Sulfatase-responsive phase-separating peptide coacervates target stress granules to reverse sorafenib resistance in hepatocellular carcinoma

Acquired drug resistance in hepatocellular carcinoma (HCC) hinders the clinical therapeutic efficacy of various drugs, but efficient intervention strategies remain scarce. In this study, we reported a coacervate-fusion strategy for inhibiting membraneless organelle stress granules (SGs) via stimuli-induced peptide droplets to reverse sorafenib resistance (SFR) in HCC. SGs are coacervated from translation-stalled mRNAs and RNA-binding proteins, including Ras-GAP SH3 domain-binding proteins (G3BPs), and play a critical role in SFR. The peptide droplets YsF-L^SG are formed by liquid-liquid separation (LLPS) of the sulfatase-responsive peptides YsF and YsF-FGDF containing the G3BP ligand. Characterizations in solution reveal that, upon exposure to arylsulfatase A (ARSA), the peptides YsF and YsF-FGDF undergo LLPS and form agglomerate droplets YsF-L^SG. Investigations of HCC-SFR cells confirm that the YsF-L^SG mixtures are efficiently internalized via clathrin-mediated endocytosis, experience ARSA-responsive hydrolysis in lysosomes and lysosomal escape, and undergo in situ LLPS into droplets. Notably, in situ-formed coacervates YsF-L^SG recruit G3BP2 and target SGs with high tumor permeability. YsF-L^SG coacervates enhance sorafenib-triggered apoptosis by relieving SGs-mediated inhibition of p38-Caspase-3 signaling and thus reversing SFR of HCC cells. Further investigations in HCC cell-derived xenograft (CDX) models confirm that YsF-L^SG peptide coacervates significantly reverse SFR through SGs-targeting and apoptosis-restoring mechanisms. Critically, the combination of the YsF-L^SG peptide coacervates with sorafenib more effectively inhibits HCC-SFR growth and has a stronger antitumor effect accompanied by good biosafety. This study highlights the reversal of HCC-SFR via fusion between internal and external coacervates, offering a new approach for overcoming cancer drug resistance. STATEMENT OF SIGNIFICANCE: Design and application of peptide-based coacervates targeting SGs to overcome drug resistance have rarely been studied. Combining the advantages of in situ formulation of coacervate peptide droplets with SGs-targeting property, we developed YsF-L^SG peptide mixtures that target SGs through in situ sulfatase-responsive LLPS into droplets for reversing the SFR of HCC. YsF-L^SG peptide mixtures present high tumor-permeability and SGs-coalescence potential, undergo CME-involved uptake, experience ARSA sulfatase-responsivity and lysosomal escape, and exhibit potent tumor-killing advantage in HCC-SFR cells and CDX mice model. YsF-L^SG peptide mixtures reverse SFR of HCC through G3BP2-recruited, SGs-targeting and apoptosis-restored mechanisms. This provides a new strategy for developing enzyme-induced LLPS peptide coacervates with drug resistance-reversal capacity.