A Biomimetic Multi-Component Subunit Vaccine via Ratiometric Loading of Hierarchical Hydrogels.

The development of subunit vaccines that mimic the molecular complexity of attenuated vaccines has been limited by the difficulty of intracellular co-delivery of multiple chemically diverse payloads at controllable concentrations. We report on hierarchical hydrogel depots employing simple poly(propylene sulfone) homopolymers to enable ratiometric loading of a protein antigen and four physicochemically distinct adjuvants in a hierarchical manner. The optimized vaccine consisted of immunostimulants either adsorbed to or encapsulated within nanogels, which were capable of noncovalent anchoring to subcutaneous tissues. These 5-component nanogel vaccines demonstrated enhanced humoral and cell-mediated immune responses compared to formulations with standard single adjuvant and antigen pairing. The use of a single simple homopolymer capable of rapid and stable loading and intracellular delivery of diverse molecular cargoes holds promise for facile development and optimization of scalable subunit vaccines and complex therapeutic formulations for a wide range of biomedical applications.

Inhibiting the Keap1/Nrf2 Protein-Protein Interaction with Protein-Like Polymers.

Successful and selective inhibition of the cytosolic protein-protein interaction (PPI) between nuclear factor erythroid 2-related factor 2 (Nrf2) and Kelch-like ECH-associating protein 1 (Keap1) can enhance the antioxidant response, with the potential for a therapeutic effect in a range of settings including in neurodegenerative disease (ND). Small molecule inhibitors have been developed, yet many have off-target effects, or are otherwise limited by poor cellular permeability. Peptide-based strategies have also been attempted to enhance specificity, yet face challenges due to susceptibility to degradation and lack of cellular penetration. Herein, these barriers are overcome utilizing a polymer-based proteomimetics. The protein-like polymer (PLP) consists of a synthetic, lipophilic polymer backbone displaying water soluble Keap1-binding peptides on each monomer unit forming a brush polymer architecture. The PLPs are capable of engaging Keap1 and displacing the cellular protective transcription factor Nrf2, which then translocates to the nucleus, activating the antioxidant response element (ARE). PLPs exhibit increased Keap1 binding affinity by several orders of magnitude compared to free peptides, maintain serum stability, are cell-penetrant, and selectively activate the ARE pathway in cells, including in primary cortical neuronal cultures. Keap1/Nrf2-inhibitory PLPs have the potential to impact the treatment of disease states associated with dysregulation of oxidative stress, such as NDs.

Controlled adsorption of multiple bioactive proteins enables targeted mast cell nanotherapy.

Protein adsorption onto nanomaterials often results in denaturation and loss of bioactivity. Controlling the adsorption process to maintain the protein structure and function has potential for a range of applications. Here we report that self-assembled poly(propylene sulfone) (PPSU) nanoparticles support the controlled formation of multicomponent enzyme and antibody coatings and maintain their bioactivity. Simulations indicate that hydrophobic patches on protein surfaces induce a site-specific dipole relaxation of PPSU assemblies to non-covalently anchor the proteins without disrupting the protein hydrogen bonding or structure. As a proof of concept, a nanotherapy employing multiple mast-cell-targeted antibodies for preventing anaphylaxis is demonstrated in a humanized mouse model. PPSU nanoparticles displaying an optimized ratio of co-adsorbed anti-Siglec-6 and anti-FcεRIα antibodies effectively inhibit mast cell activation and degranulation, preventing anaphylaxis. Protein immobilization on PPSU surfaces provides a simple and rapid platform for the development of targeted protein nanomedicines.

Reciprocal regulatory balance within the CLEC16A-RNF41 mitophagy complex depends on an intrinsically disordered protein region.

CLEC16A is an E3 ubiquitin ligase that regulates mitochondrial quality control through mitophagy and is associated with over 20 human diseases. CLEC16A forms a complex with another E3 ligase, RNF41, and a ubiquitin-specific peptidase, USP8; however, regions that regulate CLEC16A activity or the assembly of the tripartite mitophagy regulatory complex are unknown. Here, we report that CLEC16A contains an internal intrinsically disordered protein region (IDPR) that is crucial for CLEC16A function and turnover. IDPRs lack a fixed secondary structure and possess emerging yet still equivocal roles in protein stability, interactions, and enzymatic activity. We find that the internal IDPR of CLEC16A is crucial for its degradation. CLEC16A turnover was promoted by RNF41, which binds and acts upon the internal IDPR to destabilize CLEC16A. Loss of this internal IDPR also destabilized the ubiquitin-dependent tripartite CLEC16A-RNF41-USP8 complex. Finally, the presence of an internal IDPR within CLEC16A was confirmed using NMR and CD spectroscopy. Together, our studies reveal that an IDPR is essential to control the reciprocal regulatory balance between CLEC16A and RNF41, which could be targeted to improve mitochondrial health in disease.

An intrinsically disordered protein region encoded by the human disease gene CLEC16A regulates mitophagy.

CLEC16A regulates mitochondrial health through mitophagy and is associated with over 20 human diseases. However, the key structural and functional regions of CLEC16A, and their relevance for human disease, remain unknown. Here, we report that a disease-associated CLEC16A variant lacks a C-terminal intrinsically disordered protein region (IDPR) that is critical for mitochondrial quality control. IDPRs comprise nearly half of the human proteome, yet their mechanistic roles in human disease are poorly understood. Using carbon detect NMR, we find that the CLEC16A C terminus lacks secondary structure, validating the presence of an IDPR. Loss of the CLEC16A C-terminal IDPR in vivo impairs mitophagy, mitochondrial function, and glucose-stimulated insulin secretion, ultimately causing glucose intolerance. Deletion of the CLEC16A C-terminal IDPR increases CLEC16A ubiquitination and degradation, thus impairing assembly of the mitophagy regulatory machinery. Importantly, CLEC16A stability is dependent on proline bias within the C-terminal IDPR, but not amino acid sequence order or charge. Together, we elucidate how an IDPR in CLEC16A regulates mitophagy and implicate pathogenic human gene variants that disrupt IDPRs as novel contributors to diabetes and other CLEC16A-associated diseases.Abbreviations : CAS: carbon-detect amino-acid specific; IDPR: intrinsically disordered protein region; MEFs: mouse embryonic fibroblasts; NMR: nuclear magnetic resonance.

Dendritic peptide-conjugated polymeric nanovectors for non-toxic delivery of plasmid DNA and enhanced non-viral transfection of immune cells.

Plasmid DNA (pDNA) transfection is advantageous for gene therapies requiring larger genetic elements, including "all-in-one" CRISPR/Cas9 plasmids, but is limited by toxicity as well as poor intracellular release and transfection efficiency in immune cell populations. Here, we developed a synthetic non-viral gene delivery platform composed of poly(ethylene glycol)-b-poly(propylene sulfide) copolymers linked to a cationic dendritic peptide (DP) via a reduceable bond, PEG-b-PPS-ss-DP (PPDP). A library of self-assembling PPDP polymers was synthesized and screened to identify optimal constructs capable of transfecting macrophages with small (pCMV-DsRed, 4.6 kb) and large (pL-CRISPR.EFS.tRFP, 11.7 kb) plasmids. The optimized PPDP construct transfected macrophages, fibroblasts, dendritic cells, and T cells more efficiently and with less toxicity than a commercial Lipo2K reagent, regardless of pDNA size and under standard culture conditions in the presence of serum. The PPDP technology described herein is a stimuli-responsive polymeric nanovector that can be leveraged to meet diverse challenges in gene delivery.

Leveraging self-assembled nanobiomaterials for improved cancer immunotherapy.

Nanomaterials and targeted drug delivery vehicles improve the therapeutic index of drugs and permit greater control over their pharmacokinetics, biodistribution, and bioavailability. Here, nanotechnologies applied to cancer immunotherapy are discussed with a focus on current and next generation self-assembling drug delivery systems composed of lipids and/or polymers. Topics covered include the fundamental design, suitability, and inherent properties of nanomaterials that induce anti-tumor immune responses and support anti-cancer vaccination. Established active and passive targeting strategies as well as newer "indirect" methods are presented together with insights into how nanocarrier structure and surface chemistry can be leveraged for controlled delivery to the tumor microenvironment while minimizing off-target effects.

Pi-stacking Enhances Stability, Scalability of Formation, Control over Flexibility and Circulation Time of Polymeric Filaments.

Self-assembling filomicelles (FM) are of great interest to nanomedicine due to their structural flexibility, extensive systemic circulation time, and amenability to unique "cylinder-to-sphere" morphological transitions. However, current fabrication techniques for FM self-assembly are highly variable and difficult to scale. Here, we demonstrate that tetrablock copolymers composed of poly(ethylene glycol)-b-poly(propylene sulfide) (PEG-b-PPS) diblocks linked by a pi-stacking perylene bisimide (PBI) moiety permit rapid, scalable, and facile assembly of FM via the flash nanoprecipitation (FNP) method. Co-assembling the tetrablocks and PEG-b-PPS diblocks at different molar ratios resulted in mixed PBI-containing FM (mPBI-FM) with tunable length and flexibility. The flexibility of mPBI-FM can be optimized to decrease uptake by macrophages in vivo, leading to increased circulation time versus (-)PBI-FM without PBI tetrablocks after intravenous administration in mice. While PEG-b-PPS diblocks form FM within a narrow range of hydrophilic weight fractions, incorporation of pi-stacking PBI groups expanded this range to increase favorability of FM assembly. Furthermore, the aggregation-dependent fluorescence of PBI shifted during oxidation-induced "cylinder-to-sphere" transitions of mPBI-FM into micelles, resulting in a distinct emission wavelength for filamentous versus spherical nanostructures. Thus, incorporation of pi-stacking allows for rapid, scalable assembly of FM with tunable flexibility and stability for theranostic and nanomedicine applications.

The Combination of Morphology and Surface Chemistry Defines the Immunological Identity of Nanocarriers in Human Blood.

Upon exposure to blood, a corona of proteins adsorbs to nanocarrier surfaces to confer a biological identity that interfaces with the immune system. While the nanocarrier surface chemistry has long been the focus of protein corona formation, the influence of nanostructure has remained unclear despite established influences on biodistribution, clearance, and inflammation. Here, combinations of nanocarrier morphology and surface chemistry are engineered to i) achieve compositionally distinct protein coatings in human blood and ii) control protein-mediated interactions with the immune system. A library of nine PEGylated nanocarriers differing in their combination of morphology (spheres, vesicles, and cylinders) and surface chemistry (methoxy, hydroxyl, and phosphate) are synthesized to represent properties of therapeutic and biomimetic delivery vehicles. Analysis by quantitative label-free proteomic techniques reveal that specific surface chemistry and morphology combinations adsorb unique protein signatures from human blood, resulting in differential complement activation and elicitation of distinct proinflammatory cytokine responses. Furthermore, nanocarrier morphology is shown to primarily influence uptake and clearance by human monocytes, macrophages, and dendritic cells. This comprehensive analysis provides mechanistic insights into rational design choices that impact the immunological identity of nanocarriers in human blood, which can be leveraged to engineer drug delivery vehicles for precision medicine and immunotherapy.

Surface Engineering of FLT4-Targeted Nanocarriers Enhances Cell-Softening Glaucoma Therapy.

Primary open-angle glaucoma is associated with elevated intraocular pressure (IOP) that damages the optic nerve and leads to gradual vision loss. Several agents that reduce the stiffness of pressure-regulating Schlemm's canal (SC) endothelial cells, in the conventional outflow pathway of the eye, lower IOP in glaucoma patients and are approved for clinical use. However, poor drug penetration and uncontrolled biodistribution limit their efficacy and produce local adverse effects. Compared to other ocular endothelia, FLT4/VEGFR3 is expressed at elevated levels by SC endothelial cells and can be exploited for targeted drug delivery. Here, we validate FLT4 receptors as clinically relevant targets on SC cells from glaucomatous human donors and engineer polymeric self-assembled nanocarriers displaying lipid-anchored targeting ligands that optimally engage this receptor. Targeting constructs were synthesized as lipid-PEGx-peptide, differing in the number of PEG spacer units (x), and were embedded in micelles. We present a novel proteolysis assay for quantifying ligand accessibility that we employ to design and optimize our FLT4-targeting strategy for glaucoma nanotherapy. Peptide accessibility to proteases correlated with receptor-mediated targeting enhancements. Increasing the accessibility of FLT4-binding peptides enhanced nanocarrier uptake by SC cells while simultaneously decreasing the uptake by off-target vascular endothelial cells. Using a paired longitudinal IOP study in vivo, we show that this enhanced targeting of SC cells translates to IOP reductions that are sustained for a significantly longer time as compared to controls. Confocal microscopy of murine anterior segment tissue confirmed nanocarrier localization to SC within 1 h after intracameral administration. This work demonstrates that steric effects between surface-displayed ligands and PEG coronas significantly impact the targeting performance of synthetic nanocarriers across multiple biological scales. Minimizing the obstruction of modular targeting ligands by PEG measurably improved the efficacy of glaucoma nanotherapy and is an important consideration for engineering PEGylated nanocarriers for targeted drug delivery.

Just add water: hydratable, morphologically diverse nanocarrier powders for targeted delivery.

Two major obstacles that limit the widespread usage of polymeric nanocarriers include the complexity of formulation methods and their stability during storage. To address both of these issues, here we present morphologically complex nanocarriers in a hydratable powder form, which bypasses the need for expensive, harsh, and/or time-consuming nanocarrier fabrication techniques. The powders are composed of carbohydrates and self-assembling polymer amphiphiles having a low glass transition temperature. Hydration requires less than one minute and only involves the addition of aqueous media (water or saline) to rapidly obtain self-assembled micelles, worm-like micelles (i.e. filomicelles), or polymersomes from poly(ethylene glycol)-b-poly(propylene sulfide) (PEG-b-PPS) polymers. The formulated powders are highly stable, achieving hydration into monodisperse nanocarriers following >6 months of storage. Diverse drug cargoes were efficiently encapsulated during hydration, including hydrophobic small molecules for micellar morphologies, as well as individual and concurrent loading of both hydrophobic and hydrophilic molecules for vesicular morphologies. Hydrated polymersomes are shown to load hydrophilic biological macromolecules, and encapsulated enzymes retain bioactivity. Furthermore, we demonstrate that inclusion of lipid-anchored ligands in powder form permits the surface-display of targeting ligands and enhances target cell uptake, thereby extending this technology to targeted drug delivery applications. Our powder-based formulation strategy was extendable to commercially available polymer amphiphiles, including PEG-b-polystyrene and PEG-b-polycaprolactone. The formulated nanotechnologies described herein are highly modular, require minimal preparation, and remain stable in ambient long-term storage (bypassing cold chain requirements), which will enable their use in medicine (human and veterinary), research, and commercial applications from cosmetics to agriculture.

Surface chemistry-mediated modulation of adsorbed albumin folding state specifies nanocarrier clearance by distinct macrophage subsets.

Controlling nanocarrier interactions with the immune system requires a thorough understanding of the surface properties that modulate protein adsorption in biological fluids, since the resulting protein corona redefines cellular interactions with nanocarrier surfaces. Albumin is initially one of the dominant proteins to adsorb to nanocarrier surfaces, a process that is considered benign or beneficial by minimizing opsonization or inflammation. Here, we demonstrate the surface chemistry of a model nanocarrier can be engineered to stabilize or denature the three-dimensional conformation of adsorbed albumin, which respectively promotes evasion or non-specific clearance in vivo. Interestingly, certain common chemistries that have long been considered to convey stealth properties denature albumin to promote nanocarrier recognition by macrophage class A1 scavenger receptors, providing a means for their eventual removal from systemic circulation. We establish that the surface chemistry of nanocarriers can be specified to modulate adsorbed albumin structure and thereby tune clearance by macrophage scavenger receptors.

Homopolymer self-assembly of poly(propylene sulfone) hydrogels via dynamic noncovalent sulfone-sulfone bonding.

Natural biomolecules such as peptides and DNA can dynamically self-organize into diverse hierarchical structures. Mimicry of this homopolymer self-assembly using synthetic systems has remained limited but would be advantageous for the design of adaptive bio/nanomaterials. Here, we report both experiments and simulations on the dynamic network self-assembly and subsequent collapse of the synthetic homopolymer poly(propylene sulfone). The assembly is directed by dynamic noncovalent sulfone-sulfone bonds that are susceptible to solvent polarity. The hydration history, specified by the stepwise increase in water ratio within lower polarity water-miscible solvents like dimethylsulfoxide, controls the homopolymer assembly into crystalline frameworks or uniform nanostructured hydrogels of spherical, vesicular, or cylindrical morphologies. These electrostatic hydrogels have a high affinity for a wide range of organic solutes, achieving >95% encapsulation efficiency for hydrophilic small molecules and biologics. This system validates sulfone-sulfone bonding for dynamic self-assembly, presenting a robust platform for controllable gelation, nanofabrication, and molecular encapsulation.

Implicating calpain in tau-mediated toxicity in vivo.

Alzheimer's disease and other related neurodegenerative disorders known as tauopathies are characterized by the accumulation of abnormally phosphorylated and aggregated forms of the microtubule-associated protein tau. Several laboratories have identified a 17 kD proteolytic fragment of tau in degenerating neurons and in numerous cell culture models that is generated by calpain cleavage and speculated to contribute to tau toxicity. In the current study, we employed a Drosophila tauopathy model to investigate the importance of calpain-mediated tau proteolysis in contributing to tau neurotoxicity in an animal model of human neurodegenerative disease. We found that mutations that disrupted endogenous calpainA or calpainB activity in transgenic flies suppressed tau toxicity. Expression of a calpain-resistant form of tau in Drosophila revealed that mutating the putative calpain cleavage sites that produce the 17 kD fragment was sufficient to abrogate tau toxicity in vivo. Furthermore, we found significant toxicity in the fly retina associated with expression of only the 17 kD tau fragment. Collectively, our data implicate calpain-mediated proteolysis of tau as an important pathway mediating tau neurotoxicity in vivo.