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Quinine is a promising building block for creating polymer carriers for intracellular nucleic acid delivery. This is due to its ability to bind to genetic material through intercalation and electrostatic interactions and the balance of hydrophobicity and hydrophilicity dependent on the pH/charge state. Yet, studies utilizing cinchona alkaloid natural products in gene delivery are limited. Herein, we present the incorporation of a quinine functionalized monomer (Q) into block polymer architectures to form self-assembled micelles for highly efficient gene delivery. Q was incorporated into the core and/or the shell of the micelles to introduce the unique advantages of quinine to the system. We found that incorporation of Q into the core of the micelle resulted in acid-induced disassembly of the micelle and a boost in transfection efficiency by promoting endosomal escape. This effect was especially evident in the cancerous cell line, A549, which has a more acidic intracellular environment. Incorporation of Q into the shell of the micelles resulted in intercalative binding to the genetic payload as well as larger micelle-DNA complexes (micelleplexes) from the hydrophobicity of Q in the shell. These factors enable the micelleplexes to be more resistant to serum and have more persistent protein expression post-transfection. Overall, this study is the first to demonstrate the benefits of including quinine functionalities into self-assembled micelles for highly efficient gene delivery and presents a platform for inclusion of other natural products with similar properties into micellar systems.

Pressure-sensitive adhesives occupy a large role in the commercial use of polymers; however, they are typically limited to nondegradable formulations using petroleum-based materials. As the plastic and environmental crises have intensified, the need for renewable starting materials and degradable designs has similarly deepened. With that goal, we endeavored to make adhesive films from renewable terpenes as a safer and more sustainable route to PSAs. Specifically, based on our previous report of the cross-linking ability of a carveol-based carbonate through thiol-ene chemistry, we report further exploration of the adhesive possibilities of this system. A carbonate monomer of dimerized carveol was linearly polymerized with dithiothreitol via UV-initiated thiol-ene chemistry and formed into adhesive coatings, with unmodified geraniol doped in as a tackifier. We obtained a range of adhesive properties based on the ratio of exo -methylene to thiol units and reported on the degradation of the adhesive coatings.

Quinine-based polymers have previously demonstrated promising performance in delivering pDNA in cells owing to their electrostatic as well as the nonelectrostatic interactions with pDNA. Herein, we evaluate whether quinine-based polymers are versatile for delivery of mRNA and Cas9-sgRNA complexes, especially in a serum-rich environment. Both mRNA and the Cas9-sgRNA complex are potent therapeutics that are structurally, chemically, and functionally very different from pDNA. By exploring a family of 7 quinine-based polymers that vary in monomer structure and polymer composition, we tested numerous formulations (42 with pDNA, 96 with mRNA, and 48 with Cas9-sgRNA) for payload-polymer complexation and delivery to compare payload-dependent structure-activity relationships. Several formulations demonstrated performance comparable to or better than the commercially available transfection agent jetPEI. The results of this study demonstrate the potential of quinine-based as a versatile carrier platform for delivering a wide range of nucleic acid therapeutics and serving the drug delivery needs in the field genetic medicine.

Cationic polymers offer an alternative to viral vectors in nucleic acid delivery. However, the development of polymer vehicles capable of high transfection efficiency and minimal toxicity has remained elusive, and continued exploration of the vast design space is required. Traditional single polymer syntheses with large monomer bases are very time-intensive, limiting the speed at which new formulations are identified. In this work, we present an experimental method for the quick probing of the design space, utilizing a combinatorial set of 90 polymer blends, derived from 6 statistical copolymers, to deliver pDNA. This workflow facilitated rapid screening of polyplex compositions, successfully tailoring polyplex hydrophobicity, particle size, and payload binding affinity. This workflow identified blended polyplexes with high levels of transfection efficiency and cell viability relative to single copolymer controls and commercial JetPEI, indicating synergistic benefits from copolymer blending. Polyplex composition was coupled with biological outputs to guide the synthesis of single terpolymer vehicles, with high-performing polymers P10 and M20, providing superior transfection of HEK293T cells in serum-free and serum-containing media, respectively. Machine learning coupled with SHapley Additive exPlanations (SHAP) was used to identify polymer/polyplex attributes that most impact transfection efficiency, viability, and overall effective efficiency. Subsequent transfections on ARPE-19 and HDFn cells found that P10 and M20 were surpassed in performance by M10, contrasting with results in HEK293T cells. This cell type dependency reinforced the need to evaluate transfection conditions with multiple cell models to potentially identify moieties more beneficial to delivery in certain tissues. Overall, the workflow employed can be used to expedite the exploration of the polymer design space, bypassing extensive synthesis, and to develop improved polymer delivery vehicles more readily for nucleic acid therapies.

Virus detection is highly important; the last several years, since the onset of the SARS-CoV-2 pandemic, have highlighted a weakness in the field: the need for highly specialized and complex methodology for sensitive virus detection, which also manifests as sacrifices in limits of detection made to achieve simple and rapid sensing. Surface-enhanced Raman spectroscopy (SERS) has the potential to fill this gap, and two novel approaches to the development of a detection scheme are presented in this study. First, the physical entrapment of vesicular stomatitis virus (VSV) and additional virus-like particles through substrate design to localize virus analytes into SERS hotspots is explored. Then, the use of nonspecific linear polymers as affinity agents to facilitate polymer-enabled capture of the VSV for SERS detection is studied. Quantitative detection of the VSV is achieved down to 10 1 genetic copies per milliliter with an R 2 of 0.987 using the optimized physical entrapment method. Physical entrapment of two more virus-like particles is demonstrated with electron microscopy, and distinctive SERS fingerprints are shown. This study shows great promise for the further exploration of label-free virus detection methods involving thoughtful substrate design and unconventional affinity agents.

Thermoset networks are chemically cross-linked materials that exhibit high heat resistance and mechanical strength; however, the permanently cross-linked system makes end-of-life degradation difficult. Thermosets that are inherently degradable and made from renewably derived starting materials are an underexplored area in sustainable polymer chemistry. Here, we report the synthesis of novel sugar- and terpene-based monomers as the enes in thiol-ene network formation. The resulting networks showed varied mechanical properties depending on the thiol used during cross-linking, ranging from strain-at-breaks of 12 to 200%. Networks with carveol or an isosorbide-based thiol incorporated showed plastic deformation under tensile stress testing, while geraniol-containing networks demonstrated linear stress-strain behavior. The storage modulus at the rubbery plateau was highly dependent on the thiol cross-linker, showing an order of magnitude difference between commercial PETMP, DTT, and synthesized Iso2MC. Thermal degradation temperatures were low for the networks, primarily below 200 °C, and the T g values ranged from -17 to 31 °C. Networks were rapidly degraded under basic conditions, showing complete degradation after 2 days for nearly all synthesized thermosets. This library demonstrates the range of thermal and mechanical properties that can be targeted using monomers from sugars and terpenes and expands the field of renewably derived and degradable thermoset network materials.

We present the facile synthesis of a clickable polymer library with systematic variations in length, binary composition, p K a , and hydrophobicity (clog  P ) to optimize intracellular pDNA and CRISPR-Cas9 ribonucleoprotein (RNP) performance. We couple physicochemical characterization and machine learning to interpret quantitative structure-property relationships within the combinatorial design space. For the first time, we reveal unexpected disparate design parameters for nucleic acid carriers; via explainable machine learning on 432 formulations, we discover that lower polymer p K a and higher percentages of benzimidazole ethanethiol enhance pDNA delivery, yet polymer length and captamine cation identity improve RNP delivery. Closed-loop Bayesian optimization of 552 formulation ratios further enhances in vitro performance. The top three polymers yield a higher signal and stable transgene expression over 20 days in vivo , and a 1.7-fold enhancement over controls. Our facile coupling of synthesis, characterization, and machine analysis provides powerful tools to quantitate performance parameters accelerating next-generation vehicles for nucleic acid medicines., Competing Interests: The authors declare the following competing financial interest(s): Theresa M. Reineke is one of the founders of Nanite, Inc. and has an equity interest. Nanite, Inc. is one of the sponsors of this research. This interest has been reviewed and managed by the University of Minnesota in accordance with its conflict-of-interest policy. Felipe Oviedo consults for Nanite, Inc. and has an equity interest., (This journal is © The Royal Society of Chemistry.)

Nanodroplet formation is important to achieve supersaturation of active pharmaceutical ingredients (APIs) in an amorphous solid dispersion. The aim of the current study was to explore how polymer composition, architecture, molar mass, and surfactant concentration affect polymer-drug nanodroplet morphology with the breast cancer API, GDC-0810. The impact of nanodroplet size and morphology on dissolution efficacy and drug loading capacity was explored using polarized light microscopy, dynamic light scattering, and cryogenic transmission electron microscopy. Poly( N -isopropylacrylamide- stat - N,N -dimethylacrylamide) (PND) was synthesized as two linear derivatives and two bottlebrush derivatives with carboxylated or PEGylated end-groups. Hydroxypropyl methylcellulose acetate succinate grade MF (HPMCAS-MF) and poly(vinylpyrrolidone- co -vinyl acetate) (PVPVA) were included as commercial polymer controls. We report the first copolymerization synthesis of a PVPVA bottlebrush copolymer, which was the highest performing excipient in this study, maintaining 688 μg/mL GDC-0810 concentration at 60 wt % drug loading. This is likely due to strong polymer-drug noncovalent interactions and the compaction of GDC-0810 along the PVPVA bottlebrush backbone. Overall, it was observed that the most effective formulations had a hydrodynamic radius less than 25 nm with tightly compacted nanodroplet morphologies.

Bicontinuous thermotropic liquid crystal (LC) materials, e.g., double gyroid (DG) phases, have garnered significant attention due to the potential utility of their 3D network structures in wide-ranging applications. However, the utility of these materials is significantly constrained by the lack of robust molecular design rules for shape-filling amphiphiles that spontaneously adopt the saddle curvatures required to access these useful supramolecular assemblies. Toward this aim, we synthesized anomerically pure Guerbet-type glycolipids bearing cellobiose head groups and branched alkyl tails and studied their thermotropic LC self-assembly. Using a combination of differential scanning calorimetry, polarized optical microscopy, and small-angle X-ray scattering, our studies demonstrate that Guerbet cellobiosides exhibit a strong propensity to self-assemble into DG morphologies over wide thermotropic phase windows. The stabilities of these assemblies sensitively depend on the branched alkyl tail structure and the anomeric configuration of the glycolipid in a previously unrecognized manner. Complementary molecular simulations furnish detailed insights into the observed self-assembly characteristics, thus unveiling molecular motifs that foster network phase self-assembly that will enable future designs and applications of network LC materials.

Intervertebral disc (IVD) degeneration is a common pathological condition associated with low back pain. Recent evidence suggests that mesenchymal signaling cells (MSCs) promote IVD regeneration, but underlying mechanisms remain poorly defined. One postulated mechanism is via modulation of macrophage phenotypes. In this manuscript, we tested the hypothesis that MSCs produce trophic factors that alter macrophage subsets. To this end, we collected conditioned medium from human, bone marrow-derived STRO3 + MSCs. We then cultured human bone marrow-derived macrophages in MSC conditioned medium (CM) and performed single cell RNA-sequencing. Comparative analyses between macrophages cultured in hypoxic and normoxic MSC CM showed large overlap between macrophage subsets; however, we identified a unique hypoxic MSC CM-induced macrophage cluster. To determine if factors from MSC CM simulated effects of the anti-inflammatory cytokine IL-4, we integrated the data from macrophages cultured in hypoxic MSC CM with and without IL-4 addition. Integration of these data sets showed considerable overlap, demonstrating that hypoxic MSC CM simulates the effects of IL-4. Interestingly, macrophages cultured in normoxic MSC CM in the absence of IL-4 did not significantly contribute to the unique cluster within our comparison analyses and showed differential TGF-β signaling; thus, normoxic conditions did not approximate IL-4. In addition, TGF-β neutralization partially limited the effects of MSC CM. In conclusion, our study identified a unique macrophage subset induced by MSCs within hypoxic conditions and supports that MSCs alter macrophage phenotypes through TGF-β-dependent mechanisms., Competing Interests: Authors BR and TB were employed by Medtronic. Authors KC and NS were employed by Medtronic. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Koroth, Chitwood, Kumar, Lin, Reves, Boyce, Reineke, Ellingson, Johnson, Stone, Chaffin, Simha, Ogle and Bradley.)

Nucleic acid delivery with cationic polymers is a promising alternative to expensive viral-based methods; however, it often suffers from a lower performance. Herein, we present a highly efficient delivery system based on cinchona alkaloid natural products copolymerized with 2-hydroxyethyl acrylate. Cinchona alkaloids are an attractive monomer class for gene delivery applications, given their ability to bind to DNA via both electrostatics and intercalation. To uncover the structure-activity profile of the system, four structurally similar cinchona alkaloids were incorporated into polymers: quinine, quinidine, cinchonine, and cinchonidine. These polymers differed in the chain length, the presence or absence of a pendant methoxy group, and stereochemistry, all of which were found to alter gene delivery performance and the ways in which the polymers overcome biological barriers to transfection. Longer polymers that contained the methoxy-bearing cinchona alkaloids (i.e., quinine and quinidine) were found to have the best performance. These polymers exhibited the tightest DNA binding, largest and most abundant DNA-polymer complexes, and best endosomal escape thanks to their increased buffering capacity and closest nuclear proximity of the payload. Overall, this work highlights the remarkable efficiency of polymer systems that incorporate cinchona alkaloid natural products while demonstrating the profound impact that small structural changes can have on overcoming biological hurdles associated with gene delivery.

The bacterial cell envelope provides a protective barrier that is challenging for small molecules and biomolecules to cross. Given the anionic nature of both Gram-positive and Gram-negative bacterial cell envelopes, negatively charged molecules are particularly difficult to deliver into these organisms. Many strategies have been employed to penetrate bacteria, ranging from reagents such as cell-penetrating peptides, enzymes, and metal-chelating compounds to physical perturbations. While cationic polymers are known antimicrobial agents, polymers that promote the permeabilization of bacterial cells without causing high levels of toxicity and cell lysis have not yet been described. Here, we investigate four polymers that display a cationic poly(2-(dimethylamino)ethyl methacrylate ( D ) block for the internalization of an anionic adenosine triphosphate (ATP)-based chemical probe into Escherichia coli and Bacillus subtilis . We evaluated two polymer architectures, linear and micellar, to determine how shape and hydrophobicity affect internalization efficiency. We found that, in addition to these reagents successfully promoting probe internalization, the probe-labeled cells were able to continue to grow and divide. The micellar structures in particular were highly effective for the delivery of the negatively charged chemical probe. Finally, we demonstrated that these cationic polymers could act as general permeabilization reagents, promoting the entry of other molecules, such as antibiotics.

Nucleic acid-based medicines and vaccines are becoming an important part of our therapeutic toolbox. One key genetic medicine is antisense oligonucleotides (ASOs), which are short single-stranded nucleic acids that downregulate protein production by binding to mRNA. However, ASOs cannot enter the cell without a delivery vehicle. Diblock polymers containing cationic and hydrophobic blocks self-assemble into micelles that have shown improved delivery compared to linear nonmicelle variants. Yet synthetic and characterization bottlenecks have hindered rapid screening and optimization. In this study, we aim to develop a method to increase throughput and discovery of new micelle systems by mixing diblock polymers together to rapidly form new micelle formulations. We synthesized diblocks containing an n -butyl acrylate block chain extended with cationic moieties amino ethyl acrylamide (A), dimethyl amino ethyl acrylamide (D), or morpholino ethyl acrylamide (M). These diblocks were then self-assembled into homomicelles (A100, D100, and M100)), mixed micelles comprising 2 homomicelles (MixR%+R'%), and blended diblock micelles comprising 2 diblocks blended into one micelle (BldR%R'%) and tested for ASO delivery. Interestingly, we observed that mixing or blending M with A (BldA50M50 and MixA50+M50) did not improve transfection efficiency compared to A100; however, when M was mixed with D, there was a significant increase in transfection efficacy for the mixed micelle MixD50+M50 compared to D100. We further examined mixed and blended D systems at different ratios. We observed a large increase in transfection and minimal change in toxicity when M was mixed with D at a low percentage of D incorporation in mixed diblock micelles (i.e., BldD20M80) compared to D100 and MixD20+M80. To understand the cellular mechanisms that may result in these differences, we added proton pump inhibitor Bafilomycin-A1 (Baf-A1) to the transfection experiments. Formulations that contain D decreased in performance in the presence of Baf-A1, indicating that micelles with D rely on the proton sponge effect for endosomal escape more than micelles with A. This result supports our conclusion that M is able to modulate transfection of D, but not with A. This research shows that polymer blending in a manner similar to that of lipids can significantly boost transfection efficiency and is a facile way to increase throughput of testing, optimization, and successful formulation identification for polymeric nucleic acid delivery systems.

Herein, we examine the ASO-mediated gene silencing efficiency of pH-responsive micelles, by incorporating 2-(diisopropylamino)ethyl methacrylate (DIP) into the micelle core and comparing physical and biological properties with non-pH-responsive micelles. Additionally, the lipophilic effect of the micelle cores was examined in both types of micelles. Varying lipophilicity was achieved by varying alkyl monomer chain lengths─butyl (4), lauryl (12), and stearyl (18) methacrylate. Each of the micelles formed within our family offered the added benefit of well-defined and uniform templates for loading antisense oligonucleotide (ASO) payloads. Overall, the micelles followed previously established trends of outperforming their linear polymer (nonmicelle) analogs and ASO only control. More specifically, the highest performing micelles were the pH-responsive micelles with longer alkyl chains or higher lipophilicity─D-DIP+LMA and D-DIP+SMA (∼90% silencing). These two micelles demonstrated silencing efficiencies similar to Jet-PEI and Lipofectamine 2000 and caused lower toxicity than Lipofectamine 2000. The shortest alkyl chain pH-responsive micelle, D-DIP+BMA (64%), displayed strong gene silencing similar to that about that of its non-pH-responsive micelle, D-BMA (68%), and the pH-responsive micelle without an alkyl chain incorporated, D-DIP (59%). This work illuminates a minimum alkyl chain length dependence to allow gene silencing within our micelle family. However, including only longer alkyl chains into the micelle core without the pH-responsive unit DIP had a hindering effect, thus demonstrating the requirement of the DIP unit when including longer alkyl chain lengths. This work demonstrates the exemplary gene silencing efficiencies of polymeric micelles and uncovers the relationship between pH responsiveness and performance with lipophilic polymer micelles for enhancing ASO-mediated gene silencing.

Simultaneous ring-opening copolymerization is a powerful strategy for the synthesis of highly functional copolymers from different types of cyclic monomers. Although copolymers are essential to the plastics industry, environmental concerns associated with current fossil-fuel-based synthetic polymers have led to an increasing interest in the use of renewable feedstock for polymer synthesis. Herein, we report a scalable synthetic platform to afford unique polysaccharides with different pendant functional groups from biomass-derived levoglucosan and ε-caprolactone via cationic ring-opening copolymerization (cROCOP). Biocompatible and recyclable bismuth triflate was identified as the optimal catalyst for cROCOP of levoglucosan. Copolymers from tribenzyl levoglucosan and ε-caprolactone, as well as from tribenzyl and triallyl levoglucosan, were successfully synthesized. The tribenzyl levoglucosan monomer composition ranged from 16% to 64% in the copolymers with ε-caprolactone and 22% to 79% in the copolymers with triallyl levoglucosan. The allylic levoglucosan copolymer can be utilized as a renewably derived scaffold to modify copolymer properties and create other polymer architectures via postpolymerization modification. Monomer reactivity ratios were determined to investigate the copolymer microstructure, indicating that levoglucosan-based copolymers have a gradient architecture. Additionally, we demonstrated that the copolymer glass transition temperature ( T g , ranging from -44.3 to 33.8 °C), thermal stability, and crystallization behavior could be tuned based on the copolymer composition. Overall, this work underscores the utility of levoglucosan as a bioderived feedstock for the development of functional sugar-based copolymers with applications ranging from sustainable materials to biomaterials.

Quinine is a promising natural product building block for polymer-based nucleic acid delivery vehicles as its structure enables DNA binding through both intercalation and electrostatic interactions. However, studies exploring the potential of quinine-based polymers for nucleic acid delivery applications (transfection) are limited. In this work, we used a hydroquinine-functionalized monomer, HQ, with 2-hydroxyethyl acrylate to create a family of seven polymers (HQ-X, X = mole percentage of HQ), with mole percentages of HQ ranging from 12 to 100%. We developed a flow cytometer-based assay for studying the polymer-pDNA complexes (polyplex particles) directly and demonstrate that polymer composition and monomer structure influence polyplex characteristics such as the pDNA loading and the extent of adsorption of serum proteins on polyplex particles. Biological delivery experiments revealed that maximum transgene expression, outperforming commercial controls, was achieved with HQ-25 and HQ-35 as these two variants sustained gene expression over 96 h. HQ-44, HQ-60, and HQ-100 were not successful in inducing transgene expression, despite being able to deliver pDNA into the cells, highlighting that the release of pDNA is likely the bottleneck in transfection for polymers with higher HQ content. Using confocal imaging, we quantified the extent of colocalization between pDNA and lysosomes, proving the remarkable endosomal escape capabilities of the HQ-X polymers. Overall, this study demonstrates the advantages of HQ-X polymers as well as provides guiding principles for improving the monomer structure and polymer composition, supporting the development of the next generation of polymer-based nucleic acid delivery vehicles harnessing the power of natural products., Competing Interests: The authors declare no competing financial interest., (© 2023 The Authors. Published by American Chemical Society.)

We present the synthesis, characterization and radical ring-opening polymerization (rROP) capabilities of thionoisochromanone (TIC), a fungi-derivable thionolactone. TIC is the first reported six-membered thionolactone to readily homopolymerize under free radical conditions without the presence of a dormant comonomer or repeated initiation. Even more, the resulting polymer is fully degradable under mild, basic conditions. Computations providing molecular-level insights into the mechanistic and energetic details of polymerization identified a unique S , S , O -orthoester intermediate that leads to a sustained chain-end. This sustained chain-end allowed for the synthesis of a block copolymer of TIC and styrene under entirely free radical conditions without explicit radical control methods such as reversible addition-fragmentation chain transfer polymerization (RAFT). We also report the statistical copolymerization of ring-retained TIC and styrene, confirmed by elemental analysis and energy-dispersive X-ray spectroscopy (EDX). Computations into the energetic details of copolymerization indicate kinetic drivers for ring-retaining behavior. This work provides the first example of a sustainable feedstock for rROP and provides the field with the first six-membered monomer susceptible to rROP, expanding the monomer scope to aid our fundamental understanding of thionolactone rROP behavior., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

The presence of a nearby tethered functional group (G, G = tertiary amide or amine) can significantly impact the rate of cleavage of an Si-O bond. We report here an in situ 1 H NMR spectroscopic investigation of the relative rates of cleavage of model substrates containing two different Si-O substructures, namely alkoxydisiloxanes [GRO-Si(Me 2 )-O-SiMe 3 ] and carbodisiloxanes [GR-Si(Me 2 )-O-SiMe 3 ]. The trends in the relative rates (which slowed with increasing chain length, with a notable exception) of alkoxydisiloxane hydrolyses were probed via computation. The results correlated well with the experimental data. In contrast to the hydrolysis of the alkoxydisiloxanes, the carbodisiloxanes were not fully hydrolyzed, but rather formed an equilibrium mixture of starting asymmetric disiloxane, two silanols, and a new symmetrical disiloxane. We also uncovered a facile siloxy-metathesis reaction of an incoming silanol with the carbodisiloxane substrate [e.g., Me 2 NR-Si(Me 2 )-O-SiMe 3 + HOSiEt 3 ⇋ Me 2 NR-Si(Me 2 )-O-SiEt 3 + HOSiMe 3 ] facilitated by the pendant dimethylamino group, a process that was also probed by computation.

A cationic unimolecular bottlebrush polymer with chemically modified end-groups was synthesized to understand the impact of hydrophilicity on colloidal stability, nucleic acid delivery performance, and toxicity. The bottlebrush polymer template was synthesized using grafting-through techniques and was therefore composed of a polynorbornene backbone with poly(2-(dimethylamino)ethyl methacrylate) side chains with dodecyl trithiocarbonate end-groups. Postpolymerization modification was performed to fully remove the end-groups or install hydroxy and methoxy poly(ethylene glycol) functional groups on the bottlebrush exterior. The bottlebrush family was preformulated with biological payloads of pDNA and CRISPR-Cas9 RNP in both water and PBS to understand binding, aggregation kinetics, cytotoxicity, and delivery efficacy. Increasing end-group hydrophilicity and preformulation of bottleplexes in PBS increased colloidal stability and cellular viability; however, this did not always result in increased transfection efficiency. The bottlebrush family exemplifies how formulation conditions, polymer loading, and end-group functionality of bottlebrushes can be tuned to balance expression with cytotoxicity ratios and result in enhanced overall performance.

Antisense oligonucleotides (ASOs) are an important emerging therapeutic; however, they struggle to enter cells without a delivery vehicle, such as a cationic polymer. To understand the role of polymer architecture for ASO delivery, five linear polymers and five diblock polymers (capable of self-assembly into micelles) were synthesized with varying cationic groups. After complexation of each polymer/micelle with ASO, it was found that less bulky cationic moieties transfected the ASO more effectively. Interestingly, however the ASO internalization trend was the opposite of the transfection trend for cationic moiety, indicating internalization is not the major factor in determining transfection efficiency for this series. Micelleplexes (micelle-ASO complexes) generally enable higher transfection efficacy as compared to polyplexes (linear polymer-ASO complexes). Additionally, the order of addition of cells and complexes was explored. Linear polyplexes showed better transfection efficiency in adhered cells, whereas micelleplexes delivered the ASO more efficiently when the cells and micelleplexes were added simultaneously. This phenomenon may be due to increased cell-complex interactions as micelleplexes have increased colloidal stability compared to polyplexes. These findings emphasize the importance of polymer composition and architecture in governing the cellular interactions necessary for transfection, thus allowing advancement in the design principles for nonviral nucleic acid delivery formulations.

Herein, we examine the complexation and biological delivery of a short single-stranded antisense oligonucleotide (ASO) payload with four polymer derivatives that form two architectural variants (polyplexes and micelleplexes): a homopolymer poly(2-dimethylaminoethyl methacrylate) (D), a diblock polymer poly(ethylene glycol)methylether methacrylate- block -poly(2-dimethylaminoethyl methacrylate) (O b D), and two micelle-forming variants, poly(2-dimethylaminoethyl methacrylate)- block -poly( n -butyl methacrylate) (DB) and poly(ethylene glycol)methylether methacrylate- block -poly(2-dimethylaminoethyl methacrylate)- block -poly( n -butyl methacrylate) (O b DB). Both polyplexes and micelleplexes complexed ASOs, and the incorporation of an O b brush enhances colloidal stability. Micellplexes are templated by the size and shape of the unloaded micelle and that micelle-ASO complexation is not sensitive to formulation/mixing order, allowing ease, versatility, and reproducibility in packaging short oligonucleotides. The DB micelleplexes promoted the largest gene silencing, internalization, and tolerable toxicity while the O b DB micelleplexes displayed enhanced colloidal stability and highly efficient payload trafficking despite having lower cellular uptake. Overall, this work demonstrates that cationic micelles are superior delivery vehicles for ASOs denoting the importance of vehicle architecture in biological performance.

Polymer-based gene delivery relies on the binding, protection, and final release of nucleic acid cargo using polycations. Engineering polymeric vectors, by exploring novel topologies and cationic moieties, is a promising avenue to improve their performance, which hinges on the development of simple synthetic methods that allow facile preparation. In this work, we focus on cationic micelles formed from block polymers, which are examined as promising gene compaction agents and carriers. In this study, we report the synthesis and assembly of six amphiphilic poly( n -butyl acrylate)- b -poly(cationic acrylamide) diblock polymers with different types of cationic groups ((dialkyl)amine, morpholine, or imidazole) in their hydrophilic corona. The polycations were obtained through the parallel postpolymerization modification of a poly( n -butyl acrylate)- b -poly(pentafluorophenyl acrylate) reactive scaffold, which granted diblock polymers with equivalent degrees of polymerization and subsequent quantitative functionalization with cations of different p K a . Ultrasound-assisted direct dissolution of the polycations in different aqueous buffers (pH = 1-7) afforded micellar structures with low size dispersities and hydrodynamic radii below 100 nm. The formation and properties of micelle-DNA complexes ("micelleplexes") were explored via DLS, zeta potential, and dye-exclusion assays revealing that binding is influenced by the cation type present in the micelle corona where bulkiness and p K a are the drivers of micelleplex formation. Combining parallel synthesis strategies with simple direct dissolution formulation opens opportunities to optimize and expand the range of micelle delivery vehicles available by facile tuning of the composition of the cationic micelle corona.

Transforming how plastics are made, unmade, and remade through innovative research and diverse partnerships that together foster environmental stewardship is critically important to a sustainable future. Designing, preparing, and implementing polymers derived from renewable resources for a wide range of advanced applications that promote future economic development, energy efficiency, and environmental sustainability are all central to these efforts. In this Chemical Reviews contribution, we take a comprehensive, integrated approach to summarize important and impactful contributions to this broad research arena. The Review highlights signature accomplishments across a broad research portfolio and is organized into four wide-ranging research themes that address the topic in a comprehensive manner: Feedstocks, Polymerization Processes and Techniques, Intended Use, and End of Use. We emphasize those successes that benefitted from collaborative engagements across disciplinary lines.

We report the facile synthesis and characterization of 1,6-α linked functional stereoregular polysaccharides from biomass-derived levoglucosan via cationic ring-opening polymerization (cROP). Levoglucosan is a bicyclic acetal with rich hydroxyl functionality, which can be synthetically modified to install a variety of pendant groups for tailored properties. We have employed biocompatible and recyclable metal triflate catalysts - scandium and bismuth triflate - for green cROP of levoglucosan derivatives, even at very low catalyst loadings of 0.5 mol%. Combined experimental and computational studies provided key kinetic, thermodynamic, and mechanistic insights into the cROP of these derivatives with metal triflates. Computational studies reveal that ring-opening of levoglucosan derivatives is preferred at the 1,6 anhydro linkage and cROP proceeds in a regio- and stereo-specific manner to form 1,6-α glycosidic linkages. DFT calculations also show that biocompatible metal triflates efficiently coordinate with levoglucosan derivatives as compared to the highly toxic PF 5 used previously. Post-polymerization modification of levoglucosan-based polysaccharides is readily performed via UV-initiated thiol-ene click reactions. The reported levoglucosan based polymers exhibit good thermal stability ( T d > 250 °C) and a wide glass transition temperature ( T g ) window (<-150 °C to 32 °C) that is accessible with thioglycerol and lauryl mercaptan pendant groups. This work demonstrates the utility of levoglucosan as a renewably-derived scaffold, enabling facile access to tailored polysaccharides that could be important in many applications ranging from sustainable materials to biologically active polymers., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

The development of polymers that can replace engineered viral vectors in clinical gene therapy has proven elusive despite the vast portfolios of multifunctional polymers generated by advances in polymer synthesis. Functional delivery of payloads such as plasmids (pDNA) and ribonucleoproteins (RNP) to various cellular populations and tissue types requires design precision. Herein, we systematically screen a combinatorially designed library of 43 well-defined polymers, ultimately identifying a lead polycationic vehicle (P38) for efficient pDNA delivery. Further, we demonstrate the versatility of P38 in codelivering spCas9 RNP and pDNA payloads to mediate homology-directed repair as well as in facilitating efficient pDNA delivery in ARPE-19 cells. P38 achieves nuclear import of pDNA and eludes lysosomal processing far more effectively than a structural analogue that does not deliver pDNA as efficiently. To reveal the physicochemical drivers of P38's gene delivery performance, SHapley Additive exPlanations (SHAP) are computed for nine polyplex features, and a causal model is applied to evaluate the average treatment effect of the most important features selected by SHAP. Our machine learning interpretability and causal inference approach derives structure-function relationships underlying delivery efficiency, polyplex uptake, and cellular viability and probes the overlap in polymer design criteria between RNP and pDNA payloads. Together, combinatorial polymer synthesis, parallelized biological screening, and machine learning establish that pDNA delivery demands careful tuning of polycation protonation equilibria while RNP payloads are delivered most efficaciously by polymers that deprotonate cooperatively via hydrophobic interactions. These payload-specific design guidelines will inform further design of bespoke polymers for specific therapeutic contexts., Competing Interests: The authors declare the following competing financial interest(s): Theresa M. Reineke is one of the founders of Nanite, Inc. and has an equity interest. Nanite, Inc. is one of the sponsors of this research. This interest has been reviewed and managed by the University of Minnesota in accordance with its conflict-of-interest policy. Felipe Oviedo consults for Nanite, Inc. and has an equity interest., (© 2022 The Authors. Published by American Chemical Society.)

The advent of genome editing has transformed the therapeutic landscape for several debilitating diseases, and the clinical outlook for gene therapeutics has never been more promising. The therapeutic potential of nucleic acids has been limited by a reliance on engineered viral vectors for delivery. Chemically defined polymers can remediate technological, regulatory, and clinical challenges associated with viral modes of gene delivery. Because of their scalability, versatility, and exquisite tunability, polymers are ideal biomaterial platforms for delivering nucleic acid payloads efficiently while minimizing immune response and cellular toxicity. While polymeric gene delivery has progressed significantly in the past four decades, clinical translation of polymeric vehicles faces several formidable challenges. The aim of our Account is to illustrate diverse concepts in designing polymeric vectors towards meeting therapeutic goals of in vivo and ex vivo gene therapy. Here, we highlight several classes of polymers employed in gene delivery and summarize the recent work on understanding the contributions of chemical and architectural design parameters. We touch upon characterization methods used to visualize and understand events transpiring at the interfaces between polymer, nucleic acids, and the physiological environment. We conclude that interdisciplinary approaches and methodologies motivated by fundamental questions are key to designing high-performing polymeric vehicles for gene therapy.

The incorporation of renewable feedstocks into polymer backbones is of great importance in modern polymer science. We report the synthesis of 1,3-diyne polymers derived from the bispropargyl ethers of isosorbide, isomannide, and isoidide. The dialkyne monomers can be polymerized through an adaptation of the Glaser-Hay coupling using a nickel(II) cocatalyst. These well-defined diyne polymers bear an iodoalkyne end group, afforded through an unanticipated reductive elimination pathway, and display glass transition temperatures ( T g ) from 55 to 64 °C. Fully saturated, analogous polyethers can be prepared from the hydrogenation of the diyne polymers, and these show T g values between -10 and -2 °C. Both the 1,3-diyne polymers and the saturated analogues display similar trends in their T g values vis-à-vis the stereochemical features of the isohexide unit within the backbone. This polymerization provided access to two series of isohexide-based polyethers, the thermal properties of which are influenced by the nature of the 2,4-hexadiynyl and hexamethylene linkers as well as the relative configuration of the bicyclic subunit in the backbone. The reported method represents an important step toward accessing well-defined polyethers from renewable feedstocks using readily available catalysts and convenient ambient conditions.

Cationic polymer vehicles have emerged as promising platforms for nucleic acid delivery because of their scalability, biocompatibility, and chemical versatility. Advancements in synthetic polymer chemistry allow us to precisely tune chemical functionality with various macromolecular architectures to increase the efficacy of nonviral-based gene delivery. Herein, we demonstrate the first cationic bottlebrush polymer-mediated pDNA delivery by comparing unimolecular, synthetically defined bottlebrush polymers to their linear building blocks. We successfully synthesized poly(2-(dimethylamino)ethyl methacrylate) (pDMAEMA) bottlebrushes through ring-opening metathesis polymerization to afford four bottlebrush polymers with systematic increases in backbone degree of polymerization ( N bb = 13, 20, 26, and 37), while keeping the side-chain degree of polymerization constant ( N sc = 57). Physical and chemical properties were characterized, and subsequently, the toxicity and delivery efficiency of pDNA into HEK293 cells were evaluated. The bottlebrush-pDNA complex (bottleplex) with the highest N bb , BB&#95;37, displayed up to a 60-fold increase in %EGFP+ cells in comparison to linear macromonomer. Additionally, we observed a trend of increasing EGFP expression with increasing polymer molecular weight. Bottleplexes and polyplexes both displayed high pDNA internalization as measured via payload enumeration per cell; however, quantitative confocal analysis revealed that bottlebrushes were able to shuttle pDNA into and around the nucleus more successfully than pDNA delivered via linear analogues. Overall, a canonical cationic monomer, such as DMAEMA, synthesized in the form of cationic bottlebrush polymers proved to be far more efficient in functional pDNA delivery and expression than linear pDMAEMA. This work underscores the importance of architectural modifications and the potential of bottlebrushes to serve as effective biomacromolecule delivery vehicles.

Two renewable, structurally analogous monomers, isosorbide undecenoate (IU) and glucarodilactone undecenoate (GDLU) reacted with pentaerythritol tetrakis(3-mercaptopropionate) (PETT) via thiol-ene photopolymerization to form IU-PETT and GDLU-PETT thermosets. Despite their chemical similarity, uniaxial tensile testing showed that GDLU-PETT exhibited a strain-hardening behavior and is significantly tougher than IU-PETT. To understand this observation, in situ tensile testing and wide-angle X-ray scattering experiments (WAXS) were conducted. While the 2D WAXS patterns of IU-PETT displayed an isotropic halo during uniaxial deformation, they exhibited a change from an isotropic halo to a pair of scattering arcs for the GDLU-PETT samples. Density functional theory calculations further revealed that the GDLU alkyl chains are less angled than the IU alkyl chains. Based on these results, we postulate that the GDLU molecules can more easily order and align during uniaxial deformation, hence increasing intermolecular interactions between the GDLU molecules and contributing to the observed strain hardening behavior of their thermosets. This study exemplifies how molecules with subtle differences in their chemical structures can alter the structures and thermophysical properties of the resulting polymers in unpredictable ways.

Bottlebrush polymers have great potential as vehicles to noncovalently sequester, stabilize, and deliver hydrophobic small molecule actives. To this end, we synthesized a poly( N -isopropylacrylamide- stat - N , N -dimethylacrylamide) bottlebrush copolymer using ring-opening metathesis polymerization and developed a facile method to control the thermoresponsive properties using postpolymerization modification. Six increasingly hydrophilic end-groups were installed, yielding cloud point temperature control over a range of 22-42 °C. Solubility enhancement of the antiseizure medication, phenytoin, increased significantly with the hydrophilicity of the end-group moiety. Notably, carboxylated bottlebrush copolymers solubilized formulations with higher drug loadings than linear copolymers because they exist as unimolecular nanoparticles with a synthetically defined density of polymer chains that are more stable in solution. This work provides the first investigation of bottlebrush polymers for hydrophobic noncovalent sequestration and solubilization of pharmaceuticals.

We report the rapid, one-pot synthesis of functional polycarbonates derived from renewable alcohols (i.e., glucose tetraacetate, acetyl isosorbide, lauryl alcohol, and ethanol) and a cyclic carbonate bearing an imidazolecarboxylate. This tandem functionalization/ring-opening polymerization strategy can be performed on multigram scale and eliminates the need for rigorous purification and specialized equipment. A wide range of glass transition temperatures ( T g ) was accessible from these renewable pendant groups (>75 °C T g window). We also synthesized several statistical copolycarbonates to show the thermal properties can be tailored with this tandem method. Additionally, we demonstrate a circular polymer economy via chemical recycling to a cyclic carbonate precursor. This work may facilitate development of sustainable polycarbonates with tailored properties that work toward eliminating plastic waste streams.

Polymeric vehicles that efficiently package and controllably release nucleic acids enable the development of safer and more efficacious strategies in genetic and polynucleotide therapies. Developing delivery platforms that endogenously monitor the molecular interactions, which facilitate binding and release of nucleic acids in cells, would aid in the rational design of more effective vectors for clinical applications. Here, we report the facile synthesis of a copolymer containing quinine and 2-hydroxyethyl acrylate that effectively compacts plasmid DNA (pDNA) through electrostatic binding and intercalation. This polymer system poly(quinine- co -HEA) packages pDNA and shows exceptional cellular internalization, transgene expression, and low cytotoxicity compared to commercial controls for several human cell lines, including HeLa, HEK 293T, K562, and keratinocytes (N/TERTs). Using quinine as an endogenous reporter for pDNA intercalation, Raman imaging revealed that proteins inside cells facilitate the unpackaging of polymer-DNA complexes (polyplexes) and the release of their cargo. Our work showcases the ability of this quinine copolymer reporter to not only facilitate effective gene delivery but also enable diagnostic monitoring of polymer-pDNA binding interactions on the molecular scale via Raman imaging. The use of Raman chemical imaging in the field of gene delivery yields unprecedented insight into the unpackaging behavior of polyplexes in cells and provides a methodology to assess and design more efficient delivery vehicles for gene-based therapies., Competing Interests: Competing interest statement: A US Provisional Application (62/536,427) on this work has been filed., (Copyright © 2020 the Author(s). Published by PNAS.)

Chemically defined vectors such as cationic polymers are versatile alternatives to engineered viruses for the delivery of genome-editing payloads. However, their clinical translation hinges on rapidly exploring vast chemical design spaces and deriving structure-function relationships governing delivery performance. Here, we discovered a polymer for efficient intracellular ribonucleoprotein (RNP) delivery through combinatorial polymer design and parallelized experimental workflows. A chemically diverse library of 43 statistical copolymers was synthesized via combinatorial RAFT polymerization, realizing systematic variations in physicochemical properties. We selected cationic monomers that varied in their p K a values (8.1-9.2), steric bulk, and lipophilicity of their alkyl substituents. Co-monomers of varying hydrophilicity were also incorporated, enabling elucidation of the roles of protonation equilibria and hydrophobic-hydrophilic balance in vehicular properties and performance. We screened our multiparametric vector library through image cytometry and rapidly uncovered a hit polymer (P38), which outperforms state-of-the-art commercial transfection reagents, achieving nearly 60% editing efficiency via nonhomologous end-joining. Structure-function correlations underlying editing efficiency, cellular toxicity, and RNP uptake were probed through machine learning approaches to uncover the physicochemical basis of P38's performance. Although cellular toxicity and RNP uptake were solely determined by polyplex size distribution and protonation degree, respectively, these two polyplex design parameters were found to be inconsequential for enhancing editing efficiency. Instead, polymer hydrophobicity and the Hill coefficient, a parameter describing cooperativity-enhanced polymer deprotonation, were identified as the critical determinants of editing efficiency. Combinatorial synthesis and high-throughput characterization methodologies coupled with data science approaches enabled the rapid discovery of a polymeric vehicle that would have otherwise remained inaccessible to chemical intuition. The statistically derived design rules elucidated herein will guide the synthesis and optimization of future polymer libraries tailored for therapeutic applications of RNP-based genome editing.

A linear, methacrylamide polymer affinity agent was explored to capture two mycotoxins, deoxynivalenol (DON) and ochratoxin A (OTA), for multiplex surface-enhanced Raman scattering (SERS) detection. These mycotoxins are naturally occurring small molecules from fungi that can be dangerous at low concentrations. SERS detection was completed for each polymer-toxin complex at concentrations relevant to current safety regulation by the FDA: 1 ppm for DON and 5 ppb for OTA. Visibly distinguishable vibrational modes were observed in the multiplex spectra that were attributed to each mycotoxin individually, thus, not requiring any additional chemometric analysis. Density functional theory (DFT) was used to model DON and OTA to accurately label the vibrational modes in the experimental spectra as well as provide insight on the binding between both targets and the affinity agent. Fully modeled vibrations of these toxins are novel contributions due to OTA never being modeled and only a few published vibrational modes of DON. DFT guides empirical observations regarding hydrogen bonding at multiple sites of each mycotoxin target molecule through the amine groups on the polymer, confirming the capabilities of a single polymer affinity agent to facilitate multiplex detection of a class of molecules through less-specific interactions than traditional affinity agents., Competing Interests: Conflicts of interest The authors have no conflicts to declare.

Recently, protein therapeutics have gained significant attention as a result of their enhanced selectivity and diminished side effects compared to traditional small-molecule drugs. Despite their advantages, protein formulations typically suffer from stability issues because of aggregation and denaturation during production and storage, often resulting in detrimental immune responses. Surfactants can be used to stabilize and protect proteins in solution by preventing protein adsorption onto interfaces or by forming protective structures in solution. Herein, a detailed structure-activity relationship study is described, demonstrating the role that hydrophobic tail length plays in surfactant-mediated stabilization of the model therapeutic protein IgG. The FM1000 series, originating from a surfactant scaffold that allows for easy structure modulation, was synthesized by a simple 2-step procedure. First, phenylalanine was acylated with a variety of acyl chlorides of differing lengths to yield n -acyl phenylalanine, which was then coupled to Jeffamine M1000, a polyethylene glycol-based amine, to yield the final surfactant. With this FM1000 series, it was observed that the 14 carbon-long tail surfactant (14FM1000) was optimal at preventing IgG aggregation compared to surfactants with tails that were longer or shorter. Using a combination of dynamic surface tensiometry and quartz crystal microbalance with dissipation, it was hypothesized that 14FM1000 was able to prevent IgG adsorption, and therefore aggregation, by adsorbing appreciably onto surfaces quickly. 14FM1000 had the fastest rate of initial adsorption compared to the other surfactants studied. Short-tail surfactants were slow to and did not adsorb appreciably onto surfaces, allowing IgG adsorption. Although long-tail surfactants were also slow to adsorb, allowing IgG to adsorb and aggregate, their equilibrium adsorption was strong. Additionally, 14FM1000 was the most reversibly adsorbed surfactant, likely improving its ability to desorb and adsorb quickly to transient surfaces, therefore protecting the IgG at each new hydrophobic surface and preventing aggregation. By understanding the structure-activity relationship between surfactants and protein stabilization, we move toward more efficient design of future surfactants increasing the stability and utility of important protein therapeutics.

Using molecular dynamics simulations and transferable force fields, we designed a series of symmetric triblock amphiphiles (or high-χ block oligomers) comprising incompatible sugar-based (A) and hydrocarbon (B) blocks that can self-assemble into ordered nanostructures with sub-1 nm domains and full domain pitches as small as 1.2 nm. Depending on the chain length and block sequence, the ordered morphologies include lamellae, perforated lamellae, and hexagonally perforated lamellae. The self-assembly of these amphiphiles bears some similarities, but also some differences, to those formed by symmetric triblock polymers. In lamellae formed by ABA amphiphiles, the fraction of B blocks "bridging" adjacent polar domains is nearly unity, much higher than that found for symmetric triblock polymers, and the bridging molecules adopt elongated conformations. In contrast, "looping" conformations are prevalent for A blocks of BAB amphiphiles. Above the order-disorder transition temperature, the disordered states are locally well-segregated yet the B blocks of ABA amphiphiles are significantly less stretched than in the lamellar phases. Analysis of both hydrogen-bonded and nonpolar clusters reveals the bicontinuous nature of these network phases. This simulation study furnishes detailed insights into structure-property relationships for mesophase formation on the 1 nm length scale that will aid further miniaturization for numerous applications.

The improvement of nonviral gene therapies relies to a large extent on understanding many fundamental physical and biological properties of these systems. This includes interactions of synthetic delivery systems with the cell and mechanisms of trafficking delivery vehicles, which remain poorly understood on both the extra- and intracellular levels. In this study, the mechanisms of cellular internalization and trafficking of polymer-based nanoparticle complexes consisting of polycations and nucleic acids, termed polyplexes, have been observed in detail at the cellular level. For the first time evidence has been obtained that filopodia, actin projections that radiate out from the surface of cells, serve as a route for the direct endocytosis of polyplexes. Confocal microscopy images demonstrated that filopodia on HeLa cells detect external polyplexes and extend into the extracellular milieu to internalize these particles. Polyplexes are observed to be internalized into membrane-bound vesicles (i.e., clathrin-coated pits and caveolae) directly within filopodial projections and are subsequently transported along actin to the main cell body for potential delivery of the nucleic acids to the nucleus. The kinetics and speed of polyplex trafficking have also been measured. The polyplex-loaded vesicles were also discovered to traffic between two cells within filopodial bridges. These findings provide novel insight into the early events of cellular contact with polyplexes through filopodial-based interactions in addition to endocytic vesicle trafficking-an important fundamental discovery to enable advancement of nonviral gene editing, nucleic acid therapies, and biomedical materials.

Amorphous solid dispersions of polymers and drugs have been shown to improve supersaturation maintenance of poorly water-soluble drugs. Herein, amorphous spray-dried dispersions (SDDs) of poly(acrylic acid)-polystyrene (PS- b -PAA) diblock copolymers with differing degrees of polymerization were prepared in aggregated and nonaggregated states with the Biopharmaceutical Classification System Class II drug, probucol (PBC). Specifically, PS 90 - b -PAA 15 , PS 90 - b -PAA 80 , PS 38 - b -PAA 220 , and PS 38 - b -PAA 320 amphiphilic block polymers that covered a compositional range in the area of oral drug delivery were prepared to examine the role of molecular weight and controlled aggregation in promoting drug supersaturation and maintenance. In addition, hydrophilic homopolymers PAA 20 , PAA 96 , PAA 226 , and PAA 392 were prepared as controls to evaluate the role of the block copolymer-based SDDs in PBC solubilization. Characterization such as powder X-ray diffraction, scanning electron microscopy, and dissolution tests under nonsink conditions were then performed to evaluate the SDDs. When comparing the block copolymer systems, polymers that were preaggregated into micellular structures prior to spray drying with the drug promoted higher drug solubility and maintenance than when the drug was formulated with molecularly dissolved PS-PAA block polymer. Interestingly, the aggregated PS 90 - b -PAA 80 SDD with 25 wt % PBC achieved 100% burst release and maintained full supersaturation of PBC at pH 6.5 (physiological pH in the small intestine). Dissolution studies conducted at the pH of the stomach (pH = 1.2) show that a minimal amount of drug (∼10 μg/mL) was released, which could be used for protecting drugs from acidic environments (stomach) before reaching the small intestine. To evaluate drug bioavailability, in vitro Caco-2 cell assays were performed, which reveal that PAA-based excipients do not hinder drug permeation across the epithelial membrane and that PS 90 - b -PAA 80 SDD with 25 wt % PBC achieved the highest membrane permeability coefficient. This work demonstrates that block copolymer-based SDDs capable of preaggregating into nanostructures may be a tunable drug-delivery platform that can improve solubility and supersaturation maintenance of Class II pharmaceutics while also not prohibiting bioavailability through model intestinal membranes. Indeed, this concept may be extended to accommodate a myriad of pharmaceutical and excipient structures.

Cellular delivery of biomacromolecules is vital to medical research and therapeutic development. Cationic polymers are promising and affordable candidate vehicles for these precious payloads. However, the impact of polycation architecture and solution assembly on the biological mechanisms and efficacy of these vehicles has not been clearly defined. In this study, four polymers containing the same cationic poly(2-(dimethylamino)ethyl methacrylate) (D) block but placed in different architectures have been synthesized, characterized, and compared for cargo binding and biological performance. The D homopolymer and its diblock copolymer poly(ethylene glycol)- block -poly(2-(dimethylamino) ethyl methacrylate) (OD) readily encapsulate pDNA to form polyplexes. Two amphiphilic block polymer variants, poly(2-(dimethylamino)ethyl methacrylate)- block -poly( n -butyl methacrylate) (DB) and poly(ethylene glycol)- block -poly(2-(dimethylamino)ethyl methacrylate)- block -poly( n -butyl methacrylate) (ODB), self-assemble into micelles, which template pDNA winding around the cationic corona to form micelleplexes. Micelleplexes were found to have superior delivery efficiency compared to polyplexes and detailed physicochemical and biological characterizations were performed to pinpoint the mechanisms by testing hypotheses related to cellular internalization, intracellular trafficking, and pDNA unpackaging. For the first time, we find that the higher concentration of amines housed in micelleplexes stimulates both cellular internalization and potential endosomal escape, and the physical motif of pDNA winding into micelleplexes, reminiscent of DNA compaction by histones in chromatin, preserves the pDNA secondary structure in its native B form. This likely allows greater payload accessibility for protein expression with micelleplexes compared to polyplexes, which tightly condense pDNA and significantly distort its helicity. This work provides important guidance for the design of successful biomolecular delivery systems via optimizing the physicochemical properties.

The field of gene therapy, which aims to treat patients by modulating gene expression, has come to fruition and has landed several landmark FDA approvals. Most gene therapies currently rely on viral vectors to deliver nucleic acid cargo into cells, but there is significant interest in moving toward chemical-based methods, such as polymer-based vectors, due to their low cost, immunocompatibility, and tunability. The full potential of polymer-based delivery systems has yet to be realized, however, because most polymeric transfection reagents are either too inefficient or too toxic for use in the clinic. In this Account, we describe developments in carbohydrate-based cationic polymers, termed glycopolymers, for enhanced nonviral gene delivery. As ubiquitous components of biological systems, carbohydrates are a rich class of compounds that can be harnessed to improve the biocompatibility of non-native polymers, such as linear polyamines used for promoting transfection. Reineke et al. developed a new class of carbohydrate-based polymers called poly(glycoamidoamine)s (PGAAs) by step-growth polymerization of linear monosaccharides with linear ethyleneamines. These glycopolymers were shown to be both efficient and biocompatible transfection reagents. Systematic modifications of the structural components of the PGAA system revealed structure-activity relationships important to its function, including its ability to degrade in situ. Expanding upon the development of step-growth glycopolymers, monosaccharides, such as glucose, were functionalized as vinyl-based monomers for the formation of diblock copolymers via radical addition-fragmentation chain-transfer (RAFT) polymerization. Upon complexation with plasmid DNA, the glucose-containing block creates a hydrophilic shell that promotes colloidal stability as effectively as PEG functionalization. An N-acetyl-d-galactosamine variant of this diblock polymer yields colloidally stable particles that show increased receptor-mediated uptake by liver hepatocytes in vitro and promotes liver targeting in mice. Finally, the disaccharide trehalose was incorporated into polycationic structures using both step-growth and RAFT techniques. It was shown that these trehalose-based copolymers imparted increased colloidal stability and yielded plasmid and siRNA polyplexes that resist aggregation upon lyophilization and reconstitution in water. The aforementioned series of glycopolymers use carbohydrates to promote effective and safe delivery of nucleic acid cargo into a variety of human cells types by promoting vehicle degradation, tissue-targeting, colloidal stabilization, and stability toward lyophilization to extend shelf life. Work is currently underway to translate the use of glycopolymers for safe and efficient delivery of nucleic acid cargo for gene therapy and gene editing applications.

Genome editing therapies hold great promise for the cure of monogenic and other diseases; however, the application of nonviral gene delivery methods is limited by both a lack of fundamental knowledge of interactions of the gene-carrier in complex animals and biocompatibility. Herein, we characterize nonviral gene delivery vehicle formulations that are based on diblock polycations containing a hydrophilic and neutral glucose block chain extended with cationic secondary amines of three lengths, poly(methacrylamido glucopyranose- block-2-methylaminoethyl methacrylate) [P(MAG- b-MAEMt)-1, -2, -3]. These polymers were formulated with plasmid DNA to prepare polyelectrolyte complexes (polyplexes). In addition, two controls, P(EG- b-MAEMt) and P(MAEMt), were synthesized, formulated into polyplexes and the ex vivo hemocompatibility, or blood compatibility, and in vivo biodistribution of the formulations were compared to the glycopolymers. While both polymer structure and N/P (amine to phosphate) ratio were important factors affecting hemocompatibility, N/P ratio played a stronger role in determining polyplex biodistribution. P(EG- b-MAEMt) and P(MAEMt) lysed red blood cells at both high and low N/P formulations while P(MAG- b-MAEMt) did not significantly lyse cells at either formulation at short and medium polymer lengths. Conversely, P(MAG- b-MAEMt) did not affect coagulation at N/P = 5, but significantly delayed coagulation at N/P = 15. P(EG- b-MAEMt) and P(MAEMt) did not affect coagulation at either formulation. After polymer and pDNA cargo distribution was observed in vivo, P(EG- b-MAEMt) N/P = 5 and P(MAG- b-MAEMt) N/P = 5 both dissociated and deposited polymer in the liver, while pDNA cargo from P(MAG- b-MAEMt) N/P = 15 was found in the liver, lungs, and spleen. The contrast between P(MAG- b-MAEMt) at N/P = 5 and 15 demonstrates that polyplex stability in the blood can be improved with N/P ratio and potentially aid polyplex biodistribution through simply varying the formulation ratios.

Isosorbide is a rigid, sugar-derived building block that has shown promise in high-performance materials, albeit with a lack of available controlled polymerization methods. To this end, we provide mechanistic insights into the cationic and quasi-zwitterionic ring-opening polymerization (ROP) of an annulated isosorbide derivative (1,4:2,5:3,6-trianhydro-d-mannitol, 5). Ring-opening selectivity of this tricyclic ether was achieved, and the polymerization is selectively directed toward different macromolecular architectures, allowing for formation of either linear or cyclic polymers. Notably, straightforward recycling of unreacted monomer can be accomplished via sublimation. This work provides the first platform for tailored polymer architectures from isosorbide via ROP.

Fast, efficient, and inexpensive methods for delivering functional nucleic acids to primary human cell types are needed to advance regenerative medicine and cell therapies. Plasmid-based gene editing (such as with CRISPR-Cas9) can require the delivery of plasmids that are large (∼9.5-13 kbp) in comparison to common reporter plasmids (∼5-8 kbp). To develop more efficient plasmid delivery vehicles, we investigated the effect of plasmid size on the transfection of primary human dermal fibroblasts (HDFs) and induced pluripotent stem cells (iPSCs) using a heparin-treated trehalose-containing polycation (Tr4-heparin). Transfections with 4.7 kbp to 10 kbp plasmids exhibited high rates of polyplex internalization with both plasmid sizes. However, transfection with the large plasmid was nearly eliminated in HDFs and significantly reduced in iPSCs. Molecular additives were used to probe intracellular barriers to transfection. Chloroquine treatments were used to destabilize endosomes, and dexamethasone and thymidine were used to destabilize the nuclear envelope. Destabilizing the nuclear envelope resulted in significantly increased large-plasmid-transfection, indicating that nuclear localization may be more difficult for large plasmids. To demonstrate the potential clinical utility of this formulation, HDFs and iPSCs were treated with to dexamethasone-Tr4-heparin polyplexes encoding dCas9-VP64, synthetic transcription activator, targeted to collagen type VII. These transfections enhanced collagen expression in HDFs and iPSCs by 5- and 20-fold, respectively, compared to an untransfected control and were the more effective than the Lipofectamine 2000 control. Functional plasmid transfection efficiency can be significantly improved by nuclear destabilization, which could lead to improved development of nonviral vehicles for ex vivo CRISPR-Cas9 gene editing.

Recessive dystrophic epidermolysis bullosa (RDEB), an inherited disease featuring blistering wounds, causes constant inflammation that leads to the eventual development of an aggressive form of squamous cell carcinoma (RDEB SCC). The persistence of inflammatory chemokines such as MCP-1 and Il-8 in RDEB wounds may foster RDEB SCC carcinogenesis. We report the production of ternary composite nanofibers containing pullulan, chondroitin sulfate, and tannic acid as RDEB wound dressings. The swellable fibers are stable to hydration and absorb ∼500% their weight in water. The fibers remove ∼99% of MCP-1 from solution in <2 h. Scavenged media did not promote RDEB SCC migration.

The covalent attachment of polymers to the surface of proteins and nanoparticles has been widely employed in the development of biomedical platforms capable of delaying or diminishing immune surveillance. The most widely employed polymer for these applications has been poly(ethylene glycol) (PEG), yet recent evidence has suggested that other polymer architectures and compositions provide significantly better in vitro and in vivo properties of protein-polymer hybrid materials. Moreover, few direct comparisons of PEG to these polymers have been reported. Here we describe the assembly and characterization of a series of polymer conjugates of a representative immunogenic viruslike particle (VLP) using (poly(oligo(ethylene glycol) methacrylate), poly(methacrylamido glucopyranose), and PEG, and an investigation of their ability to shield the protein from antibody recognition as a function of polymer loading density, chain length, architecture, and conjugation site. Increasing chain length and loading density were both found to significantly diminish antibody recognition of the VLP conjugates; the conformation adopted by different polymer architectures was also found to greatly influence antibody recognition. A direct comparison of these conjugates to PEGylated VLPs in vivo showed that all formulations gave rise to similar antibody titers that were significantly diminished relative to unmodified particles. Interestingly, the quality of the antibody response was impacted by the properties of the conjugate, with differences in observed affinity and avidity suggesting a complex dependence on loading density, chain length, and architecture.