A Highly Efficient One-for-All Nanodroplet for Ultrasound Imaging-Guided and Cavitation-Enhanced Photothermal Therapy

Abstract

Background: Photothermal therapy (PTT) has attracted considerable attention for cancer treatment as it is highly controllable and minimally invasive. Various multifunctional nanosystems have been fabricated in an "all-in-one" form to guide and enhance PTT by integrating imaging and therapeutic functions. However, the complex fabrication of nanosystems and their high cost limit its clinical translation.

Materials and methods: Herein, a high efficient "one-for-all" nanodroplet with a simple composition but owning multiple capabilities was developed to achieve ultrasound (US) imaging-guided and cavitation-enhanced PTT. Perfluoropentane (PFP) nanodroplet with a polypyrrole (PPy) shell (PFP@PPy nanodroplet) was synthesized via ultrasonic emulsification and in situ oxidative polymerization. After characterization of the morphology, its photothermal effect, phase transition performance, as well as its capabilities of enhancing US imaging and acoustic cavitation were examined. Moreover, the antitumor efficacy of the combined therapy with PTT and acoustic cavitation via the PFP@PPy nanodroplets was studied both in vitro and in vivo.

Results: The nanodroplets exhibited good stability, high biocompatibility, broad optical absorption over the visible and near-infrared (NIR) range, excellent photothermal conversion with an efficiency of 60.1% and activatable liquid-gas phase transition performance. Upon NIR laser and US irradiation, the phase transition of PFP cores into microbubbles significantly enhanced US imaging and acoustic cavitation both in vitro and in vivo. More importantly, the acoustic cavitation enhanced significantly the antitumor efficacy of PTT as compared to PTT alone thanks to the cavitation-mediated cell destruction, which demonstrated a substantial increase in cell detachment, 81.1% cell death in vitro and 99.5% tumor inhibition in vivo.

Conclusion: The PFP@PPy nanodroplet as a "one-for-all" theranostic agent achieved highly efficient US imaging-guided and cavitation-enhanced cancer therapy, and has considerable potential to provide cancer theranostics in the future.

Keywords: acoustic cavitation; one-for-all nanodroplet; photothermal therapy; theranostics; ultrasound imaging.

Scheme 1

Schematic illustration showing (A) the synthetic process of “one-for-all”-type theranostic PFP@PPy NDs, and (B) corresponding mechanisms of US imaging-guided and cavitation-enhanced cancer therapy that combined photothermal and cavitation effects via the PFP@PPy NDs.

Figure 1

Characterization of PFP@PPy nanodroplets. (A) TEM images. (B) The size distribution, determined by the DLS. (C) The absorption spectra of PFP nanodroplets, PPy nanoparticles and PFP@PPy nanodroplets. (D) Cytotoxicity of PFP@PPy nanodroplets at different volume concentrations with and without NIR laser irradiation. **p < 0.01.

Figure 2

Photothermal temperature curves of PFP@PPy nanodroplets over time (0-10 min) with (A) different volume concentrations (NIR laser: 1 W/cm²) and (B) different laser power densities (volume concentration of PFP: 0.001% v/v). (C) Photothermal conversion efficiency of PFP@PPy nanodroplets. (D) The liquid-gas phase transition of PFP@PPy nanodroplets (volume concentration of PFP: 0.002% v/v) was evaluated using microscopic (upper) and IR thermal images (lower) under laser irradiation (1 W/cm²) at different time points. The same concentration of PFP nanodroplets were used as the control group. Scale bar = 10 μm.

Figure 3

In vitro US imaging performance. (A) B-mode and contrast enhanced ultrasound (CEUS) imaging of PFP@PPy nanodroplets after NIR laser irradiation (1 W/cm²) or combined laser and US irradiation (1 MHz, 2.2 MPa, 50 μs and 10 Hz) for 1 min in gel phantoms. The same concentration of PFP nanodroplets were used as negative controls. The mean US intensity values in both (B) B-mode and (C) CEUS mode images. **p < 0.01. Scale bar = 1 cm.

Figure 4

Characterization of the acoustic cavitation of photothermally generated microbubbles under combined NIR laser (1 W/cm²) and US (1 MHz, 2.2 MPa, 50 μs and 10 Hz) irradiation. (A) The pictures of the SCL regions in two groups and (B) corresponding light intensities and luminous areas. Passive acoustic detection (PCD) of cavitation events, including (C) time-domain signals and (D) frequency spectrum analysis. **p < 0.01. Scale bar = 1 cm.

Figure 5

In vitro evaluation of therapeutic efficacy after various treatments by using cell-counting kit-8 (CCK-8) assay. The cultured cells were randomly divided into three groups, including the cells without nanodroplets, with PFP nanodroplets and PFP@PPy nanodroplets at the same volume concentration of PFP (0.002% v/v). **p < 0.01.

Figure 6

(A) The merged calcein-AM/PI staining fluorescent and bright-field microscopic images at different experimental conditions. (B) The quantitative analysis of integrated calcein and PI fluorescence in the cells. (C) The relative number of live/dead cells at different conditions. (D) The SCL representative image of the group I for characterizing the acoustic cavitation.

Figure 7

(A) In vivo US images of tumors before and after the combined laser and US irradiation with (I) PFP nanodroplets (control group) and (II) PFP@PPy nanodroplets (n = 3). White dashed circles indicate the region of tumors. (B) In vivo mean US intensity values of the two groups. **p < 0.01.

Figure 8

In vivo antitumor efficacy. (A) Time-dependent tumor growth curves after treatment (n = 4). (B) The photographs and (C) the mean tumor weight of the excised tumors from different groups. (D) Time-dependent body weight curves of nude mice after different treatments. (E) Representative images of H&E-stained tumor sections showing pathological changes. (Groups I) Saline, II: PFP@PPy nanodroplets, III: laser + US, IV: PFP@PPy nanodroplets + US, (V) PFP@PPy nanodroplets + laser, VI: PFP@PPy nanodroplets + laser + US).*p < 0.05,**p < 0.01.