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.

Controlled Release of Exosomes Using Atom Transfer Radical Polymerization-Based Hydrogels.

Exosomes are 30-200 nm sized extracellular vesicles that are increasingly recognized as potential drug delivery vehicles. However, exogenous exosomes are rapidly cleared from the blood upon intravenous delivery, which limits their therapeutic potential. Here, we report bioactive exosome-tethered poly(ethylene oxide)-based hydrogels for the localized delivery of therapeutic exosomes. Using cholesterol-modified DNA tethers, the lipid membrane of exosomes was functionalized with initiators to graft polymers in the presence of additional initiators and crosslinker using photoinduced atom transfer radical polymerization (ATRP). This strategy of tethering exosomes within the hydrogel network allowed their controlled release over a period of 1 month, which was much longer than physically entrapped exosomes. Exosome release profile was tuned by varying the crosslinking density of the polymer network and the use of photocleavable tethers allowed stimuli-responsive release of exosomes. The therapeutic potential of the hydrogels was assessed by evaluating the osteogenic potential of bone morphogenetic protein 2-loaded exosomes on C2C12 and MC3T3-E1 cells. Thus, ATRP-based exosome-tethered hydrogels represent a tunable platform with improved efficacy and an extended-release profile.

Atom Transfer Radical Polymerization for Biorelated Hybrid Materials.

Proteins, nucleic acids, lipid vesicles, and carbohydrates are the major classes of biomacromolecules that function to sustain life. Biology also uses post-translation modification to increase the diversity and functionality of these materials, which has inspired attaching various other types of polymers to biomacromolecules. These polymers can be naturally (carbohydrates and biomimetic polymers) or synthetically derived and have unique properties with tunable architectures. Polymers are either grafted-to or grown-from the biomacromolecule's surface, and characteristics including polymer molar mass, grafting density, and degree of branching can be controlled by changing reaction stoichiometries. The resultant conjugated products display a chimerism of properties such as polymer-induced enhancement in stability with maintained bioactivity, and while polymers are most often conjugated to proteins, they are starting to be attached to nucleic acids and lipid membranes (cells) as well. The fundamental studies with protein-polymer conjugates have improved our synthetic approaches, characterization techniques, and understanding of structure-function relationships that will lay the groundwork for creating new conjugated biomacromolecular products which could lead to breakthroughs in genetic and tissue engineering.

Biocatalytic "Oxygen-Fueled" Atom Transfer Radical Polymerization.

Atom transfer radical polymerization (ATRP) can be carried out in a flask completely open to air using a biocatalytic system composed of glucose oxidase (GOx) and horseradish peroxidase (HRP) with an active copper catalyst complex. Nanomolar concentrations of the enzymes and ppm amounts of Cu provided excellent control over the polymerization of oligo(ethylene oxide) methyl ether methacrylate (OEOMA500 ), generating polymers with high molecular weight (Mn >70 000) and low dispersities (1.13≤D≤1.27) in less than an hour. The continuous oxygen supply was necessary for the generation of radicals and polymer chain growth as demonstrated by temporal control and by inducing hypoxic conditions. In addition, the enzymatic cascade polymerization triggered by oxygen was used for a protein and DNA functionalized with initiators to form protein-b-POEOMA and DNA-b-POEOMA bioconjugates, respectively.

Automated Synthesis of Well-Defined Polymers and Biohybrids by Atom Transfer Radical Polymerization Using a DNA Synthesizer.

A DNA synthesizer was successfully employed for preparation of well-defined polymers by atom transfer radical polymerization (ATRP), in a technique termed AutoATRP. This method provides well-defined homopolymers, diblock copolymers, and biohybrids under automated photomediated ATRP conditions. PhotoATRP was selected over other ATRP methods because of mild reaction conditions, ambient temperature, tolerance to oxygen, and no need to introduce reducing agents or radical initiators. Both acrylate and methacrylate monomers were successfully polymerized with excellent control in the DNA synthesizer. Diblock copolymers were synthesized with different targeted degrees of polymerization and with high retention of chain-end functionality. Both hydrophobic and hydrophilic monomers were grafted from DNA. The DNA-polymer hybrids were characterized by SEC and DLS. The AutoATRP method provides an efficient route to prepare a range of different polymeric materials, especially polymer-biohybrids.

Open-air green-light-driven ATRP enabled by dual photoredox/copper catalysis.

Photoinduced atom transfer radical polymerization (photo-ATRP) has risen to the forefront of modern polymer chemistry as a powerful tool giving access to well-defined materials with complex architecture. However, most photo-ATRP systems can only generate radicals under biocidal UV light and are oxygen-sensitive, hindering their practical use in the synthesis of polymer biohybrids. Herein, inspired by the photoinduced electron transfer-reversible addition-fragmentation chain transfer (PET-RAFT) polymerization, we demonstrate a dual photoredox/copper catalysis that allows open-air ATRP under green light irradiation. Eosin Y was used as an organic photoredox catalyst (PC) in combination with a copper complex (X-CuII/L). The role of PC was to trigger and drive the polymerization, while X-CuII/L acted as a deactivator, providing a well-controlled polymerization. The excited PC was oxidatively quenched by X-CuII/L, generating CuI/L activator and PC˙+. The ATRP ligand (L) used in excess then reduced the PC˙+, closing the photocatalytic cycle. The continuous reduction of X-CuII/L back to CuI/L by excited PC provided high oxygen tolerance. As a result, a well-controlled and rapid ATRP could proceed even in an open vessel despite continuous oxygen diffusion. This method allowed the synthesis of polymers with narrow molecular weight distributions and controlled molecular weights using Cu catalyst and PC at ppm levels in both aqueous and organic media. A detailed comparison of photo-ATRP with PET-RAFT polymerization revealed the superiority of dual photoredox/copper catalysis under biologically relevant conditions. The kinetic studies and fluorescence measurements indicated that in the absence of the X-CuII/L complex, green light irradiation caused faster photobleaching of eosin Y, leading to inhibition of PET-RAFT polymerization. Importantly, PET-RAFT polymerizations showed significantly higher dispersity values (1.14 ≤ D ≤ 4.01) in contrast to photo-ATRP (1.15 ≤ D ≤ 1.22) under identical conditions.

Reflection on the Matyjaszewski Lab Webinar Series and the Rise of Webinars in Polymer Chemistry.

Webinar series are helping our community of polymer scientists to stay engaged and connected, despite the cancellation of in-person meetings and the periodic closure of laboratories to contain the spread of the coronavirus pandemic. The sustainable and inclusive character of these virtual events make them valuable learning and networking opportunities. As organizers of the Matyjaszewski Lab Webinar Series, we share herein our experience, highlighting the benefits of virtual meetings and providing a short guide for webinar organizers. Researchers, particularly young scientists, are encouraged to organize such virtual events to broaden their skills and strengthen their professional network.

Biocompatible photoinduced CuAAC using sodium pyruvate.

Sodium pyruvate, a natural intermediate produced during cellular metabolism, is commonly used in buffer solutions and media for biochemical applications. Here we show the use of sodium pyruvate (SP) as a reducing agent in a biocompatible aqueous photoinduced azide-alkyne cycloaddition (CuAAC) reaction. This copper(I)-catalyzed 1,3-dipolar cycloaddition is triggered by SP under UV light irradiation, exhibits oxygen tolerance and temporal control, and provides a convenient alternative to current CuAAC systems, particularly for biomolecular conjugations.

Preparation of Well-Defined Polymers and DNA-Polymer Bioconjugates via Small-Volume eATRP in the Presence of Air.

An aqueous electrochemically mediated atom transfer radical polymerization (eATRP) was performed in a small volume solution (75 µL) deposited on a screen-printed electrode (SPE). The reaction was open to air, thanks to the use of glucose oxidase (GOx) as an oxygen scavenger. Well-defined poly(2-(methylsulfinyl)ethyl acrylate) (PMSEA), poly(oligo(ethylene oxide) methyl ether methacrylate) (POEOMA), and corresponding DNA-polymer biohybrids were synthesized by the small-volume eATRP at room temperature. The reactions were simplified and polymerization rates increased by the application of the enzyme deoxygenating system and the compact electrochemical setup. Importantly, the volume of polymerization mixture was lowered to microliters, which not only decreases the cost for each reaction, but can also be potentially implemented in combinatorial chemistry and electrode-array configurations for high-throughput systems.

Rapid On-Demand Extracellular Vesicle Augmentation with Versatile Oligonucleotide Tethers.

Exosomes show potential as ideal vehicles for drug delivery because of their natural role in transferring biological cargo between cells. However, current methods to engineer exosomes without negatively impacting their function remain challenging. Manipulating exosome-secreting cells is complex and time-consuming, while direct functionalization of exosome surface proteins suffers from low specificity and low efficiency. We demonstrate a rapid, versatile, and scalable method with oligonucleotide tethers to enable diverse surface functionalization on both human and murine exosomes. These exosome surface modifiers, which range from reactive functional groups and small molecules to aptamers and large proteins, can readily and efficiently enhance native exosome properties. We show that cellular uptake of exosomes can be specifically altered with a tethered AS1411 aptamer, and targeting specificity can be altered with a tethered protein. We functionalize exosomes with an immunomodulatory protein, FasL, and demonstrate their biological activity both in vitro and in vivo. FasL-functionalized exosomes, when bioprinted on a collagen matrix, allows spatial induction of apoptosis in tumor cells and, when injected in mice, suppresses proliferation of alloreactive T cells. This oligonucleotide tethering strategy is independent of the exosome source and further circumvents the need to genetically modify exosome-secreting cells.

Synthesis of Polymer Bioconjugates via Photoinduced Atom Transfer Radical Polymerization under Blue Light Irradiation.

A rapid blue-light-induced atom transfer radical polymerization (ATRP) was conducted in a biologically friendly environment. Well-controlled polymerization of oligo(ethylene oxide) methyl ether methacrylate (OEOMA) was successfully performed in aqueous media (1X PBS) under irradiation by blue LED strips. With 10.0 mW/cm2 intensity output at 450 nm, >90% conversion was achieved in 2 h in the presence of a system comprising glucose, glucose oxidase, and sodium pyruvate. Poly-(OEOMA) was synthesized with predetermined M n and low dispersities using low ppm of Cu catalysts. Importantly, secondary structures of proteins, as analyzed by circular dichroism (CD), were preserved under blue-light irradiation due to its lower energy output. The aqueous blue-light ATRP technique was applied to biological systems by synthesizing well-defined protein-polymer and DNA-polymer hybrids by the "grafting-from" method.

Ultrasonication-Induced Aqueous Atom Transfer Radical Polymerization.

A new procedure for ultrasonication-induced atom transfer radical polymerization (sono-ATRP) in aqueous media was developed. Polymerizations of oligo(ethylene oxide) methyl ether methacrylate (OEOMA) and 2-hydroxyethyl acrylate (HEA) in water were successfully carried out in the presence of ppm amounts of CuBr2 catalyst and tris(2-pyridylmethyl)amine ligand when exposed to ultrasonication (40 kHz, 110 W) at room temperature. Aqueous sono-ATRP enabled polymerization of water-soluble monomers with excellent control over the molecular weight, dispersity, and high retention of chain-end functionality. Temporal control over the polymer chain growth was demonstrated by switching the ultrasound on/off due to the regeneration of activators by hydroxyl radicals formed by ultrasonication. The synthesis of a well-defined block copolymer and DNA-polymer biohybrid was also successful using this process.

Supramolecular guest relay using host-protein nanocavities: an application of host-induced guest protonation.

Small drug molecules and other important metabolites are delivered via a suitable carrier protein-mediated transport through a specific receptor. The process is highly coordinated and associated with complexation induced properties of deliverable molecules. To get a molecular insight, in this report, we tried to mimic the delivery process to know how the carrier protein relocates the drug molecule from the macrocyclic host cavity to its binding pocket and how the electronic and the chemical properties of the guest get altered. Bovine and human serum albumin (BSA and HSA) were used as the model carrier proteins which can snatch out 6-propanoyl-2-(N,N-dimethylamino)naphthalene (PRO), dye used as a drug model (known to bind at the drug-binding pocket of the carrier protein), from the cucurbit[7]uril (CB7) cavity, a potential drug delivery carrier. Prior to performing the fluorescence-based bio-supramolecular relocation assay using BSA and HSA, CB7 and PRO, we have investigated the effect of CB7 encapsulation and protonation on the fluorescence properties of PRO. A significant shift in the pKa value from 3.4 to 6.6 (ca. 3.2 logarithmic units) of PRO was observed upon encapsulation with CB7, which causes a huge fluorescence quenching even at neutral pH. The binding affinity of protonated and neutral PRO for CB7 also confirms a 3.2 unit shift in the acid-dissociation constant. A displacement assay using a strong CB7 binder, viz., 1,6-diaminohexane, confirms encapsulation of PRO in the CB7 cavity. Encapsulation of neutral PRO by CB7 shows a significant fluorescence enhancement accompanied by a ∼35 nm blue shift in the emission maxima.