Multivalent Scaffolds to Promote B cell Tolerance.

Autoimmune diseases are characterized by aberrant immune responses toward self-antigens. Current treatments lack specificity, promoting adverse effects by broadly suppressing the immune system. Therapies that specifically target the immune cells responsible for disease are a compelling strategy to mitigate adverse effects. Multivalent formats that display numerous binding epitopes off a single scaffold may enable selective immunomodulation by eliciting signals through pathways unique to the targeted immune cells. However, the architecture of multivalent immunotherapies can vary widely, and there is limited clinical data with which to evaluate their efficacy. Here, we set forth to review the architectural properties and functional mechanisms afforded by multivalent ligands and evaluate four multivalent scaffolds that address autoimmunity by altering B cell signaling pathways. First, we address both synthetic and natural polymer backbones functionalized with a variety of small molecule, peptide, and protein ligands for probing the effects of valency and costimulation. Then, we review nanoparticles composed entirely from immune signals which have been shown to be efficacious. Lastly, we outline multivalent liposomal nanoparticles capable of displaying high numbers of protein antigens. Taken together, these examples highlight the versatility and desirability of multivalent ligands for immunomodulation and illuminate strengths and weaknesses of multivalent scaffolds for treating autoimmunity.

Surface Presentation of Hyaluronic Acid Modulates Nanoparticle-Cell Association.

Nanoparticle (NP) drug carriers have revolutionized medicine and increased patient quality of life. Clinically approved formulations typically succeed because of reduced off-target toxicity of the cargo. However, increasing carrier accumulation at disease sites through precise targeting remains one of the biggest challenges in the field. Novel multivalent ligand presentations and self-assembled constructs can enhance cell association, but an inability to draw direct comparisons across formulations has hindered progress. Furthermore, how nanoparticle structure influences function often is unclear. In this report, we leverage the well-characterized hyaluronic acid (HA)-CD44 binding pair to investigate how the surface architecture of modified NPs impacts their association with ovarian cancer cells that overexpress CD44. We functionalized anionic liposomes with 5 kDa HA by either covalent conjugation via surface coupling or electrostatic self-assembly using the layer-by-layer (LbL) adsorption method. Comparing these two methods, we observed a consistent enhancement of NP-cell association with the self-assembly LbL technique, particularly with higher molecular weight (≥10 kDa) HA. To further optimize association, we increased the surface-available HA. We synthesized a bottlebrush glycopolymer composed of a polynorbornene backbone and pendant 5 kDa HA and layered this macromolecule onto NPs. Flow cytometry revealed that the LbL HA bottlebrush NP outperformed the LbL linear display of HA. Cellular visualization by deconvolution optical microscopy corroborated results from all three constructs. Using exogenous HA to block NP-CD44 interactions, we found the LbL HA bottlebrush NP had a 4-fold higher binding avidity than the best-performing LbL linear HA NP. We further observed that decreasing the density of HA bottlebrush side chains to 75% had minimal impact on LbL NP stability or cell association, though we did see a reduction in binding avidity with this side-chain-modified NP. Our studies indicate that LbL surfaces are highly effective for multivalent displays, and the mode in which they present a targeting ligand can be optimized for NP cell targeting.

Stereochemical Control Yields Mucin Mimetic Polymers.

All animals except sponges produce mucus. Across the animal kingdom, this hydrogel mediates surface wetting, viscosity, and protection against microbes. The primary components of mucus hydrogels are mucins-high molecular weight O-glycoproteins that adopt extended linear structures. Glycosylation is integral to mucin function, but other characteristics that give rise to their advantageous biological activities are unknown. We postulated that the extended conformation of mucins is critical for their ability to block microbial virulence phenotypes. To test this hypothesis, we developed synthetic mucin mimics that recapitulate the dense display of glycans and morphology of mucin. We varied the catalyst in a ring-opening metathesis polymerization (ROMP) to generate substituted norbornene-derived glycopolymers containing either cis- or trans-alkenes. Conformational analysis of the polymers based on allylic strain suggested that cis- rather than trans-poly(norbornene) glycopolymers would adopt linear structures that mimic mucins. High-resolution atomic force micrographs of our polymers and natively purified Muc2, Muc5AC, and Muc5B mucins revealed that cis-polymers adopt extended, mucin-like structures. The cis-polymers retained this structure in solution and were more water-soluble than their trans-analogs. Consistent with mucin's linear morphology, cis-glycopolymers were more potent binders of a bacterial virulence factor, cholera toxin. Our findings highlight the importance of the polymer backbone in mucin surrogate design and underscore the significance of the extended mucin backbone for inhibiting virulence.

Stimulated Raman scattering of polymer nanoparticles for multiplexed live-cell imaging.

A novel nanoparticle-based imaging strategy is introduced that couples biocompatible organic nanoparticles and stimulated Raman scattering (SRS) microscopy. Polymer nanoparticles with vibrational labels incorporated were readily prepared for multi-color SRS imaging with excellent photo-stability. The Raman-active polymer dots are nontoxic, rapidly enter various cell types, and are applied in multiplexed cell-type sorting.

Influence of Substituent Chain Branching on the Transfection Efficacy of Cyclopropenium-Based Polymers.

The realization of gene therapy relies on the development of delivery vectors with high efficiency and biocompatibility. With a multitude of structures accessible, the core challenge is precisely tuning vector structure to probe and optimize structure⁻property relationships. Employing a modular strategy, two pairs of cationic polymers based on the trisaminocyclopropenium (TAC) ion were synthesized where the substituents differ in the degree of alkyl chain branching. All TAC-based polymers exhibited higher transfection efficiencies than the untreated controls, with variable in vitro toxicities. Considering both cytotoxicity and transfection efficacy, an optimal nonviral vector was identified. Our studies highlight the importance of exercising precise control over polymer structure, both in terms of backbone identity and substituent nature, and the necessity of a robust, modular platform from which to study them.

Clickable Poly(ionic liquids): A Materials Platform for Transfection.

The potential applications of cationic poly(ionic liquids) range from medicine to energy storage, and the development of efficient synthetic strategies to target innovative cationic building blocks is an important goal. A post-polymerization click reaction is reported that provides facile access to trisaminocyclopropenium (TAC) ion-functionalized macromolecules of various architectures, which are the first class of polyelectrolytes that bear a formal charge on carbon. Quantitative conversions of polymers comprising pendant or main-chain secondary amines were observed for an array of TAC derivatives in three hours using near equimolar quantities of cyclopropenium chlorides. The resulting TAC polymers are biocompatible and efficient transfection agents. This robust, efficient, and orthogonal click reaction of an ionic liquid, which we term ClickabIL, allows straightforward screening of polymeric TAC derivatives. This platform provides a modular route to synthesize and study various properties of novel TAC-based polymers.

The evolution of cyclopropenium ions into functional polyelectrolytes.

Versatile polyelectrolytes with tunable physical properties have the potential to be transformative in applications such as energy storage, fuel cells and various electronic devices. Among the types of materials available for these applications, nanostructured cationic block copolyelectrolytes offer mechanical integrity and well-defined conducting paths for ionic transport. To date, most cationic polyelectrolytes bear charge formally localized on heteroatoms and lack broad modularity to tune their physical properties. To overcome these challenges, we describe herein the development of a new class of functional polyelectrolytes based on the aromatic cyclopropenium ion. We demonstrate the facile synthesis of a series of polymers and nanoparticles based on monomeric cyclopropenium building blocks incorporating various functional groups that affect physical properties. The materials exhibit high ionic conductivity and thermal stability due to the nature of the cationic moieties, thus rendering this class of new materials as an attractive alternative to develop ion-conducting membranes.