Installing a Single Monomer within Acrylic Polymers Using Photoredox Catalysis.

Incorporating exactly one monomer at a defined position during a chain polymerization is exceptionally challenging due to the statistical nature of monomer addition. Herein, photoinduced electron/energy transfer (PET) enables the incorporation of exactly one vinyl ether into polyacrylates synthesized via reversible addition-fragmentation chain transfer (RAFT) polymerization. Near-quantitative addition (>96%) of a single vinyl ether is achieved while retaining >99% of the thiocarbonylthio chain ends. Kinetic studies reveal that performing the reactions at 2 °C limits unwanted chain breaking events. Finally, the syntheses of diblock copolymers are reported where molecular weights and dispersities are well-controlled on either side of the vinyl ether. Overall, this report introduces an approach to access acrylic copolymers containing exactly one chemical handle at a defined position, enabling novel macromolecular architectures to probe structure-function properties, introduce sites for de/reconstruction, store information, etc.

Spatially-Encoding Hydrogels With DNA to Control Cell Signaling.

Patterning biomolecules in synthetic hydrogels offers routes to visualize and learn how spatially-encoded cues modulate cell behavior (e.g., proliferation, differentiation, migration, and apoptosis). However, investigating the role of multiple, spatially defined biochemical cues within a single hydrogel matrix remains challenging because of the limited number of orthogonal bioconjugation reactions available for patterning. Herein, a method to pattern multiple oligonucleotide sequences in hydrogels using thiol-yne photochemistry is introduced. Rapid hydrogel photopatterning of hydrogels with micron resolution DNA features (≈1.5 µm) and control over DNA density are achieved over centimeter-scale areas using mask-free digital photolithography. Sequence-specific DNA interactions are then used to reversibly tether biomolecules to patterned regions, demonstrating chemical control over individual patterned domains. Last, localized cell signaling is shown using patterned protein-DNA conjugates to selectively activate cells on patterned areas. Overall, this work introduces a synthetic method to achieve multiplexed micron resolution patterns of biomolecules onto hydrogel scaffolds, providing a platform to study complex spatially-encoded cellular signaling environments.

Modular Nucleic Acid Scaffolds for Synthesizing Monodisperse and Sequence-Encoded Antibody Oligomers.

Synthesizing protein oligomers that contain exact numbers of multiple different proteins in defined architectures is challenging. DNA-DNA interactions can be used to program protein assembly into oligomers; however, existing methods require changes to DNA design to achieve different numbers and oligomeric sequences of proteins. Herein, we develop a modular DNA scaffold that uses only six synthetic oligonucleotides to organize proteins into defined oligomers. As a proof-of-concept, model proteins (antibodies) are oligomerized into dimers and trimers, where antibody function is retained. Illustrating the modularity of this technique, dimer and trimer building blocks are then assembled into pentamers containing three different antibodies in an exact stoichiometry and oligomeric sequence. In sum, this report describes a generalizable method for organizing proteins into monodisperse, sequence-encoded oligomers using DNA. This advance will enable studies into how oligomeric protein sequences affect material properties in areas spanning pharmaceutical development, cascade catalysis, synthetic photosynthesis, and membrane transport.

Controlling the DNA Hybridization Chain Reaction.

A novel method for controlling the oligomerization of metastable DNA hairpins using the hybridization chain reaction (HCR) is reported. Control was achieved through the introduction of a base-pair mismatch in the duplex of the hairpins. The mismatch modification allows one to kinetically differentiate initiation versus propagation events, leading to DNA oligomers up to 10 monomers long and improving dispersities from 2.5 to 1.3-1.6. Importantly, even after two consecutive chain extensions, dispersity remained unaffected, showing that well-defined block co-oligomers can be achieved. As a proof-of-concept, this technique was then applied to hairpin monomers functionalized with a mutant green fluorescent protein to prepare protein oligomers. Taken together, this work introduces an effective method for controlling living macromolecular HCR oligomerization in a manner analogous to the controlled polymerization of small molecules.

Ultrahigh Molecular Weight Hydrophobic Acrylic and Styrenic Polymers through Organic-Phase Photoiniferter-Mediated Polymerization.

As many physical properties of polymers scale with molecular weight, the ability to achieve polymers of nearly inaccessibly high molecular weight provides an opportunity to probe the upper size limit of macromolecular phenomena. Yet many of the most stimulating macromolecular designs remain out of reach of current ultrahigh molecular weight (UHMW) polymer synthetic approaches. Herein, we show that UHMW polymers of diverse composition can be achieved by irradiation of thiocarbonylthio photoiniferters with long-wave ultraviolet or visible light in concentrated organic solution. This facile photopolymerization strategy is general to acrylic-, acrylamido-, methacrylic-, and styrenic-based monomers, enabling the synthesis of well-defined macromolecules with molecular weights in excess of 106 g/mol. The high chain-end fidelity afforded by photoiniferter polymerization conditions facilitated the design of UHMW amphiphilic block copolymers, which were found to self-assemble into micellar morphologies up to 200 nm in diameter.

DNA-Directed Protein Packing within Single Crystals.

Designed DNA-DNA interactions are investigated for their ability to modulate protein packing within single crystals of mutant green fluorescent proteins (mGFPs) functionalized with a single DNA strand (mGFP-DNA). We probe the effects of DNA sequence, length, and protein-attachment position on the formation and protein packing of mGFP-DNA crystals. Notably, when complementary mGFP-DNA conjugates are introduced to one another, crystals form with nearly identical packing parameters, regardless of sequence if the number of bases is equivalent. DNA complementarity is essential, because experiments with non-complementary sequences produce crystals with different protein arrangements. Importantly, the DNA length and its position of attachment on the protein markedly influence the formation of and protein packing within single crystals. This work shows how designed DNA interactions can be used to influence the growth and packing in X-ray diffraction quality protein single crystals and is thus an important step forward in protein crystal engineering.

Cross-Linked Aptamer-Lipid Micelles for Excellent Stability and Specificity in Target-Cell Recognition.

The specific binding ability of DNA-lipid micelles (DLMs) can be increased by the introduction of an aptamer. However, supramolecular micellar structures based on self-assemblies of amphiphilic DLMs are expected to demonstrate low stability when interacting with cell membranes under certain conditions, which could lead to a reduction in selectivity for targeting cancer cells. We herein report a straightforward cross-linking strategy that relies on a methacrylamide branch to link aptamer and lipid segments. By an efficient photoinduced polymerization process, covalently linked aptamer-lipid units help stabilize the micelle structure and enhance aptamer probe stability, further improving the targeting ability of the resulting nanoassembly. Besides the development of a facile cross-linking method, this study clarifies the relationship between aptamer-lipid concentration and the corresponding binding ability.

Polymerization-Induced Self-Assembly of Micelles Observed by Liquid Cell Transmission Electron Microscopy.

In this paper, we describe the use of liquid cell transmission electron microscopy (LCTEM) for inducing and imaging the formation of spherical micelles from amphiphilic block copolymers. Within the irradiated region of the liquid cell, diblock copolymers were produced which self-assembled, yielding a targeted spherical micellar phase via polymerization-induced self-assembly (PISA). Critically, we demonstrate that nanoparticle formation can be visualized in situ and that in the presence of excess monomer, nanoparticle growth occurs to yield sizes and morphologies consistent with standard PISA conditions. Experiments were enabled by employing automated LCTEM sample preparation and by analyzing LCTEM data with multi-object tracking algorithms designed for the detection of low-contrast materials.

Star Architecture Promoting Morphological Transitions during Polymerization-Induced Self-Assembly.

Polymerization-induced self-assembly (PISA) via reversible addition-fragmentation chain transfer (RAFT) dispersion polymerization is an effective method to produce block copolymer nano-objects of various morphologies at high solids. However, current PISA formulations have been limited to linear block copolymers. We report the synthesis of AB2 star block copolymers via RAFT aqueous dispersion polymerization of diacetone acrylamide using a poly(ethylene glycol) methyl ether bearing two chain transfer agents as the difunctional macromolecular chain transfer agent (macro-CTA), which was efficiently synthesized using 2,4,6-trichloro-1,3,5-triazine and activated esters to afford a high end functionality (97%). The star polymer architecture can significantly promote morphological transitions to obtain higher-order morphologies at both lower solids and lower degrees of polymerization of the core-forming block in comparison with its linear counterpart. This work demonstrates that polymer architecture is another important parameter that should be considered when conducting PISA synthesis to obtain complex morphologies.

Grafting-From Proteins Using Metal-Free PET-RAFT Polymerizations under Mild Visible-Light Irradiation.

We report a new strategy toward polymer-protein conjugates using a grafting-from method that employs photoinduced electron/energy transfer-reversible addition-fragmentation chain transfer (PET-RAFT) polymerization. Initial screening of reaction conditions showed rapid polymerization of acrylamides under high dilution in water using eosin Y as a photocatalyst in the presence of a tertiary amine. A lysozyme-modified chain transfer agent allowed the same conditions to be utilized for grafting-from polymerizations, and we further demonstrated the broad scope of this technique by polymerizing acrylic and styrenic monomers. Finally, retention of the RAFT end group was suggested by successful chain extension with N-isopropylacrylamide from the polymer-protein conjugates to form block copolymer-protein conjugates. This strategy should expand the capabilities of grafting-from proteins with RAFT polymerization under mild conditions to afford diverse functional materials.

Catalyst-Free Photoinduced End-Group Removal of Thiocarbonylthio Functionality.

An initiator- and catalyst-free method for polymer end-group modification has been designed. Under long-wave ultraviolet irradiation, polymers with thiocarbonylthio end groups undergo photolytic cleavage to reveal an active macroradical capable of irreversible termination with a suitable hydrogen source. This straightforward method was successfully demonstrated by the removal of a range of end groups that commonly result from reversible addition-fragmentation chain transfer or photoiniferter polymerizations, including trithiocarbonate, dithiobenzoate, xanthate, and dithiocarbamate mediating agents. This strategy proved efficient for polymers derived from acrylamido, acrylic, methacrylic, styrenic, and vinylpyrrolidone monomers.

Radical Ring-Opening Copolymerization of Cyclic Ketene Acetals and Maleimides Affords Homogeneous Incorporation of Degradable Units.

Radical copolymerization of donor-acceptor (D-A) monomer pairs has served as a versatile platform for the development of alternating copolymers. However, due to the use of conventional radical polymerization, the resulting copolymers have generally been limited to nondegradable vinyl polymers. By combining radical D-A copolymerization with radical ring-opening polymerization (rROP), we have synthesized an alternating copolymer with a high incorporation of degradable backbone units. Copolymerization of N-ethyl maleimide (NEtMI) with the cyclic ketene acetal (CKA) 2-methylene-4-phenyl-1,3-dioxolane (MPDL) was demonstrated to proceed in an alternating fashion, and controlled polymerization was achieved using reversible addition-fragmentation chain transfer (RAFT) polymerization. Spontaneous copolymerization, in the absence of an exogenous initiating source, occurred when the mixture of monomers was heated, presumably due to the large electron disparity between the comonomers. Chain-extension with styrene afforded well-defined P(MPDL-alt-NEtMI)-b-polystyrene copolymers, and degradation of the homopolymers and block copolymers showed complete breakdown of the alternating copolymer.

Closed-System One-Pot Block Copolymerization by Temperature-Modulated Monomer Segregation.

A biphasic one-pot polymerization method enables the preparation of block copolymers from monomers with similar and competitive reactivities without the addition of external materials. AB diblock copolymers were prepared by encapsulating a frozen solution of monomer B on the bottom of a reaction vessel, while the solution polymerization of monomer A was conducted in a liquid layer above. Physical separation between the solid and liquid phases permitted only homopolymerization of monomer A until heating above the melting point of the lower phase, which released monomer B, allowing the addition of the second block to occur. The triggered release of monomer B allowed for chain extension without additional deoxygenation steps or exogenous monomer addition. A method for the closed (i.e., without addition of external reagents) one-pot synthesis of block copolymers with conventional glassware using straightforward experimental techniques has thus been developed.

Role of Polymer Architecture on the Activity of Polymer-Protein Conjugates for the Treatment of Accelerated Bone Loss Disorders.

Polymers of similar molecular weights and chemical constitution but varying in their macromolecular architectures were conjugated to osteoprotegerin (OPG) to determine the effect of polymer topology on protein activity in vitro and in vivo. OPG is a protein that inhibits bone resorption by preventing the formation of mature osteoclasts from the osteoclast precursor cell. Accelerated bone loss disorders, such as osteoporosis, rheumatoid arthritis, and metastatic bone disease, occur as a result of increased osteoclastogenesis, leading to the severe weakening of the bone. OPG has shown promise as a treatment in bone disorders; however, it is rapidly cleared from circulation through rapid liver uptake, and frequent, high doses of the protein are necessary to achieve a therapeutic benefit. We aimed to improve the effectiveness of OPG by creating OPG-polymer bioconjugates, employing reversible addition-fragmentation chain transfer polymerization to create well-defined polymers with branching densities varying from linear, loosely branched to densely branched. Polymers with each of these architectures were conjugated to OPG using a "grafting-to" approach, and the bioconjugates were characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The OPG-polymer bioconjugates showed retention of activity in vitro against osteoclasts, and each bioconjugate was shown to be nontoxic. Preliminary in vivo studies further supported the nontoxic characteristics of the bioconjugates, and measurement of the bone mineral density in rats 7 days post-treatment via peripheral quantitative computed tomography suggested a slight increase in bone mineral density after administration of the loosely branched OPG-polymer bioconjugate.

Polymerization-induced thermal self-assembly (PITSA).

Polymerization-induced self-assembly (PISA) is a versatile technique to achieve a wide range of polymeric nanoparticle morphologies. Most previous examples of self-assembled soft nanoparticle synthesis by PISA rely on a growing solvophobic polymer block that leads to changes in nanoparticle architecture during polymerization in a selective solvent. However, synthesis of block copolymers with a growing stimuli-responsive block to form various nanoparticle shapes has yet to be reported. This new concept using thermoresponsive polymers is termed polymerization-induced thermal self-assembly (PITSA). A reversible addition-fragmentation chain transfer (RAFT) polymerization of N-isopropylacrylamide from a hydrophilic chain transfer agent composed of N,N-dimethylacrylamide and acrylic acid was carried out in water above the known lower critical solution temperature (LCST) of poly(N-isopropylacrylamide) (PNIPAm). After reaching a certain chain length, the growing PNIPAm self-assembled, as induced by the LCST, into block copolymer aggregates within which dispersion polymerization continued. To characterize the nanoparticles at ambient temperatures without their dissolution, the particles were crosslinked immediately following polymerization at elevated temperatures via the reaction of the acid groups with a diamine in the presence of a carbodiimide. Size exclusion chromatography was used to evaluate the unimer molecular weight distributions and reaction kinetics. Dynamic light scattering and transmission electron microscopy provided insight into the size and morphologies of the nanoparticles. The resulting block copolymers formed polymeric nanoparticles with a range of morphologies (e.g., micelles, worms, and vesicles), which were a function of the PNIPAm block length.

Efficient and Chemoselective Synthesis of ω,ω-Heterodifunctional Polymers.

We report a strategy for the preparation of semitelechelic polymers containing two distinct functionalities at one chain end by consecutive and chemoselective nucleophilic aromatic substitution reactions on 2,4,6-trichloro-1,3,5-triazine (TCT). Because of its commercial availability, well-defined nature, and ubiquity in biological applications, monomethyl ether poly(ethylene glycol) (mPEG) was chosen to demonstrate the utility of this ω,ω-heterodifunctional end-group modification strategy. TCT-functionalized mPEG underwent highly efficient ω,ω-heterodisubstitution via sequential chemoselective substitution with model thiols and amines. The efficiency of nucleophile conjugation to the polymer end group was confirmed by 1H NMR spectroscopy and matrix assisted laser desorption-ionization time-of-flight mass spectrometry. In addition, density functional theory calculations provided insight into the importance of nucleophile addition order. This route introduces TCT derivatization as a powerful and facile tool to achieve specific polymeric end-group complexity and efficient heterogeneous functionalization.

Rapid microwave-assisted sol-gel preparation of Pd-substituted LnFeO3 (Ln = Y, La): phase formation and catalytic activity.

We present a rapid microwave-assisted sol-gel approach to Pd-substituted LnFeO3 (Ln = Y, La) for applications in C-C coupling reactions. These materials could be prepared in household microwave ovens in less than 15 minutes of reaction time with the final materials displaying well-defined structure and morphology. Phase evolution was studied using time-dependent microwave heatings and then compared with the results obtained from thermogravimetric analyses. Materials were confirmed to be phase pure by laboratory and synchrotron X-ray diffraction. Substituted Pd is ionic as shown by the binding energy shift from X-ray photoelectron spectroscopy. The short heating periods required for phase purity allow these materials less time for sintering as compared to conventional solid state preparation methods, making relatively high surface areas achievable. These materials have been successfully used as catalyst precursor materials for C-C coupling reactions in which the active species is Pd(0). Pd-substituted LnFeO3 (Ln = Y, La) provides Pd(0) in solution which can be complexed by the ligand SPhos, allowing for aryl chloride coupling.