Machine learning-aided engineering of hydrolases for PET depolymerization.

Plastic waste poses an ecological challenge1-3 and enzymatic degradation offers one, potentially green and scalable, route for polyesters waste recycling4. Poly(ethylene terephthalate) (PET) accounts for 12% of global solid waste5, and a circular carbon economy for PET is theoretically attainable through rapid enzymatic depolymerization followed by repolymerization or conversion/valorization into other products6-10. Application of PET hydrolases, however, has been hampered by their lack of robustness to pH and temperature ranges, slow reaction rates and inability to directly use untreated postconsumer plastics11. Here, we use a structure-based, machine learning algorithm to engineer a robust and active PET hydrolase. Our mutant and scaffold combination (FAST-PETase: functional, active, stable and tolerant PETase) contains five mutations compared to wild-type PETase (N233K/R224Q/S121E from prediction and D186H/R280A from scaffold) and shows superior PET-hydrolytic activity relative to both wild-type and engineered alternatives12 between 30 and 50 °C and a range of pH levels. We demonstrate that untreated, postconsumer-PET from 51 different thermoformed products can all be almost completely degraded by FAST-PETase in 1 week. FAST-PETase can also depolymerize untreated, amorphous portions of a commercial water bottle and an entire thermally pretreated water bottle at 50 ºC. Finally, we demonstrate a closed-loop PET recycling process by using FAST-PETase and resynthesizing PET from the recovered monomers. Collectively, our results demonstrate a viable route for enzymatic plastic recycling at the industrial scale.

Role of PLGA Variability in Controlled Drug Release from Dexamethasone Intravitreal Implants.

Long-acting injectable formulations based on poly(lactide-co-glycolide) (PLGA) have been commercialized for over 30 years in at least 20 FDA-approved products. These formulations offer several advantages, including reduced dosing frequency, improved patient compliance, and maintenance of therapeutic levels of drug. Despite extensive studies, the inherent complexity of the PLGA copolymer still poses significant challenges associated with the development of generic formulations having drug release profiles equivalent to those of the reference listed drugs. In addition, small changes to PLGA physicochemical properties or the drug product manufacturing process can have a major impact on the drug release profile of these long-acting formulations. This work seeks to better understand how variability in the physicochemical properties of similar PLGAs affects drug release from PLGA solid implants using Ozurdex (dexamethasone intravitreal implant) as the model system. Four 50:50, acid-terminated PLGAs of similar molecular weights were used to prepare four dexamethasone intravitreal implants structurally equivalent to Ozurdex. The PLGAs were extensively characterized by using a variety of analytical techniques prior to implant manufacture using a continuous, hot-melt extrusion process. In vitro release testing of the four structurally equivalent implants was performed in both normal saline and phosphate-buffered saline (PBS), yielding drastically different results between the two methods. In normal saline, no differences in the release profiles were observed. In PBS, the drug release profiles were sensitive to small changes in the residual monomer content, carboxylic acid end group content, and blockiness of the polymers. This finding further underscores the need for a physiologically relevant in vitro release testing method as part of a robust quality control strategy for PLGA-based solid implant formulations.

Chemically Triggered Synthesis, Remodeling, and Degradation of Soft Materials.

Polymer topology dictates dynamic and mechanical properties of materials. For most polymers, topology is a static characteristic. In this article, we present a strategy to chemically trigger dynamic topology changes in polymers in response to a specific chemical stimulus. Starting with a dimerized PEG and hydrophobic linear materials, a lightly cross-linked polymer, and a cross-linked hydrogel, transformations into an amphiphilic linear polymer, lightly cross-linked and linear random copolymers, a cross-linked polymer, and three different hydrogel matrices were achieved via two controllable cross-linking reactions: reversible conjugate additions and thiol-disulfide exchange. Significantly, all the polymers, before or after topological changes, can be triggered to degrade into thiol- or amine-terminated small molecules. The controllable transformations of polymeric morphologies and their degradation herald a new generation of smart materials.

Mechanism of Polymer-Mediated Cryopreservation Using Poly(methyl glycidyl sulfoxide).

Under the right conditions, some biological systems can maintain high viability after being frozen and thawed, but many others (e.g., organs and many mammalian cells) cannot. To increase the rates of post-thaw viability and widen the library of living cells and tissues that can be stored frozen, an improved understanding of the mode of action of polymeric cryoprotectants is required. Here, we present a polymeric cryoprotectant, poly(methyl glycidyl sulfoxide) (PMGS), that achieved higher post-thaw viability for fibroblast cells than its small-molecule analogue dimethyl sulfoxide. By limiting the amount of water that freezes and facilitating cellular dehydration after ice nucleation, PMGS mitigates the mechanical and osmotic stresses that the freezing of water imparts on cells and facilitates higher-temperature vitrification of the remaining unfrozen volume. The development of PMGS advances a fundamental physical understanding of polymer-mediated cryopreservation, which enables new material design for long-term preservation of complex cellular networks and tissue.

Understanding Poly(vinyl alcohol)-Mediated Ice Recrystallization Inhibition through Ice Adsorption Measurement and pH Effects.

The development of improved cryopreservative materials is necessary to enable complete recovery of living cells and tissue after frozen storage. Remarkably, poly(vinyl alcohol) (PVA) displays some of the same cryoprotective properties as many antifreeze proteins found in cold tolerant organisms. In particular, PVA is very effective at halting the Ostwald ripening of ice, a process that mechanically damages cells and tissue. Despite the large practical importance of such a property, the mechanism by which PVA interacts with ice is poorly understood, hindering the development of improved cryoprotective materials. Herein, we quantitatively evaluated ice growth kinetics in the presence of PVA at different pH conditions and in the presence of a range of neutral salts. We demonstrated that pH, but not salt identity, alters the ability of PVA to halt ice grain coarsening. These observations are consistent with hydrogen-bonding playing a crucial role in PVA-mediated ice recrystallization inhibition. The evolution of the size distribution of ice crystals with annealing was consistent with incomplete surface coverage of ice with PVA. Binding assay measurements of dissolved fluorescently labeled PVA in an ice slurry showed that PVA interacts with ice through weak adsorption (<9%) to the ice crystal surface, which stands in contrast to fluorescently tagged type III antifreeze peptide, which binds strongly (ca. 64%) under the same conditions.

Phase behavior of electrostatically complexed polyelectrolyte gels using an embedded fluctuation model.

Nanostructured, responsive hydrogels formed due to electrostatic interactions have promise for applications such as drug delivery and tissue mimics. These physically cross-linked hydrogels are composed of an aqueous solution of oppositely charged triblocks with charged end-blocks and neutral, hydrophilic mid-blocks. Due to their electrostatic interactions, the end-blocks microphase separate and form physical cross-links that are bridged by the mid-blocks. The structure of this system was determined using a new, efficient embedded fluctuation (EF) model in conjunction with self-consistent field theory. The calculations using the EF model were validated against unapproximated field-theoretic simulations with complex Langevin sampling and were found consistent with small angle X-ray scattering (SAXS) measurements on an experimental system. Using both the EF model and SAXS, phase diagrams were generated as a function of end-block fraction and polymer concentration. Several structures were observed including a body-centered cubic sphere phase, a hexagonally packed cylinder phase, and a lamellar phase. Finally, the EF model was used to explore how parameters that directly relate to polymer chemistry can be tuned to modify the resulting phase diagram, which is of practical interest for the development of new hydrogels.

Synthetic aptamer-polymer hybrid constructs for programmed drug delivery into specific target cells.

Viruses have evolved specialized mechanisms to efficiently transport nucleic acids and other biomolecules into specific host cells. They achieve this by performing a coordinated series of complex functions, resulting in delivery that is far more efficient than existing synthetic delivery mechanisms. Inspired by these natural systems, we describe a process for synthesizing chemically defined molecular constructs that likewise achieve targeted delivery through a series of coordinated functions. We employ an efficient "click chemistry" technique to synthesize aptamer-polymer hybrids (APHs), coupling cell-targeting aptamers to block copolymers that secure a therapeutic payload in an inactive state. Upon recognizing the targeted cell-surface marker, the APH enters the host cell via endocytosis, at which point the payload is triggered to be released into the cytoplasm. After visualizing this process with coumarin dye, we demonstrate targeted killing of tumor cells with doxorubicin. Importantly, this process can be generalized to yield APHs that specifically target different surface markers.

Drug release mechanisms of high-drug-load, melt-extruded dexamethasone intravitreal implants.

Ozurdex is an FDA-approved sustained-release, biodegradable implant formulated to deliver the corticosteroid dexamethasone to the posterior segment of the eye for up to 6 months. Hot-melt extrusion is used to prepare the 0.46 mm × 6 mm, rod-shaped implant by embedding the drug in a matrix of poly(lactic-co-glycolic acid) (PLGA) in a 60:40 drug:polymer ratio by weight. In our previous work, the Ozurdex implant was carefully studied and reverse engineered to produce a compositionally and structurally equivalent implant for further analysis. In this work, the reverse-engineered implant was thoroughly characterized throughout the in vitro dissolution process to elucidate the mechanisms of controlled drug release. The implant exhibited a triphasic release profile in 37 °C normal saline with a small burst release (1-2 %), a one-week lag phase with limited release (less than 10 %), and a final phase where the remainder of the dose was released over 3-4 weeks. The limited intermolecular interaction between dexamethasone and PLGA rendered the breakdown of the polymer the dominating mechanism of controlled release. A close relationship between drug release and total implant mass loss was observed. Unique chemical and structural differences were seen between the core of the implant and the implant surface driven by diffusional limitations, autocatalytic hydrolysis, and osmotic effects.

Manufacturing dexamethasone intravitreal implants: Process control and critical quality attributes.

Over 20 long-acting injectable formulations based on poly(lactide-co-glycolide) (PLGA) have been approved by the FDA to date. PLGA is a biodegradable polymer that can extend drug release from these dosage forms for up to six months after administration. Despite the commercial success of several of these formulations, there are still a limited number of products that utilize PLGA, and there are currently no generic counterparts of these products on the market. Significant technical challenges are associated with preparation of chemically and structurally equivalent formulations that yield an equivalent drug release profile to the reference listed drug (RLD) both in vitro and in vivo. In this work, Ozurdex (dexamethasone intravitreal implant) was used as a model system to explore how the manufacturing process of PLGA-based solid implants impacts the quality and performance of the dosage form. Control of implant structural characteristics, including diameter, internal porosity, and surface roughness, was required to maintain accurate unit dose potency. Implants were prepared by a continuous hot-melt extrusion process that was thoroughly characterized to show the importance of precise feeding control to meet dimensional specifications. Five extruder die designs were evaluated using the same hot-melt extrusion process to produce five structurally-distinct implants. The structural differences did not alter the in vitro drug release profile when tested in both normal saline and phosphate-buffered saline (pH 7.4); however, implant porosity was shown to impact the mechanical strength of the implants. This work seeks to provide insight into the manufacturing process of PLGA-based solid implants to support development of future novel and generic drug products.

A general approach to controlling the surface composition of poly(ethylene oxide)-based block copolymers for antifouling coatings.

To control the surface properties of a polystyrene-block-poly(ethylene oxide) diblock copolymer, perfluorinated chemical moieties were specifically incorporated into the block copolymer backbone. A polystyrene-block-poly[(ethylene oxide)-stat-(allyl glycidyl ether)] [PS-b-P(EO-stat-AGE)] statistical diblock terpolymer was synthesized with varying incorporations of allyl glycidyl ether (AGE) in the poly(ethylene oxide) block from 0 to 17 mol %. The pendant alkenes of the AGE repeat units were subsequently functionalized by thiol-ene chemistry with 1H,1H,2H,2H-perfluorooctanethiol, yielding fluorocarbon-functionalized AGE (fAGE) repeat units. (1)H NMR spectroscopy and size-exclusion chromatography indicated well-defined structures with complete functionalization of the pendant alkenes. The surfaces of the polymer films were characterized after spray coating by X-ray photoelectron spectroscopy (XPS) and near-edge X-ray absorption fine structure spectroscopy (NEXAFS), showing that the P(EO-stat-fAGE) block starts to compete with polystyrene to populate the surface after only 1 mol % incorporation of fAGE. Increasing the incorporation of fAGE led to an increased amount of perfluorocarbons on the surface and a decrease in the concentration of PS. At a fAGE incorporation of 8 mol %, PS was not detected at the surface, as measured by NEXAFS spectroscopy. Water contact angles measured by the captive-air-bubble technique showed the underwater surfaces to be dynamic, with advancing and receding contact angles varying by >20°. Protein adsorption studies demonstrated that the fluorinated surfaces effectively prevent nonspecific binding of proteins relative to an unmodified PS-b-PEO diblock copolymer. In biological systems, settlement of spores of the green macroalga Ulva was significantly lower for the fAGE-incorporated polymers compared to the unmodified diblock and a polydimethylsiloxane elastomer standard. Furthermore, the attachment strength of sporelings (young plants) of Ulva was also reduced for the fAGE-containing polymers, affirming their potential as fouling-release coatings.

Impact of Macromonomer Molar Mass and Feed Composition on Branch Distributions in Model Graft Copolymerizations.

Graft polymers are useful in a versatile range of material applications. Understanding how changes to the grafted architecture, such as the grafting density (z), the side-chain degree of polymerization (Nsc), and the backbone degree of polymerization (Nbb), affect polymer properties is critical for accurately tuning material performance. For graft-through copolymerizations, changes to Nsc and z are controlled by the macromonomer degree of polymerization (NMM) and the initial fraction of the macromonomer in the feed (fMM0), respectively. We show that changes to these parameters can influence the copolymerization reactivity ratios and, in turn, impact the side-chain distribution along a graft polymer backbone. Poly((±)-lactide) macromonomers with NMM values as low as ca. 1 and as high as 72 were copolymerized with a small-molecule dimethyl ester norbornene comonomer over a range of fMM0 values (0.1 ≤ fMM0 ≤ 0.8) using ring-opening metathesis polymerization (ROMP). Monomer conversion was determined using 1H nuclear magnetic resonance spectroscopy, and the data were fit with terminal and nonterminal copolymerization models. The results from this work provide essential information for manipulating Nsc and z while maintaining synthetic control over the side-chain distribution for graft-through copolymerizations.

Aerobic radical polymerization mediated by microbial metabolism.

Performing radical polymerizations under ambient conditions is a major challenge because molecular oxygen is an effective radical quencher. Here we show that the facultative electrogen Shewanella oneidensis can control metal-catalysed living radical polymerizations under apparent aerobic conditions by first consuming dissolved oxygen via aerobic respiration, and then directing extracellular electron flux to a metal catalyst. In both open and closed containers, S. oneidensis enabled living radical polymerizations without requiring the preremoval of oxygen. Polymerization activity was closely tied to S. oneidensis anaerobic metabolism through specific extracellular electron transfer proteins and was effective for a variety of monomers using low (parts per million) concentrations of metal catalysts. Finally, polymerizations survived repeated challenges of oxygen exposure and could be initiated using lyophilized or spent (recycled) cells. Overall, our results demonstrate how the unique ability of S. oneidensis to use both oxygen and metals as respiratory electron acceptors can be leveraged to address salient challenges in polymer synthesis.

Coordination-Assisted Self-Assembled Polypeptide Nanogels to Selectively Combat Bacterial Infection.

In the present scenario, the invention of bacteria-selective antimicrobial agent comprising negligible toxicity and hemolytic effect is a great challenge. To surmount this challenge, here, a series of polypeptide nanogels (PNGs) have been fabricated by a coordination-assisted self-assembly of a mannose-conjugated antimicrobial polypeptide, poly(arginine-r-valine)-mannose (poly(Arg-r-Val)-M2), with Zn2+ ions. The fabricated PNGs are spherical in shape with a unique structural appearance similar to that of Taxus baccata fruits. PNGs, with a unique structural arrangement and threshold surface charge density, selectively interact with the bacterial membrane and exhibit potent antimicrobial activity, as reflected in their lower minimum inhibitory concentration values (varies from 2 to 16 µg/mL). PNGs show a remarkably high binding constant, 6.02 × 105 M-1 (from isothermal titration calorimetry, ITC), with the bacterial membrane which manifests its potent bactericidal effect. PNGs are nontoxic against mammalian and red blood cells as reflected from their higher cell viability and insignificant hemolytic effect. PNGs are taken up by the bacterial membrane and selectively undergo structural deformation (scrutinized by ITC) followed by an exposure of free poly(Arg-r-Val)-M2 molecules. The free poly(Arg-r-Val)-M2 molecules are enforced to lyse the bacterial membrane (visualized by cryo-transmission electron microscopy) followed by the diffusion of the cytoplasmic component out of the membrane which culminates in the final death of the bacterium.

Mussel-Inspired Anchoring of Polymer Loops That Provide Superior Surface Lubrication and Antifouling Properties.

We describe robustly anchored triblock copolymers that adopt loop conformations on surfaces and endow them with unprecedented lubricating and antifouling properties. The triblocks have two end blocks with catechol-anchoring groups and a looping poly(ethylene oxide) (PEO) midblock. The loops mediate strong steric repulsion between two mica surfaces. When sheared at constant speeds of ∼2.5 µm/s, the surfaces exhibit an extremely low friction coefficient of ∼0.002-0.004 without any signs of damage up to pressures of ∼2-3 MPa that are close to most biological bearing systems. Moreover, the polymer loops enhance inhibition of cell adhesion and proliferation compared to polymers in the random coil or brush conformations. These results demonstrate that strongly anchored polymer loops are effective for high lubrication and low cell adhesion and represent a promising candidate for the development of specialized high-performance biomedical coatings.

Hierarchically ordered nanopatterns for spatial control of biomolecules.

The development and study of a benchtop, high-throughput, and inexpensive fabrication strategy to obtain hierarchical patterns of biomolecules with sub-50 nm resolution is presented. A diblock copolymer of polystyrene-b-poly(ethylene oxide), PS-b-PEO, is synthesized with biotin capping the PEO block and 4-bromostyrene copolymerized within the polystyrene block at 5 wt %. These two handles allow thin films of the block copolymer to be postfunctionalized with biotinylated biomolecules of interest and to obtain micropatterns of nanoscale-ordered films via photolithography. The design of this single polymer further allows access to two distinct superficial nanopatterns (lines and dots), where the PEO cylinders are oriented parallel or perpendicular to the substrate. Moreover, we present a strategy to obtain hierarchical mixed morphologies: a thin-film coating of cylinders both parallel and perpendicular to the substrate can be obtained by tuning the solvent annealing and irradiation conditions.

Renewable-resource thermoplastic elastomers based on polylactide and polymenthide.

An alpha,omega-functionalized polymenthide was synthesized by the ring-opening polymerization of menthide in the presence of diethylene glycol with diethyl zinc as the catalyst. Termination with water afforded the dihydroxy polymenthide. The reaction of this telechelic polymer with triethylaluminum formed the corresponding aluminum alkoxide macroinitiator that was used for the controlled polymerization of lactide to yield biorenewable polylactide-b-polymenthide-b-polylactide triblock copolymers. The molecular weight and chemical composition were easily adjusted by the monomer-to-initiator ratios. Microphase separation in these triblock copolymers was confirmed by small-angle X-ray scattering and differential scanning calorimetry. A representative triblock was prepared with a hexagonally packed cylindrical morphology as determined by small-angle X-ray scattering, and tensile testing was employed to assess the mechanical behavior. On the basis of the ultimate elongations and elastic recovery, these triblock copolymers behaved as thermoplastic elastomers.