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Objective: Prepare a multifunctional ultrasound molecular probe, hyaluronic acid-mediated cell-penetrating peptide-modified 10-hydroxycamptothecin-loaded phase-transformation lipid nanoparticles (HA/CPPs-10-HCPT-NPs), and to combine HA/CPPs-10-HCPT-NPs with low-intensity focused ultrasound (LIFU) for precision theranostics against hepatocellular carcinoma (HCC). Methods: HA/CPPs-10-HCPT-NPs were prepared using thin-film dispersion, ultrasound emulsification, and electrostatic effects. HA/CPPs-10-HCPT-NPs were characterized for particle size, zeta potential, encapsulation efficiency and drug-loading efficiency. In vitro , HA/CPPs-10-HCPT-NPs were tested for acoustic droplet vaporization (ADV) at different time points/acoustic intensities; the ability of HA/CPPs-10-HCPT-NPs to target SMMC-7721 cells was detected by confocal laser scanning microscopy (CLSM); the penetrating ability of CG-TAT-GC-modified NPs was verified by CLSM in a 3D multicellular tumor spheroid (MCTS) model; the effect of HA/CPPs-10-HCPT-NPs combined with LIFU on killing SMMC-7721 cells was measured by CCK-8 and flow cytometry. In vivo , the tumor-target efficiency of HA/CPPs-10-HCPT-NPs was evaluated by a small-animal fluorescence imaging system and CLSM; the enhanced ultrasound imaging efficiency of HA/CPPs-10-HCPT-NPs combined with LIFU was measured by an ultrasound imaging analyzer; the therapeutic effect of HA/CPPs-10-HCPT-NPs combined with LIFU was evaluated by tumor volume, tumor inhibition rate, and staining (hematoxylin and eosin (H &amp; E), proliferating cell nuclear antigen (PCNA) and TUNEL). Results: Mean particle size and mean zeta potential of HA/CPPs-10-HCPT-NPs were 284.2±13.3 nm and - 16.55±1.50 mV, respectively. HA/CPPs-10-HCPT-NPs could bind to SMMC-7721 cells more readily than CPPs-10-HCPT-NPs. Penetration depth into 3D MCTS of HA/CPPs-10-HCPT-NPs was 2.76-fold larger than that of NPs without CG-TAT-GC. HA/CPPs-10-HCPT-NPs could enhance ultrasound imaging by undergoing ADV triggered by LIFU. HA/CPPs-10-HCPT-NPs+LIFU group demonstrated significantly higher efficiency of anti-proliferation and apoptosis percentage than all other groups. In mouse liver tumor xenografts, HA/CPPs-10-HCPT-NPs could target tumor sites and enhance ultrasound imaging under LIFU. HA/CPPs-10-HCPT-NPs+LIFU group had a significantly smaller tumor volume, lower proliferative index (PI), and higher tumor inhibition and apoptotic index (AI) than all other groups. Conclusions: Combined application of HA/CPPs-10-HCPT-NPs and LIFU should be a valuable and promising strategy for precise HCC theranostics.

theranostics

Cell-penetrating Peptide-modified Targeted Drug-loaded Phase-transformation Lipid Nanoparticles Combined with Low-intensity Focused Ultrasound for Precision Theranostics against Hepatocellular Carcinoma