Monitoring Protein Complexation with Polyphosphazene Polyelectrolyte Using Automated Dynamic Light Scattering Titration and Asymmetric Flow Field Flow Fractionation and Protein Recognition Immunoassay.

Polyphosphazenes represent a class of intrinsically flexible polyelectrolytes with potent immunoadjuvant activity, which is enabled through non-covalent self-assembly with antigenic proteins by charge complexation. The formation of supramolecular complexes between polyphosphazene adjuvant, poly[di(carboxylatophenoxy)phosphazene] (PCPP), and a model vaccine antigen, hen egg lysozyme, was studied under physiological conditions using automated dynamic light scattering titration, asymmetric flow field flow fractionation (AF4), enzyme-linked immunosorbent assay (ELISA), and fluorescent quenching methods. Three regimes of self-assembly were observed covering complexation of PCPP with lysozyme in the nano-scale range, multi-chain complexes, and larger aggregates with complexes characterized by a maximum loading of over six hundred protein molecules per PCPP chain and dissociation constant in the micromolar range (Kd = 7 × 10-6 mol/L). The antigenicity of PCPP bound lysozyme, when compared to equivalent lysozyme solutions, was largely retained for all complexes, but observed a dramatic reduction for heavily aggregated systems. Routes to control the complexation regimes with elevated NaCl or KCl salt concentrations indicate ion-specific effects, such that more smaller-size complexes are present at higher NaCl, counterintuitive with respect to PCPP solubility arguments. While the order of mixing shows a prominent effect at lower stoichiometries of mixing, higher NaCl salt reduces the effect all together.

Advances in the Synthesis of Preceramic Polymers for the Formation of Silicon-Based and Ultrahigh-Temperature Non-Oxide Ceramics.

Preceramic polymers (PCPs) are a group of specialty macromolecules that serve as precursors for generating inorganics, including ceramic carbides, nitrides, and borides. PCPs represent interesting synthetic challenges for chemists due to the elements incorporated into their structure. This group of polymers is also of interest to engineers as PCPs enable the processing of polymer-derived ceramic products including high-performance ceramic fibers and composites. These finished ceramic materials are of growing significance for applications that experience extreme operating environments (e.g., aerospace propulsion and high-speed atmospheric flight). This Review provides an overview of advances in the synthesis and postpolymerization modification of macromolecules forming nonoxide ceramics. These PCPs include polycarbosilanes, polysilanes, polysilazanes, and precursors for ultrahigh-temperature ceramics. Following our review of PCP synthetic chemistry, we provide examples of the application and processing of these polymers, including their use in fiber spinning, composite fabrication, and additive manufacturing. The principal objective of this Review is to provide a resource that bridges the disciplines of synthetic chemistry and ceramic engineering while providing both insights and inspiration for future collaborative work that will ultimately drive the PCP field forward.

Protein Binding and Release by Polymeric Cell-Penetrating Peptide Mimics.

There is significant potential in exploiting antibody specificity to develop new therapeutic treatments. However, intracellular protein delivery is a paramount challenge because of the difficulty in transporting large, polar molecules across cell membranes. Cell-penetrating peptide mimics (CPPMs) are synthetic polymers that are versatile materials for intracellular delivery of biological molecules, including nucleic acids and proteins, with superior performance compared to their natural counterparts and commercially available peptide-based reagents. Studies have demonstrated that noncovalent complexation with these synthetic carriers is necessary for the delivery of proteins, but the fundamental interactions dominating CPPM-protein complexation are not well understood. Beyond these interactions, the mechanism of release for many noncovalent carriers is not well established. Herein, interactions expected to be critical in CPPM-protein binding and unbinding were explored, including hydrogen bonding, electrostatics, and hydrophobic interactions. Despite the guanidinium-rich functionality of these polymeric carriers, hydrogen bonding was shown not to be a dominant interaction in CPPM-protein binding. Fluorescence quenching assays were used to decouple the effect of electrostatic and hydrophobic interactions between amphiphilic CPPMs and proteins. Furthermore, by conducting competition assays with other proteins, unbinding of protein cargoes from CPPM-protein complexes was demonstrated and provided insight into mechanisms of protein release. This work offers understanding toward the role of carrier and cargo binding and unbinding in intracellular outcomes. In turn, an improved fundamental understanding of noncovalent polymer-protein complexation will enable more effective methods for intracellular protein delivery.

Tuning Net Charge in Aliphatic Polycarbonates Alters Solubility and Protein Complexation Behavior.

A synthetic strategy yielded polyelectrolytes and polyampholytes with tunable net charge for complexation and protein binding. Organocatalytic ring-opening polymerizations yielded aliphatic polycarbonates that were functionalized with both carboxylate and ammonium side chains in a post-polymerization, radical-mediated thiol-ene reaction. Incorporating net charge into the polymer architecture altered the chain dimensions in phosphate buffered solution in a manner consistent with self-complexation and complexation behavior with model proteins. A net cationic polyampholyte with 5% of carboxylate side chains formed large clusters rather than small complexes with bovine serum albumin, while 50% carboxylate polyampholyte was insoluble. Overall, the aliphatic polycarbonates with varying net charge exhibited different macrophase solution behaviors when mixed with protein, where self-complexation appears to compete with protein binding and larger-scale complexation.

Non-Covalent Carrier Hydrophobicity as a Universal Predictor of Intracellular Protein Activity.

Over the past decade, extensive optimization of polymeric cell-penetrating peptide (CPP) mimics (CPPMs) by our group has generated a substantial library of broadly effective carriers which circumvent the need for covalent conjugation often required by CPPs. In this study, design rules learned from CPPM development were applied to reverse-engineer the first library of simple amphiphilic block copolypeptides for non-covalent protein delivery, namely, poly(alanine-block-arginine), poly(phenylalanine-block-arginine), and poly(tryptophan-block-arginine). This new CPP library was screened for enhanced green fluorescent protein and Cre recombinase delivery alongside a library of CPPMs featuring equivalent side-chain configurations. Due to the added hydrophobicity imparted by the polymer backbone as compared to the polypeptide backbone, side-chain functionality was not a universal predictor of carrier performance. Rather, overall carrier hydrophobicity predicted the top performers for both internalization and activity of protein cargoes, regardless of backbone identity. Furthermore, comparison of protein uptake and function revealed carriers which facilitated high gene recombination despite remarkably low Cre internalization, leading us to formalize the concept of intracellular availability (IA) of the delivered cargo. IA, a measure of cargo activity per quantity of cargo internalized, provides valuable insight into the physical relationship between cellular internalization and bioavailability, which can be affected by bottlenecks such as endosomal escape and cargo release. Importantly, carriers with maximal IA existed within a narrow hydrophobicity window, more hydrophilic than those exhibiting maximal cargo uptake. Hydrophobicity may be used as a scaffold-independent predictor of protein uptake, function, and IA, enabling identification of new, effective carriers which would be overlooked by uptake-based screening methods.

The Role of Cargo Binding Strength in Polymer-Mediated Intracellular Protein Delivery.

Delivering proteins into the intracellular environment is a critical step toward probing vital cellular processes for the purposes of ultimately developing new therapeutics. Polymeric carriers are widely used to facilitate protein delivery with guanidinium-rich macromolecules leading the way within this category. Although binding interactions between natural proteins and synthetic polymers have been studied extensively, the relationship between polymer-protein binding and intracellular delivery is seldom explored. Elucidating the role of cargo binding in delivery is a promising direction that is expected to provide new insights that further optimize intracellular protein delivery. Herein, model polymeric carriers called protein transduction domain mimics (PTDMs) were studied for their ability to bind to a variety of protein cargoes, including an antibody, where the proteins encompassed a range of sizes (∼16-151 kDa) and isoelectric points (4.7-11.4). The PTDM-protein complexes were also delivered into Jurkat T cells in an attempt to establish a general correlation between binding ability and delivery outcomes. Binding assays resulted in a vast range of dissociation constants (Kd), which spanned from 3.5 to 4820 nM and indicated a variety of binding strengths between PTDM and protein. More significantly, PTDMs preferentially bound certain types of proteins over others, such as the antibody fragment over the whole antibody. Furthermore, increased PTDM-protein binding affinity did not correlate with protein delivery, suggesting that the successful internalization of complexes is independent of binding equilibrium. Although binding did not correlate with internalization here, the potential for binding affinity to impact other aspects of delivery, like cargo functionality inside the cell, remains an open possibility.

Associative and Dissociative Processes in Non-Covalent Polymer-Mediated Intracellular Protein Delivery.

Functional protein delivery has created new opportunities for studying intracellular processes and discovering new therapeutics. To that end, researchers have pursued intracellular protein delivery by using an increasing number of methods. This focus review will highlight polymeric carriers that non-covalently bind and deliver protein cargo in vitro. The correlation between polymer-protein binding and delivery as well as the correlation between complex-membrane binding and delivery is reviewed. Finally, binding and its relation to the intracellular function of the protein post-delivery is considered. The purpose of this review is to evaluate the role that binding interactions play in the non-covalent protein-delivery landscape. Presently, the literature does not adequately resolve how binding throughout the process ultimately affects delivery. The field does contain preliminary insights that are expected to impact future delivery applications when developed further.

Optimal Hydrophobicity in Ring-Opening Metathesis Polymerization-Based Protein Mimics Required for siRNA Internalization.

Exploring the role of polymer structure for the internalization of biologically relevant cargo, specifically siRNA, is of critical importance to the development of improved delivery reagents. Herein, we report guanidinium-rich protein transduction domain mimics (PTDMs) based on a ring-opening metathesis polymerization scaffold containing tunable hydrophobic moieties that promote siRNA internalization. Structure-activity relationships using Jurkat T cells and HeLa cells were explored to determine how the length of the hydrophobic block and the hydrophobic side chain compositions of these PTDMs impacted siRNA internalization. To explore the hydrophobic block length, two different series of diblock copolymers were synthesized: one series with symmetric block lengths and one with asymmetric block lengths. At similar cationic block lengths, asymmetric and symmetric PTDMs promoted siRNA internalization in the same percentages of the cell population regardless of the hydrophobic block length; however, with 20 repeat units of cationic charge, the asymmetric block length had greater siRNA internalization, highlighting the nontrivial relationships between hydrophobicity and overall cationic charge. To further probe how the hydrophobic side chains impacted siRNA internalization, an additional series of asymmetric PTDMs was synthesized that featured a fixed hydrophobic block length of five repeat units that contained either dimethyl (dMe), methyl phenyl (MePh), or diphenyl (dPh) side chains and varied cationic block lengths. This series was further expanded to incorporate hydrophobic blocks consisting of diethyl (dEt), diisobutyl (diBu), and dicyclohexyl (dCy) based repeat units to better define the hydrophobic window for which our PTDMs had optimal activity. High-performance liquid chromatography retention times quantified the relative hydrophobicities of the noncationic building blocks. PTDMs containing the MePh, diBu, and dPh hydrophobic blocks were shown to have superior siRNA internalization capabilities compared to their more and less hydrophobic counterparts, demonstrating a critical window of relative hydrophobicity for optimal internalization. This better understanding of how hydrophobicity impacts PTDM-induced internalization efficiencies will help guide the development of future delivery reagents.