Surface Engineered Biomolecular Condensates for Targeted Cell Cytotoxicity.

Biomolecular condensates have attracted attention in membraneless cellular organization, bioreactor design, drug delivery, cellular regulation, and tissue engineering. However, the openness of their interfaces poses challenges for precise enzymatic control. Here, a two-step interfacial engineering strategy is developed to construct a decanoic acid (DA) membrane on decalysine/polyinosinic acid biomolecular condensates. This membrane reduces interfacial mobility. It also enhances enrichment of hydrophobic small molecules (e.g., Nile Red). Crucially, it imposes molecular-weight-dependent spatial control over biomacromolecules: species ≤60 kDa (e.g., single-stranded DNA (ssDNA), Horseradish Peroxidase (HRP), lipase) enrich within the microdroplet interior, while high-molecular-weight alkaline phosphatase (ALP) localizes at the interface. This spatial regulation significantly modulates enzymatic kinetics, boosting catalytic activity for both lipase and ALP within DA-coated condensates. Specifically, interfacial ALP accelerates dephosphorylation of N-(9-fluorenylmethoxycarbonyl)-L-tyrosine-(O)-phosphate (Fmoc-TyrP) and subsequent nanofiber growth, altering the condensate's internal physical environment and triggering release of enriched biomacromolecules like ssDNA. In cell co-culture, DA-coated condensates efficiently deliver ALP on HeLa cell membranes; subsequent Fmoc-TyrP addition induced apoptosis, reducing cell viability to 5%, compared to 50% with uncoated condensates. This work establishes a foundation for precision biocatalysis and targeted therapeutic platforms using engineered condensates, enabling functional customization of synthetic organelles.