Carrier-Free Nanoassembly Suppresses Phase Separation via Ribosome-Inspired Crowding Control for Enhanced Chemo-Immunotherapy.

Chemotherapy can induce tumor cell death and stimulate immunity, yet its efficacy is often compromised by the immunosuppressive tumor microenvironment dominated by M2-like tumor-associated macrophages (TAMs). Redirecting TAMs toward an M1-like phenotype through mammalian target of rapamycin (mTOR)-mediated metabolic modulation has therefore emerged as a promising therapeutic strategy. To this end, we developed a carrier-free nanoassembly (SOP) composed of the mTOR inhibitor OSI-027 and the chemotherapeutic agent SN-38. SOP harnesses ribosome-inspired crowding control to suppress liquid-liquid phase separation (LLPS), thereby facilitating M2 reprogramming and enhancing antitumor efficacy. Following intravenous administration, SOP accumulated in tumor tissues with 2-fold greater efficiency than free drugs. Once internalized, SOP released SN-38 to induce tumor cell death and immunogenic responses, while OSI-027 simultaneously inhibited mTORC1/2 signaling and reduced ribosome abundance-key crowding agents driving LLPS. The resulting disruption of LLPS promoted TAM repolarization toward M1, establishing a synergistic interplay between OSI-027 and SN-38. This dual action translated into robust therapeutic outcomes across diverse models, including cell-derived xenografts, patient-derived xenografts, and KPC (LSL-KrasG12D/+; LSL-Trp53R172H/+; Pdx-1-Cre) mice. Moreover, SOP suppressed resistance-related gene expression, overcoming chemotherapy resistance. In summary, this carrier-free nanoassembly not only reprograms the immunosuppressive tumor microenvironment but also introduces LLPS suppression as a mechanism for TAM regulation, positioning SOP as a potent platform for enhanced chemo-immunotherapy.