A Tumor-Selective Self-Assembling Network of Poly(amino acid) Induces Cascading Bystander Cytotoxicity through Microvesicle Fission Amplification

Signal transduction is essential for tumor progression and therapeutic response. Extracellular vesicles (EVs) have emerged as critical mediators of intercellular communication and therapeutic delivery. However, current EV-based strategies that rely on <i>ex vivo</i> manipulation encounter significant limitations, including nonspecific biodistribution, limited tumor accumulation, and diminished biological activity, underscoring the need for the localized production of therapeutic EVs within tumors. To address this, a rationally designed poly(_D-amino acid) copolymer, methoxy poly(ethylene glycol)-<i>block</i>-poly(_D-lysine₁₀)-<i>block</i>-poly(<i>O</i>-phospho-_D-tyrosine₅-<i>co</i>-_D-alanine₂₅) (EG₄₅-_D-K-_D-pYA), is developed, which undergoes alkaline phosphatase (ALP)-catalyzed self-assembly to form a tumor-localized, positively charged network. This assembly triggers the formation of localized microvesicles (MVs) and initiates a self-sustaining fission cascade, amplifying the cytotoxic signaling. Upon ALP catalysis, 88.3% of the phosphate group in EG₄₅-_D-K-_D-pYA is hydrolyzed within 24 h, shifting zeta potential from -4.95 to +25.0 mV and increasing β-sheet content from 4.0% to 46.0%, promoting a morphological transition from nanoparticle to network. EG₄₅-_D-K-_D-pYA disrupts tumor cell membranes, mitochondria, and nuclei, inducing potent tumor-selective cytotoxicity (IC₅₀ = 0.58 μM at 24 h) and <i>in situ</i> generation of positively charged MVs (ζ = +32.9 mV). These MVs, enriched with membrane fragments, poly(amino acid)s, and mitochondrial and nuclear components, further mediate tumor-selective cell death and sustain cytotoxic propagation across four MV generations (P₀-P₄), with cytotoxicity rates of 53.13%, 28.71%, 36.35%, 24.69%, and 7.05%, respectively. EG₄₅-_D-K-_D-pYA achieves 90.1% tumor growth inhibition and markedly suppresses metastases by forming network <i>in vivo</i>. This study presents a tumor-selective therapeutic approach that integrates enzyme-triggered self-assembly with MV-driven fission amplification to induce robust bystander cytotoxicity and sustained tumor suppression.