Hybrid Bonding Bottlebrush Polymers Grafted from a Supramolecular Polymer Backbone.

Bottlebrush polymers, macromolecules consisting of dense polymer side chains grafted from a central polymer backbone, have unique properties resulting from this well-defined molecular architecture. With the advent of controlled radical polymerization techniques, access to these architectures has become more readily available. However, synthetic challenges remain, including the need for intermediate purification, the use of toxic solvents, and challenges with achieving long bottlebrush architectures due to backbone entanglements. Herein, we report hybrid bonding bottlebrush polymers (systems integrating covalent and noncovalent bonding of structural units) consisting of poly(sodium 4-styrenesulfonate) (p(NaSS)) brushes grafted from a peptide amphiphile (PA) supramolecular polymer backbone. This was achieved using photoinitiated electron/energy transfer-reversible addition-fragmentation chain transfer (PET-RAFT) polymerization in water. The structure of the hybrid bonding bottlebrush architecture was characterized using cryogenic transmission electron microscopy, and its properties were probed using rheological measurements. We observed that hybrid bonding bottlebrush polymers were able to organize into block architectures containing domains with high brush grafting density and others with no observable brushes. This finding is possibly a result of dynamic behavior unique to supramolecular polymer backbones, enabling molecular exchange or translational diffusion of monomers along the length of the assemblies. The hybrid bottlebrush polymers exhibited higher solution viscosity at moderate shear, protected supramolecular polymer backbones from disassembly at high shear, and supported self-healing capabilities, depending on grafting densities. Our results demonstrate an opportunity for novel properties in easily synthesized bottlebrush polymer architectures built with supramolecular polymers that might be useful in biomedical applications or for aqueous lubrication.

Single-cell coating with biomimetic extracellular nanofiber matrices.

Cell therapies offer great promise in the treatment of diseases and tissue regeneration, but their clinical use has many challenges including survival, optimal performance in their intended function, or localization at sites where they are needed for effective outcomes. We report here on a method to coat a biodegradable matrix of biomimetic nanofibers on single cells that could have specific functions ranging from cell signaling to targeting and helping cells survive when used for therapies. The fibers are composed of peptide amphiphile (PA) molecules that self-assemble into supramolecular nanoscale filaments. The PA nanofibers were able to create a mesh-like coating for a wide range of cell lineages with nearly 100 % efficiency, without interrupting the natural cellular phenotype or functions. The targeting abilities of this system were assessed in vitro using human primary regulatory T (hTreg) cells coated with PAs displaying a vascular cell adhesion protein 1 (VCAM-1) targeting motif. This approach provides a biocompatible method for single-cell coating that does not negatively alter cellular phenotype, binding capacity, or immunosuppressive functionality, with potential utility across a broad spectrum of cell therapies. STATEMENT OF SIGNIFICANCE: Cell therapies hold great promise in the treatment of diseases and tissue regeneration, but their clinical use has been limited by cell survival, targeting, and function. We report here a method to coat single cells with a biodegradable matrix of biomimetic nanofibers composed of peptide amphiphile (PA) molecules. The nanofibers were able to coat cells, such as human primary regulatory T cells, with nearly 100 % efficiency, without interrupting the natural cellular phenotype or functions. The approach provides a biocompatible method for single-cell coating that does not negatively alter cellular phenotype, binding capacity, or immunosuppressive functionality, with potential utility across a broad spectrum of cell therapies.

CLIP-Seq analysis enables the design of protective ribosomal RNA bait oligonucleotides against C9ORF72 ALS/FTD poly-GR pathophysiology.

Amyotrophic lateral sclerosis and frontotemporal dementia patients with a hexanucleotide repeat expansion in C9ORF72 (C9-HRE) accumulate poly-GR and poly-PR aggregates. The pathogenicity of these arginine-rich dipeptide repeats (R-DPRs) is thought to be driven by their propensity to bind low-complexity domains of multivalent proteins. However, the ability of R-DPRs to bind native RNA and the significance of this interaction remain unclear. Here, we used computational and experimental approaches to characterize the physicochemical properties of R-DPRs and their interaction with RNA. We find that poly-GR predominantly binds ribosomal RNA (rRNA) in cells and exhibits an interaction that is predicted to be energetically stronger than that for associated ribosomal proteins. Critically, modified rRNA "bait" oligonucleotides restore poly-GR-associated ribosomal deficits and ameliorate poly-GR toxicity in patient neurons and Drosophila models. Our work strengthens the hypothesis that ribosomal function is impaired by R-DPRs, highlights a role for direct rRNA binding in mediating ribosomal dysfunction, and presents a strategy for protecting against C9-HRE pathophysiological mechanisms.

De novo amyloid peptides with subtle sequence variations differ in their self-assembly and nanomechanical properties.

Proteinaceous amyloids are well known for their widespread pathological roles but lately have emerged also as key components in several biological functions. The remarkable ability of amyloid fibers to form tightly packed conformations in a cross ß-sheet arrangement manifests in their robust enzymatic and structural stabilities. These characteristics of amyloids make them attractive for designing proteinaceous biomaterials for various biomedical and pharmaceutical applications. In order to design customizable and tunable amyloid nanomaterials, it is imperative to understand the sensitivity of the peptide sequence for subtle changes based on amino acid position and chemistry. Here we report our results from four rationally-designed amyloidogenic decapeptides that subtly differ in hydrophobicity and polarity at positions 5 and 6. We show that making the two positions hydrophobic renders the peptide with enhanced aggregation and material properties while introducing polar residues in position 5 dramatically changes the structure and nanomechanical properties of the fibrils formed. A charged residue at position 6, however, abrogates amyloid formation. In sum, we show that subtle changes in the sequence do not make the peptide innocuous but rather sensitive to aggregation, reflected in the biophysical and nanomechanical properties of the fibrils. We conclude that tolerance of peptide amyloid for changes in the sequence, however small they may be, should not be neglected for the effective design of customizable amyloid nanomaterials.

Role of supramolecular polymers in photo-actuation of spiropyran hydrogels.

Supramolecular-covalent hybrid polymers have been shown to be interesting systems to generate robotic functions in soft materials in response to external stimuli. In recent work supramolecular components were found to enhance the speed of reversible bending deformations and locomotion when exposed to light. The role of morphology in the supramolecular phases integrated into these hybrid materials remains unclear. We report here on supramolecular-covalent hybrid materials that incorporate either high-aspect-ratio peptide amphiphile (PA) ribbons and fibers, or low-aspect-ratio spherical peptide amphiphile micelles into photo-active spiropyran polymeric matrices. We found that the high-aspect-ratio morphologies not only play a significant role in providing mechanical reinforcement to the matrix but also enhance photo-actuation for both light driven volumetric contraction and expansion of spiropyran hydrogels. Molecular dynamics simulations indicate that water within the high-aspect-ratio supramolecular polymers exhibits a faster draining rate as compared to those in spherical micelles, which suggests that the high-aspect-ratio supramolecular polymers effectively facilitate the transport of trapped water molecules by functioning as channels and therefore enhancing actuation of the hybrid system. Our simulations provide a useful strategy for the design of new functional hybrid architectures and materials with the aim of accelerating response and enhancing actuation by facilitating water diffusion at the nanoscopic level.

Supramolecular Nanofibers Block SARS-CoV-2 Entry into Human Host Cells.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection relies on its spike protein binding to angiotensin-converting enzyme 2 (ACE2) on host cells to initiate cellular entry. Blocking the interactions between the spike protein and ACE2 offers promising therapeutic opportunities to prevent infection. We report here on peptide amphiphile supramolecular nanofibers that display a sequence from ACE2 in order to promote interactions with the SARS-CoV-2 spike receptor binding domain. We demonstrate that displaying this sequence on the surface of supramolecular assemblies preserves its α-helical conformation and blocks the entry of a pseudovirus and its two variants into human host cells. We also found that the chemical stability of the bioactive structures was enhanced in the supramolecular environment relative to the unassembled peptide molecules. These findings reveal unique advantages of supramolecular peptide therapies to prevent viral infections and more broadly for other targets as well.

Topical application of an irreversible small molecule inhibitor of lysyl oxidases ameliorates skin scarring and fibrosis.

Scarring is a lifelong consequence of skin injury, with scar stiffness and poor appearance presenting physical and psychological barriers to a return to normal life. Lysyl oxidases are a family of enzymes that play a critical role in scar formation and maintenance. Lysyl oxidases stabilize the main component of scar tissue, collagen, and drive scar stiffness and appearance. Here we describe the development and characterisation of an irreversible lysyl oxidase inhibitor, PXS-6302. PXS-6302 is ideally suited for skin treatment, readily penetrating the skin when applied as a cream and abolishing lysyl oxidase activity. In murine models of injury and fibrosis, topical application reduces collagen deposition and cross-linking. Topical application of PXS-6302 after injury also significantly improves scar appearance without reducing tissue strength in porcine injury models. PXS-6302 therefore represents a promising therapeutic to ameliorate scar formation, with potentially broader applications in other fibrotic diseases.

A Perspective on the History and Current Opportunities of Aqueous RAFT Polymerization.

Reversible addition-fragmentation chain transfer (RAFT) polymerization has proven itself as a powerful polymerization technique affording facile control of molecular weight, molecular weight distribution, architecture, and chain end groups - while maintaining a high level of tolerance for solvent and monomer functional groups. RAFT is highly suited to water as a polymerization solvent, with aqueous RAFT now utilized for applications such as controlled synthesis of ultra-high molecular weight polymers, polymerization induced self-assembly, and biocompatible polymerizations, among others. Water as a solvent represents a non-toxic, cheap, and environmentally friendly alternative to organic solvents traditionally utilized for polymerizations. This, coupled with the benefits of RAFT polymerization, makes for a powerful combination in polymer science. This perspective provides a historical account of the initial developments of aqueous RAFT polymerization at the University of Southern Mississippi from the McCormick Research Group, details practical considerations for conducting aqueous RAFT polymerizations, and highlights some of the recent advances aqueous RAFT polymerization can provide. Finally, some of the future opportunities that this versatile polymerization technique in an aqueous environment can offer are discussed, and it is anticipated that the aqueous RAFT polymerization field will continue to realize these, and other exciting opportunities into the future.

Peptide Amphiphile Supramolecular Nanofibers Designed to Target Abdominal Aortic Aneurysms.

An abdominal aortic aneurysm (AAA) is a localized dilation of the aorta located in the abdomen that poses a severe risk of death when ruptured. The cause of AAA is not fully understood, but degradation of medial elastin due to elastolytic matrix metalloproteinases is a key step leading to aortic dilation. Current therapeutic interventions are limited to surgical repair to prevent catastrophic rupture. Here, we report the development of injectable supramolecular nanofibers using peptide amphiphile molecules designed to localize to AAA by targeting fragmented elastin, matrix metalloproteinase 2 (MMP-2), and membrane type 1 matrix metalloproteinase. We designed four targeting peptide sequences from X-ray crystallographic data and incorporated them into PA molecules via solid phase peptide synthesis. After coassembling targeted and diluent PAs at different molar ratios, we assessed their ability to form nanofibers using transmission electron microscopy and to localize to AAA in male and female Sprague-Dawley rats using light sheet fluorescence microscopy. We found that three formulations of the PA nanofibers were able to localize to AAA tissue, but the MMP-2 targeting PA substantially outperformed the other nanofibers. Additionally, we demonstrated that the MMP-2 targeting PA nanofibers had an optimal dose of 5 mg (∼12 mg/kg). Our results show that there was not a significant difference in targeting between male and female Sprague-Dawley rats. Given the ability of the MMP-2 targeting PA nanofiber to localize to AAA tissue, future studies will investigate potential diagnostic and targeted drug delivery applications for AAA.

Self-Assembled Peptide Amphiphile Nanofibers for Controlled Therapeutic Delivery to the Atherosclerotic Niche.

Atherosclerotic plaque remains the leading contributor to cardiovascular disease and requires invasive surgical procedures for its removal. Nanomedicine offers a minimally invasive approach to alleviate plaque burden by targeted therapeutic delivery. However, nanocarriers are limited without the ability to sense and respond to the diseased microenvironment. In this study, targeted self-assembled peptide amphiphile (PA) nanofibers were developed that cleave in response to biochemical cues expressed in atherosclerotic lesions-reactive oxygen species (ROS) and intracellular glutathione-to deliver a liver X receptor agonist (LXR) to enhance macrophage cholesterol efflux. The PAs released LXR in response to physiological levels of ROS and reducing agents and could be co-assembled with plaque-targeting PAs to form nanofibers. The resulting LXR PA nanofibers promoted cholesterol efflux from macrophages in vitro as well as LXR alone and with lower cytotoxicity. Further, the ApoA1-LXR PA nanofibers targeted plaque within an atherosclerotic mouse model in vivo and activated ATP-binding cassette A1 (ABCA1) expression as well as LXR alone with reduced liver toxicity. Taken together, these results demonstrate the potential of self-assembled PA nanofibers for controlled therapeutic delivery to the atherosclerotic niche.

Enhanced Detection of Desmoplasia by Targeted Delivery of Iron Oxide Nanoparticles to the Tumour-Specific Extracellular Matrix.

Diagnostic imaging of aggressive cancer with a high stroma content may benefit from the use of imaging contrast agents targeted with peptides that have high binding affinity to the extracellular matrix (ECM). In this study, we report the use of superparamagnetic iron-oxide nanoparticles (IO-NP) conjugated to a nonapeptide, CSGRRSSKC (CSG), which specifically binds to the laminin-nidogen-1 complex in tumours. We show that CSG-IO-NP accumulate in tumours, predominantly in the tumour ECM, following intravenous injection into a murine model of pancreatic neuroendocrine tumour (PNET). In contrast, a control untargeted IO-NP consistently show poor tumour uptake, and IO-NP conjugated to a pentapeptide. CREKA that bind fibrin clots in blood vessels show restricted uptake in the angiogenic vessels of the tumours. CSG-IO-NP show three-fold higher intratumoral accumulation compared to CREKA-IO-NP. Magnetic resonance imaging (MRI) T2-weighted scans and T2 relaxation times indicate significant uptake of CSG-IO-NP irrespective of tumour size, whereas the uptake of CREKA-IO-NP is only consistent in small tumours of less than 3 mm in diameter. Larger tumours with significantly reduced tumour blood vessels show a lack of CREKA-IO-NP uptake. Our data suggest CSG-IO-NP are particularly useful for detecting stroma in early and advanced solid tumours.

Intravenous Delivery of Lung-Targeted Nanofibers for Pulmonary Hypertension in Mice.

Pulmonary hypertension is a highly morbid disease with no cure. Available treatments are limited by systemic adverse effects due to non-specific biodistribution. Self-assembled peptide amphiphile (PA) nanofibers are biocompatible nanomaterials that can be modified to recognize specific biological markers to provide targeted drug delivery and reduce off-target toxicity. Here, PA nanofibers that target the angiotensin I-converting enzyme and the receptor for advanced glycation end-products (RAGE) are developed, as both proteins are overexpressed in the lung with pulmonary hypertension. It is demonstrated that intravenous delivery of RAGE-targeted nanofibers containing the targeting epitope LVFFAED (LVFF) significantly accumulated within the lung in a chronic hypoxia-induced pulmonary hypertension mouse model. Using 3D light sheet fluorescence microscopy, it is shown that LVFF nanofiber localization is specific to the diseased pulmonary tissue with immunofluorescence analysis demonstrating colocalization of the targeted nanofiber to RAGE in the hypoxic lung. Furthermore, biodistribution studies show that significantly more LVFF nanofibers localized to the lung compared to major off-target organs. Targeted nanofibers are retained within the pulmonary tissue for 24 h after injection. Collectively, these data demonstrate the potential of a RAGE-targeted nanomaterial as a drug delivery platform to treat pulmonary hypertension.

Development of novel nanofibers targeted to smoke-injured lungs.

Smoke inhalation injury is associated with significant mortality and current therapies remain supportive. The purpose of our study was to identify proteins upregulated in the lung after smoke inhalation injury and develop peptide amphiphile nanofibers that target these proteins. We hypothesize that nanofibers targeted to angiotensin-converting enzyme or receptor for advanced glycation end products will localize to smoke-injured lungs. METHODS: Five targeting sequences were incorporated into peptide amphiphile monomers methodically to optimize nanofiber formation. Nanofiber formation was assessed by conventional transmission electron microscopy. Rats received 8 min of wood smoke. Levels of angiotensin-converting enzyme and receptor for advanced glycation end products were evaluated by immunofluorescence. Rats received the targeted nanofiber 23 h after injury via tail vein injection. Nanofiber localization was determined by fluorescence quantification. RESULTS: Peptide amphiphile purity (>95%) and nanofiber formation were confirmed. Target proteins were increased in smoke inhalation versus sham (p < 0.001). After smoke inhalation and injection of targeted nanofibers, we found a 10-fold increase in angiotensin-converting enzyme-targeted nanofiber localization to lung (p < 0.001) versus sham with minimal localization of non-targeted nanofiber (p < 0.001). CONCLUSIONS: We synthesized, characterized, and evaluated systemically delivered targeted nanofibers that localized to the site of smoke inhalation injury in vivo. Angiotensin-converting enzyme-targeted nanofibers serve as the foundation for developing a novel nanotherapeutic that treats smoke inhalation lung injury.

Superstructured Biomaterials Formed by Exchange Dynamics and Host-Guest Interactions in Supramolecular Polymers.

Dynamic and reversible assembly of molecules is ubiquitous in the hierarchical superstructures of living systems and plays a key role in cellular functions. Recent work from the laboratory reported on the reversible formation of such superstructures in systems of peptide amphiphiles conjugated to oligonucleotides and electrostatically complimentary peptide sequences. Here, a supramolecular system is reported upon where exchange dynamics and host-guest interactions between ß-cyclodextrin and adamantane on peptide amphiphiles lead to superstructure formation. Superstructure formation with bundled nanoribbons generates a mechanically robust hydrogel with a highly porous architecture that can be 3D printed. Functionalization of the porous superstructured material with a biological signal results in a matrix with significant in vitro bioactivity toward neurons that could be used as a supramolecular model to design novel biomaterials.

Allomelanin: A Biopolymer of Intrinsic Microporosity.

Melanin is a ubiquitous natural pigment found in a diverse array of organisms. Allomelanin is a class of nitrogen-free melanin often found in fungi. Herein, we find artificial allomelanin analogues exhibit high intrinsic microporosity and describe an approach for further increasing and tuning that porosity. Notably, the synthetic method involves an oxidative polymerization of 1,8-DHN in water, negating the need for multiple complex templating steps and avoiding expensive or complex chemical precursors. The well-defined morphologies of these nanomaterials were elucidated by a combination of electron microscopy and scattering methods, yielding to high-resolution 3D reconstruction based on small-angle X-ray scattering (SAXS) results. Synthetic allomelanin nanoparticles exhibit high BET areas, up to 860 m2/g, and are capable of ammonia capture up to 17.0 mmol/g at 1 bar. In addition, these nanomaterials can adsorb nerve agent simulants in solution and as a coating on fabrics with high breathability where they prevent breakthrough. We also confirmed that naturally derived fungal melanin can adsorb nerve gas simulants in solution efficiently despite lower porosity than synthetic analogues. Our approach inspires further analysis of yet to be discovered biological materials of this class where melanins with intrinsic microporosity may be linked to evolutionary advantages in relevant organisms and may in turn inspire the design of new high surface area materials.

Design of materials with supramolecular polymers.

One hundred years ago Hermann Staudinger was strongly criticized by his scientific peers for his macromolecular hypothesis, but today it is hard to imagine a world without polymers. His hypothesis described polymers as macromolecules composed of large numbers of structural units connected by covalent bonds. In the 1990s the concept of supramolecular polymers emerged in the scientific literature as discrete entities of large molar mass comparable to that of classical polymers but built through non-covalent bonds among monomers. Supramolecular polymers exist in biological systems, and potentially blend the physical properties of covalent polymers with unique features such as high degrees of internal order within the polymeric structure, defined shapes, and novel dynamics. This trend article provides a summary of seminal contributions in supramolecular polymerization and provides recent examples from the Stupp laboratory to demonstrate the potential applications of an exciting class of materials composed fully or partially of supramolecular polymers. In closing, we provide our perspective on future opportunities provided by this field at the onset of a second century of polymers. It is our objective here to demonstrate that this second century could be as prosperous, if not more so, than the preceding one.

Proapoptotic Peptide Brush Polymer Nanoparticles via Photoinitiated Polymerization-Induced Self-Assembly.

Herein, we report the photoinitiated polymerization-induced self-assembly (photo-PISA) of spherical micelles consisting of proapoptotic peptide-polymer amphiphiles. The one-pot synthetic approach yielded micellar nanoparticles at high concentrations and at scale (150 mg mL-1 ) with tunable peptide loadings up to 48 wt. %. The size of the micellar nanoparticles was tuned by varying the lengths of hydrophobic and hydrophilic building blocks. Critically, the peptide-functionalized nanoparticles imbued the proapoptotic "KLA" peptides (amino acid sequence: KLAKLAKKLAKLAK) with two key properties otherwise not inherent to the sequence: 1) proteolytic resistance compared to the oligopeptide alone; 2) significantly enhanced cell uptake by multivalent display of KLA peptide brushes. The result was demonstrated improved apoptosis efficiency in HeLa cells. These results highlight the potential of photo-PISA in the large-scale synthesis of functional, proteolytically resistant peptide-polymer conjugates for intracellular delivery.

Development of Optimized Tissue-Factor-Targeted Peptide Amphiphile Nanofibers to Slow Noncompressible Torso Hemorrhage.

Noncompressible torso hemorrhage accounts for a significant portion of preventable trauma deaths. We report here on the development of injectable, targeted supramolecular nanotherapeutics based on peptide amphiphile (PA) molecules that are designed to target tissue factor (TF) and, therefore, selectively localize to sites of injury to slow hemorrhage. Eight TF-targeting sequences were identified, synthesized into PA molecules, coassembled with nontargeted backbone PA at various weight percentages, and characterized via circular dichroism spectroscopy, transmission electron microscopy, and X-ray scattering. Following intravenous injection in a rat liver hemorrhage model, two of these PA nanofiber coassemblies exhibited the most specific localization to the site of injury compared to controls (p < 0.05), as quantified using immunofluorescence imaging of injured liver and uninjured organs. To determine if the nanofibers were targeting TF in vivo, a mouse saphenous vein laser injury model was performed and showed that TF-targeted nanofibers colocalized with fibrin, demonstrating increased levels of nanofiber at TF-rich sites. Thromboelastograms obtained using samples of heparinized rat whole blood containing TF demonstrated that no clots were formed in the absence of TF-targeted nanofibers. Lastly, both PA nanofiber coassemblies decreased blood loss in comparison to sham and backbone nanofiber controls by 35-59% (p < 0.05). These data demonstrate an optimal TF-targeted nanofiber that localizes selectively to sites of injury and TF exposure, and, interestingly, reduces blood loss. This research represents a promising initial phase in the development of a TF-targeted injectable therapeutic to reduce preventable deaths from hemorrhage.

Semiconductor Quantum Dots Are Efficient and Recyclable Photocatalysts for Aqueous PET-RAFT Polymerization.

This Letter describes the use of CdSe quantum dots (QDs) as photocatalysts for photoinduced electron transfer reversible addition-fragmentation chain transfer (PET-RAFT) polymerization of a series of aqueous acrylamides and acrylates. The high colloidal solubility and photostability of these QDs allowed polymerization to occur with high efficiency (>90% conversion in 2.5 h), low dispersity (PDI < 1.1), and ultralow catalyst loading (<0.5 ppm). The use of protein concentrators enabled the removal of the photocatalyst from the polymer and monomer with tolerable metal contamination (8.41 ug/g). These isolated QDs could be recycled for four separate polymerizations without a significant decrease in efficiency. By changing the pore size of the protein concentrators, the QDs and polymer could be separated from the remaining monomer, allowing for the synthesis of block copolymers using a single batch of QDs with minimal purification steps and demonstrating the fidelity of chain ends.