Engineering exosome polymer hybrids by atom transfer radical polymerization

Exosomes are emerging as ideal drug delivery vehicles due to their biological origin and ability to transfer cargo between cells. However, rapid clearance of exogenous exosomes from the circulation as well as aggregation of exosomes and shedding of surface proteins during storage limit their clinical translation. Here, we demonstrate highly controlled and reversible functionalization of exosome surfaces with well-defined polymers that modulate the exosome’s physiochemical and pharmacokinetic properties. Using cholesterol-modified DNA tethers and complementary DNA block copolymers, exosome surfaces were engineered with different biocompatible polymers. Additionally, polymers were directly grafted from the exosome surface using biocompatible photo-mediated atom transfer radical polymerization (ATRP). These exosome polymer hybrids (EPHs) exhibited enhanced stability under various storage conditions and in the presence of proteolytic enzymes. Tuning of the polymer length and surface loading allowed precise control over exosome surface interactions, cellular uptake, and preserved bioactivity. EPHs show fourfold higher blood circulation time without altering tissue distribution profiles. Our results highlight the potential of precise nanoengineering of exosomes toward developing advanced drug and therapeutic delivery systems using modern ATRP methods.

Exosomes are a subclass of lipid bilayer-enclosed extracellular vesicles (EVs) that play a crucial role in intercellular communication (1–3). They are secreted by most cell types in the body and are known to interact with recipient cells in several ways, including surface receptor interactions, membrane fusion, receptor-mediated endocytosis, phagocytosis and/or micropinocytosis (4, 5). Their nanoscopic size (30 to 150 nm), high biocompatibility, low immunogenicity (depending on the cell source), and ability to cross biological barriers, including the blood–brain barrier, make them an ideal vehicle for exogenous drug delivery (6–9). Over the last decade, multiple studies have shown effective utility of exosomes for the delivery of small molecule drugs, proteins, nucleic acids and nanoparticles for the treatment of several diseases (10–12). Although initial progress toward their clinical translation has been made, the need for a more robust platform persists. The therapeutic potential of exosomes is largely restricted due to their low exogenous drug-loading efficiency and limited ex vivo stability (13, 14). Moreover, systemically administered exosomes suffer from rapid clearance from blood in 2 to 20 min postinjection that is poorly suited for longer therapeutic action (15, 16).Engineering exosomes to incorporate nonnative moieties or materials can augment their therapeutic capabilities (17–19). While bioengineering methods by genetically modifying the exosome-secreting cells have been explored, such approaches require careful design and expensive reagents, yet suffer from low incorporation efficiency and limited scalability. Alternatively, ex vivo engineering of exosome surfaces with synthetic macromolecules is a powerful approach to easily modulate their surface interactions and consequently alter or enhance their biochemical and physicochemical properties.Here, we create a polymer-based platform that expands the structural repertoire of engineered exosomes and addresses the shortcomings of the ex vivo and in vivo stability of exosome-based therapeutics. We combine our previously reported method for rapid and on-demand functionalization of exosomes through DNA tethers (20) with atom transfer radical polymerization (ATRP) techniques (21–23) to engineer exosome polymer hybrids (EPHs). These EPHs display significantly enhanced stability and pharmacokinetics. We explore the preparation methods for EPHs by either tethering preformed DNA block copolymers (DNABCPs) onto the exosome membrane (“grafting-to”) or by grafting polymers directly from the exosomal surface (“grafting-from”) (Fig. 1) using DNA initiators. These membrane-tethering approaches allow precise control over the polymer length, composition, and loading on the exosome surface and thereby show minimal effect on the accessibility of surface proteins or other membrane-tethered agents that may be used for targeted delivery. We show that the cellular uptake and bioactivity of native and drug-loaded exosomes are preserved following polymer functionalization. Tethered polymers enhance the stability of exosomes under different storage conditions, including in the presence of proteolytic enzymes. The blood circulation half-lives of EPHs are significantly increased using different polymers, while maintaining their intrinsic tissue-targeting properties.Open in a separate windowFig. 1.Preparation of EPHs using DNA tethers. Chol-DNA embeds into the exosome membrane to form Exo-ssDNA (single-stranded DNA) species with DNA strands orienting outward. Hybridization of complementary DNA block copolymer (DNA′-Polymer) to the DNA tethers on Ex-ssDNA species generates Exo-dsDNA-Polymer (Exo-Polymer) by the “grafting-to” strategy. Alternatively, for the “grafting-from” strategy, a complementary DNA initiator (DNA′-Initiator) functionalized with the α-bromoisobutyrate group is hybridized with the DNA tethers, followed by surface-initiated ATRP to prepare Exo-Polymer species.