Three birds with one stone: co-encapsulation of diclofenac and DL-menthol for realizing enhanced energy deposition, glycolysis inhibition and anti-inflammation in HIFU surgery

Abstract

Despite attracting increasing attention in clinic, non-invasive high-intensity focused ultrasound (HIFU) surgery still commonly suffers from tumor recurrence and even matastasis due to the generation of thermo-resistance in non-apoptotic tumor cells and adverse therapy-induced inflammation with enhanced secretion of growth factors in irradiated region. In this work, inspired by the intrinsic property that the expression of thermo-resistant heat shock proteins (HSPs) is highly dependent with adenosine triphosphate (ATP), dual-functionalized diclofenac (DC) with anti-inflammation and glycolysis-inhibition abilities was successfully co-encapsulated with phase-change dl-menthol (DLM) in poly(lactic-co-glycolic acid) nanoparticles (DC/DLM@PLGA NPs) to realize improved HIFU surgery without causing adverse inflammation. Both in vitro and in vivo studies demonstrated the great potential of DC/DLM@PLGA NPs for serving as an efficient synergistic agent for HIFU surgery, which can not only amplify HIFU ablation efficacy through DLM vaporization-induced energy deposition but also simultaneously sensitize tumor cells to hyperthermia by glycolysis inhibition as well as diminished inflammation. Thus, our study provides an efficient strategy for simultaneously improving the curative efficiency and diminishing the harmful inflammatory responses of clinical HIFU surgery.

Keywords: Anti-inflammation; Diclofenac; Glycolysis inhibition; High-intensity focused ultrasound; Phase-change.

Scheme 1

Schematic illustration of sensitizing tumor cells to HIFU by tumor glycolysis inhibition via DC/DLM@PLGA NPs. a The synthesis process of PLGA NPs encapsulating DLM and DC; b principles of enhancing HIFU-mediated anti-tumor efficacy and decreased inflammation

Fig. 1

a SEM and b TEM images of DC/DLM@PLGA NPs. c hydrodynamic diameter distribution (inset: Tyndall effect of DC/DLM@PLGA NPs) and d zeta potential. e Microscopy images of DC/DLM@PLGA NPs under heating for 60 s. f TG curve of DC/DLM@PLGA NPs and g UV-vis spectra. h Stability of DC/DLM@PLGA NPs in various media. i Temperature elevation curves of different NPs under HIFU irradiation (power: 25 W; duty cycle: 50%; 3 s on and 3 s off). (Inset: experimental setup for monitoring temperature change by an infrared camera)

Fig. 2

a Schematic illustration of DC release from DC/DLM@PLGA NPs under HIFU irradiation. b DC release profiles under different temperatures and c DC release performances from PLGA NPs triggered by HIFU irradiation. d TEM image of typical DC/DLM@PLGA NPs after HIFU irradiation. e Sustained ultrasonic contrast images of DC/DLM@PLGA NPs (10 mg/mL, 3 mL) in a 60 °C water bath

Fig. 3

a Hemolysis test of DC/DLM@PLGA NPs at different concentrations. b MTT and c CCK-8 assays. d Schematic diagram of experimental setup for 4T1 cells exposed to 50% duty cycle HIFU at 25 W. e Cytotoxicity of different DC/DLM@PLGA NPs with gradient concentrations. f Intracellular glucose contents of 4T1 cells and HUVECs after co-incubation with DC/DLM@PLGA NPs for 24 or 48 h. g Live/Dead fluorescent staining of 4T1 cells in PBS, DC/DLM@PLGA NPs, and DC/DLM@PLGA NPs + HIFU groups (60 and 120 s), Scale bar: 100 μm. h Intracellular ATP content measurement and i analysis of HSPs expression after treatment with DC/DLM@PLGA NPs, untreated cell served as a control group. j Schematic representation of the process of DC-induced reduction in protein synthesis

Fig. 4

a Schematic diagram of experimental setup for in vitro pig liver ablation. b Digital photographs of the ablation areas of pig livers after injection of 200 µL solution of PBS, PLGA, DLM@PLGA, or DC/DLM@PLGA NPs and irradiation with HIFU at 25 W and 50% duty cycle for 2 min. c Corresponding calculation of the ablation volumes. d H&E histological staining of pork liver. Scale bar: 50 μm

Fig. 5

a Schematic illustration for experimental design. b Changes in tumor volumes in different groups of tumor-bearing mice after treatment (*p < 0.05, **p < 0.01). c Typical images to tumor tissues. d Average tumor weight obtained on the 14th day (*p < 0.05, **p < 0.01); (e) Typical H&E, TUNEL and Ki67 staining of tumor slices. Scale bar: 50 μm

Fig. 6

Detection of serum inflammatory cytokines, including a TNF-α, b IL-6 and c IL-1β in BALB/c mice after different treatments (*p < 0.05, **p < 0.01). d Immunohistochemical staining of typical inflammatory cytokines. Scale bar: 50 μm