Enzymatic Regioselection for the Synthesis and Biodegradation of Porphysome Nanovesicles

Abstract

We recently reported that porphyrin–phospholipid conjugates can self-assemble into “porphysome” nanovesicles composed of a dense porphyrin bilayer. Porphysomes exhibit structurally driven nanoscale optical properties and have intrinsic capabilities for multimodal imaging, drug delivery, and photothermal therapy. Previous studies were based on porphysomes formed from a mixture of two chemically similar phospholipid–porphyrin regioisomers. However, use of a mixture of regioisomers is far from ideal for robust nanoparticle characterization in vitro and in vivo. In general, difficulties in synthesizing, detecting, and distinguishing phospholipid regioisomers have prevented examination of their in vivo fate until now. To our knowledge, the results reported herein are the first to demonstrate in vivo enzymatic biodegradability for any intrinsically optically active nanoparticle, a feature that might be important when considering the use of new nanomaterials in human clinical trials. Chemically modified phospholipids have proven useful for diverse biotechnology applications. Phospholipids can be labeled at various positions on their head group or side chain. While head-group modification can readily be achieved using the primary amine group of phosphatidylethanolamine, side-chain modification is less straightforward but is appropriate for conjugating more hydrophobic ligands while maintaining an amphipathic phospholipid character. Recently, phospholipids modified with cholesterol, retinoic acid, and porphyrin side chains have been developed that display useful properties for drug-delivery, immunological, and biophotonic applications. Synthesis of single sidechain-modified phospholipids is often affected by acyl migration of the side chains. The resulting regioisomers (e.g., Figure 1a) have similar structures, which make their separation impractical and their detection challenging or impossible using techniques such as HPLC, NMR spectroscopy, and mass spectrometry. Regioselective phospholipid side-chain modification has been achieved using a number of techniques. Synthesis of modified phospholipids has been performed in multistep reactions using a modified glycerol backbone, with protecting groups sometimes being required. Acylation of lyso-