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Phospholipid nanoshells, for example, liposomes, provide a versatile enabling platform for the development of nanometer-sized biosensors and molecular delivery systems. Utilization of phospholipid nanoshells is limited by the inherent instability in complex biological environments, where the phospholipid nanoshell may disassemble and degrade, thus releasing the contents and destroying sensor function. Polymer scaffold stabilization (PSS), wherein the phospholipid nanoshells are prepared by partitioning reactive monomers into the lipid bilayer lamella followed by radical polymerization, has emerged to increase phospholipid nanoshell stability. In this work, we investigated the effects of three different radical initiator conditions to fabricate stable PSS-phospholipid nanoshells yet retain the activity of encapsulated model fluorescent sensor proteins. To identify nondestructive initiation conditions, UV photoinitiation, neutral redox initiation, and thermal initiation were investigated as a function of PSS-phospholipid nanoshell stabilization and fluorescence emission intensity of enhanced green fluorescent protein (eGFP) and tandem dimer Tomato (td-Tomato). All three initiator approaches yielded comparably stable PSS-phospholipid nanoshells, although slight variations in PSS-phospholipid nanoshell size were observed, ranging from ca. 140 nm for unstabilized phospholipid nanoshells to 300-500 nm for PSS-phospholipid nanoshells. Fluorescence emission intensity of encapsulated eGFP was completely attenuated under thermal initiation (0% vs control), moderately attenuated under UV photoinitiation (40 ± 4% vs control), and unaffected by neutral redox initiation (97 ± 3% vs control). Fluorescence emission intensity of encapsulated td-Tomato was significantly attenuated under thermal initiation (13 ± 3% vs control), moderately attenuated UV photoinitiation (64 ± 5% vs control), and unaffected by neutral redox initiation (98% ± 4% vs control). Therefore, the neutral redox initiation method provides a significant advancement toward the preparation of protein-functionalized PSS-phospholipid nanoshells. These results should help to guide future applications and designs of biosensor platforms using PSS-phospholipid nanoshells and other polymer systems employing protein transducers.

Enhanced Fluorescent Protein Activity in Polymer Scaffold-Stabilized Phospholipid Nanoshells Using Neutral Redox Initiator Polymerization Conditions