti-tumor activities against OS both in vitro and in vivo.

Conclusions: Peptide-based semiconducting polymer nanoparticles permit efficient NIR-II fluorescence/NIR-I PA dual-modal imaging and targeted PTT/PDT for OS.

Keywords: Dual-modal imaging; Osteosarcoma-targeted; Photodynamic therapy; Photothermal therapy.

Scheme 1

Schematic illustration of the preparation process of SPN-PT and NIR-II fluorescence/NIR-I PA imaging and targeted PTT/PDT against osteosarcoma

Fig. 1

Characterization of SPN-PT photophysical properties. A Representative TEM image and DLS analysis of SPN-PT. Scale bar = 500 nm. B Stability of averaged diameters of SPN-PT in PBS, MEM and FBS during the storage for 30 days. C Absorption spectra (red) and corresponding fluorescence emission spectra (black) of SPN-PT. D The UV absorption standard curve of SPN-PT aqueous solution at 700 nm. E Representative NIR-II fluorescence images (upper) and NIR-II fluorescence intensities with the concentrations of SPN-PT under 808 nm excitation with a 980 nm filter. F PA imaging spectra (680 to 820 nm) of SPN-PT (100 µg mL⁻¹). G Representative PA images (upper) and the fitting curve of PA intensity of SPN-PT at 695 nm against concentrations (lower). H Normalized UV absorption stability of SPN-PT and Ce6 at 700 nm and 640 nm, respectively. The solutions were irradiated by a 635 nm laser at 0.75 W cm⁻². I Photostability monitored of SPN-PT in PBS, MEM, FBS under continuous 808 nm laser excitation for 30 min. (a.u. arbitrary unit). Data were shown as mean ± SD, n = 3

Fig. 2

Photothermal and photodynamic performance of SPN-PT. A Photothermal conversion behavior of different concentrations of SPN-PT under laser irradiation (635 nm, 0.45 W cm⁻²). B IR thermal images of SPN-PT at concentrations corresponding to A irradiated by laser (635 nm, 0.45 W cm⁻²). Temperature elevation curves (C) and IR thermal images (D) of SPN-PT (100 µg ml⁻¹) irradiated by different power densities of laser (635 nm, 0.05 W cm⁻² - 0.60 W cm⁻²). E Temperature change curves of SPN-PT (100 µg mL⁻¹) over 5 ON/OFF cycles employing 635 nm laser (0.5 W cm⁻²) followed by passive cooling every 5 min. F The photothermal heating and cooling curves of SPN-PT and PBS under the 635 nm laser irradiation (0.6 W cm⁻²). G Linear time data versus negative natural logarithm was obtained from the cooling period. Absorption spectra change of DPBF incubated with SPN-PT and irradiated by 635 nm laser at 0.5 W cm⁻² (H) and 0.75 W cm⁻² (I). J Absorption intensity changes of DPBF at 419 nm in the presence of SPN-PT (50 µg mL⁻¹) with the irradiated time of 635 nm laser (0.5 W cm⁻², red; 0.75 W cm⁻², black)

Fig. 3

Cellular uptake of FITC-PT and FITC-SP. A Representative CLSM images of 143B cells and of 4T1 cells incubated with FITC-PT (20 µg mL⁻¹) for different time. Scale bar = 20 μm. Representative images and quantitative statistics of NIR-II fluorescence intensities of 143B or 4T1 cells incubated with different concentrations of SPN-PT (from left to right: 10 µg mL⁻¹, 20 µg mL⁻¹, 40 µg mL⁻¹) for 12 h (B) and 24 h (C). Flow cytometry analysis of 143B cells incubated with FITC-PT (20 µg mL⁻¹) (D) and FITC-SP (20 µg mL⁻¹) (E) for different time (from left to right: Con (control), 15 min, 30 min, 2 h, 4 h). Data were shown as mean ± SD, n = 3

Fig. 4

In vitro cytotoxic effects and PDT effects of SPN-PT. A Cell viability of 143B, MG63 and 4T1 cells determined by MTT assays after SPN-PT incubation (blue) or SPN-PT+laser treatments (635 nm, 0.75 W cm⁻², 5 min per well) (red). B Flow cytometry of 143B cells co-stained with Annexin V-FITC and PI after different treatments, from left to right: PBS, laser, SPN-PT (16 µg mL⁻¹), SPN-PT and laser irradiation (16 µg mL⁻¹) (635 nm, 0.5 W cm⁻², 5 min). C Representative CLSM images of live and dead 143B cells received different treatments: PBS (first row), SPN-PT (16 µg mL⁻¹) (second row), SPN-PT+laser (16 µg mL⁻¹) (635 nm, 0.5 W cm⁻², 5 min) (third row). Necrotic cell nuclei could be stained by PI in red, while live cells were only stained by Calcein-AM in green. Scale bar = 50 μm. D CLSM images of ¹O₂ generated by in 143B cells irradiated by 635 nm laser (0.5 W cm⁻²), with (down) or without (up) incubation of SPN-PT for different time. BF: bright field. Scale bar = 20 μm. Data were shown as mean ± SD, n ≥ 3

Fig. 5

In vivo imaging using SPN-PT. A NIR-II fluorescence images of 143B xenograft mice at different time points post injection of SPN-PT (upper, 100 µg mL⁻¹, 100 µL, i.v.) or SPN-SP (lower, 100 µg mL⁻¹, 100 µL, i.v.). The fluorescence images were acquired under the excitation of 808 nm with a 980 nm filter. Scale bar = 1 cm. B Monitoring of NIR-II fluorescence intensities collected within the tumor region (red circle in A) of 143B tumor bearing mice. Data were shown as mean ± SD, n = 3. (p < 0.05, *; p < 0.01, **; p < 0.001, ***; p < 0.0001, ****). C Two-dimensional (upper) and three-dimensional (lower) PA imaging of 143B tumors in living mice injected with SPN-PT (100 µg mL⁻¹, 100 µL, i.v.) at 1 h, 4 h, 8 h, 24 h. The PA images were acquired at 695 nm. D Average PA intensities within the whole tumor area (white circle in C). (i.v. intravenous injection). Data were shown as mean ± SD, n = 3

Fig. 6

SPN-PT-based therapeutic effects. A IR thermal images of 143B xenograft mice treated with PBS + 635 nm laser (0.75 W cm⁻²) (upper row), SPN-SP (100 µg mL⁻¹, 100 µL) + 635 nm laser (0.75 W cm⁻²) (second row), and SPN-PT (100 µg mL⁻¹, 100 µL) + 635 nm laser (0.75 W cm⁻²) (lower row) for 6 min. B Corresponding temperature changes in the region of tumors from (A). C Representative morphology images of 143B tumor bearing mice received different treatments on 0 d (upper row) and 15 d (lower row). Scale bar = 1 cm. Monitoring of tumor volumes (D) and body weight of mice F after application of different treatments within the 15 days’ therapeutic procedure. E Representative photograph of tumors excised from mice on 15 d post treatments. G H&E results of tumors obtained from four groups on 24 h post treatments. Scale bar = 200 μm. Data were shown as mean ± SD, n ≥ 3. (p < 0.05, *; p < 0.01, **; p < 0.001, ***; p < 0.0001, ****)

Fig. 7

Biocompatibility evaluation of SPN-PT-based therapies. H&E staining of major organs (heart, liver, spleen, lung, kidney) excised from mice at the endpoint of the experimental therapies. Scale bar = 200 μm