Liquid–liquid phase separation for microencapsulation of native cytokine to enhance immune activation

Abstract

Therapeutic cytokines have achieved remarkable success in combination immunotherapy against malignant tumors. Nonetheless, their short half-life in the bloodstream leads to poor compliance and hinders their clinical effectiveness. While some pegylated cytokines have been developed to extend their circulation half-life, structural modifications often alter cytokines’ receptor affinities, reduce their activity, and even pose the risk of reversing their clinical effects. Therefore, there is an urgent need to develop new long-acting cytokines with stable blood concentration profiles and high activity. Inspired by the biomimetic partition process of liquid–liquid phase separation (LLPS) in living cells, we designed a method for microencapsulating cytokines into polymer microparticles driven by LLPS, enabling sustained delivery of native cytokines with high activity. Initially, we created a PEG/dextran aqueous biphasic system by mixing porous microparticles loaded with dextran-70 kDa and a PEG-20 kDa solution. Next, we introduced GM-CSF as a model cytokine into the biphasic system, allowing it to be distributed into the dextran-rich phase under the driving force of LLPS. We then sealed the porous microparticles to complete the microencapsulation of GM-CSF, resulting in GM-CSF/LLPS-MP. GM-CSF/LLPS-MP demonstrated a consistent release of native GM-CSF over a two-week period, promoting dendritic cell differentiation and function. Moreover, it enhanced the synergistic inhibitory effect of GM-CSF and PD-1 antibodies on melanoma tumors compared to GM-CSF solution. These findings offer proof of concept that liquid–liquid phase separation is an effective method for achieving the microencapsulation of native cytokines, thereby enhancing immune activation.