Ensembles of synthetic polymers mimic biological fluids.

Recently a report by Ruan et al. in Nature described how relatively simple random heteropolymers can replicate the properties of biological fluids. These polymers capture the segmental-level interactions between proteins and could enhance folding of membrane proteins, improve stability, and enable DNA sequestration in a chemistry specific manner.

Self-Assembling Polypeptides in Complex Coacervation.

Intracellular compartmentalization plays a pivotal role in cellular function, with membrane-bound organelles and membrane-less biomolecular "condensates" playing key roles. These condensates, formed through liquid-liquid phase separation (LLPS), enable selective compartmentalization without the barrier of a lipid bilayer, thereby facilitating rapid formation and dissolution in response to stimuli. Intrinsically disordered proteins (IDPs) or proteins with intrinsically disordered regions (IDRs), which are often rich in charged and polar amino acid sequences, scaffold many condensates, often in conjunction with RNA.Comprehending the impact of IDP/IDR sequences on phase separation poses a challenge due to the extensive chemical diversity resulting from the myriad amino acids and post-translational modifications. To tackle this hurdle, one approach has been to investigate LLPS in simplified polypeptide systems, which offer a narrower scope within the chemical space for exploration. This strategy is supported by studies that have demonstrated how IDP function can largely be understood based on general chemical features, such as clusters or patterns of charged amino acids, rather than residue-level effects, and the ways in which these kinds of motifs give rise to an ensemble of conformations.Our laboratory has utilized complex coacervates assembled from oppositely charged polypeptides as a simplified material analogue to the complexity of liquid-liquid phase separated biological condensates. Complex coacervation is an associative LLPS that occurs due to the electrostatic complexation of oppositely charged macro-ions. This process is believed to be driven by the entropic gains resulting from the release of bound counterions and the reorganization of water upon complex formation. Apart from their direct applicability to IDPs, polypeptides also serve as excellent model polymers for investigating molecular interactions due to the wide range of available side-chain functionalities and the capacity to finely regulate their sequence, thus enabling precise control over interactions with guest molecules.Here, we discuss fundamental studies examining how charge patterning, hydrophobicity, chirality, and architecture affect the phase separation of polypeptide-based complex coacervates. These efforts have leveraged a combination of experimental and computational approaches that provide insight into molecular level interactions. We also examine how these parameters affect the ability of complex coacervates to incorporate globular proteins and viruses. These efforts couple directly with our fundamental studies into coacervate formation, as such "guest" molecules should not be considered as experiencing simple encapsulation and are instead active participants in the electrostatic assembly of coacervate materials. Interestingly, we observed trends in the incorporation of proteins and viruses into coacervates formed using different chain length polypeptides that are not well explained by simple electrostatic arguments and may be the result of more complex interactions between globular and polymeric species. Additionally, we describe experimental evidence supporting the potential for complex coacervates to improve the thermal stability of embedded biomolecules, such as viral vaccines.Ultimately, peptide-based coacervates have the potential to help unravel the physics behind biological condensates, while paving the way for innovative methods in compartmentalization, purification, and biomolecule stabilization. These advancements could have implications spanning medicine to biocatalysis.

Design Rules for the Sequestration of Viruses into Polypeptide Complex Coacervates.

Encapsulation is a strategy that has been used to facilitate the delivery and increase the stability of proteins and viruses. Here, we investigate the encapsulation of viruses via complex coacervation, which is a liquid-liquid phase separation resulting from the complexation of oppositely charged polymers. In particular, we utilized polypeptide-based coacervates and explored the effects of peptide chemistry, chain length, charge patterning, and hydrophobicity to better understand the effects of the coacervating polypeptides on virus incorporation. Our study utilized two nonenveloped viruses, porcine parvovirus (PPV) and human rhinovirus (HRV). PPV has a higher charge density than HRV, and they both appear to be relatively hydrophobic. These viruses were compared to characterize how the charge, hydrophobicity, and patterning of chemistry on the surface of the virus capsid affects encapsulation. Consistent with the electrostatic nature of complex coacervation, our results suggest that electrostatic effects associated with the net charge of both the virus and polypeptide dominated the potential for incorporating the virus into a coacervate, with clustering of charges also playing a significant role. Additionally, the hydrophobicity of a virus appears to determine the degree to which increasing the hydrophobicity of the coacervating peptides can enhance virus uptake. Nonintuitive trends in uptake were observed with regard to both charge patterning and polypeptide chain length, with these parameters having a significant effect on the range of coacervate compositions over which virus incorporation was observed. These results provide insights into biophysical mechanisms, where sequence effects can control the uptake of proteins or viruses into biological condensates and provide insights for use in formulation strategies.

Conjugated Polyelectrolyte-Based Complex Fluids as Aqueous Exciton Transport Networks.

The ability to assemble artificial systems that mimic aspects of natural light-harvesting functions is fascinating and attractive for materials design. Given the complexity of such a system, a simple design pathway is desirable. Here, we argue that associative phase separation of oppositely charged conjugated polyelectrolytes (CPEs) can provide such a path in an environmentally benign medium: water. We find that complexation between an exciton-donor and acceptor CPE leads to formation of a complex fluid. We interrogate exciton transfer from the donor to the acceptor CPE within the complex fluid and find that transfer is highly efficient. We also find that excess molecular ions can tune the modulus of the inter-CPE complex fluid. Even at high ion concentrations, CPEs remain complexed with significantly delocalized electronic wavefunctions. Our work lays the rational foundation for complex, tunable aqueous light-harvesting systems via the intrinsic thermodynamics of associative phase separation.

The utility of c-Met as a diagnostic tissue biomarker in primary colorectal cancer.

The transmembrane protein, c-Met, is thought to be overexpressed and activated in colorectal cancer (CRC). This study explored its potential as a diagnostic tissue biomarker for CRC in a large human CRC tissue collection obtained from a randomized clinical trial. Tissue microarrays of matched normal colorectal epithelium and primary cancer were prepared from specimens obtained from 280 patients recruited to the MRC CLASICC trial (ISRCTN 74883561) and interrogated using immunohistochemistry for c-Met expression. The distribution and intensity of immunopositivity was graded using a validated, semi-quantifiable score, and differences in median scores analysed using the Wilcoxon signed-rank test. A receiver operating characteristic (ROC) curve was plotted to measure the diagnostic accuracy of c-Met as a biomarker in CRC. Epithelial cell membrane expression of c-Met differed significantly between CRC and normal colorectal tissue: median 12.00 (Interquartile range (IQR) 6-15) versus median 6.00 (IQR 2.70-12.00) respectively (P = <.0001). ROC-AUC analysis of c-Met expression yielded a CRC diagnostic probability of 0.66 (95% CI: 0.61 to 0.70; P < .0001). A score of ≥14.50 showed high specificity at 85.32% (95% CI 80.33%-89.45%) but sensitivity of only 30.92% (CI 25.37%-36.90%). Thus c-Met is consistently overexpressed in human CRC as compared to normal colorectal epithelium tissue. c-Met expression may have a role in diagnosis and prognostication if combined with other biomarkers.

Luminal Bioavailability of Orally Administered ω-3 PUFAs in the Distal Small Intestine, and Associated Changes to the Ileal Microbiome, in Humans with a Temporary Ileostomy.

BACKGROUND: Oral administration of purified omega-3 (ω-3) PUFAs is associated with changes to the fecal microbiome. However, it is not known whether this effect is associated with increased PUFA concentrations in the gut. OBJECTIVES: We investigated the luminal bioavailability of oral ω-3 PUFAs (daily dose 1 g EPA and 1g DHA free fatty acid equivalents as triglycerides in soft-gel capsules, twice daily) and changes to the gut microbiome, in the ileum. METHODS: Ileostomy fluid (IF) and blood were obtained at baseline, after first capsule dosing (median 2 h), and at a similar time after final dosing on day 28, in 11 individuals (median age 63 y) with a temporary ileostomy. Fatty acids were measured by LC-tandem MS. The ileal microbiome was characterized by 16S rRNA PCR and Illumina sequencing. RESULTS: There was a mean 6.0 ± 9.8-fold and 6.6 ± 9.6-fold increase in ileal EPA and DHA concentrations (primary outcome), respectively, at 28 d, which was associated with increased RBC ω-3 PUFA content (P ≤ 0.05). The first oral dose did not increase the ileal ω-3 PUFA concentration except in 4 individuals, who displayed high luminal EPA and DHA concentrations, which reduced to concentrations similar to the overall study population at day 28, suggesting physiological adaptation. Bacteroides, Clostridium, and Streptococcus were abundant bacterial genera in the ileum. Ileal microbiome variability over time and between individuals was large, with no consistent change associated with acute ω-3 PUFA dosing. However, high concentrations of EPA and DHA in IF on day 28 were associated with higher abundance of Bacteroides (r2 > 0.86, P < 0.05) and reduced abundance of other genera, including Actinomyces (r2 > 0.94, P < 0.05). CONCLUSIONS: Oral administration of ω-3 PUFAs leads to increased luminal ω-3 PUFA concentrations and changes to the microbiome, in the ileum of individuals with a temporary ileostomy. This study is registered on the ISRCTN registry as ISRCTN14530452.

Protein Encapsulation Using Complex Coacervates: What Nature Has to Teach Us.

Protein encapsulation is a growing area of interest, particularly in the fields of food science and medicine. The sequestration of protein cargoes is achieved using a variety of methods, each with benefits and drawbacks. One of the most significant challenges associated with protein encapsulation is achieving high loading while maintaining protein viability. This difficulty is exacerbated because many encapsulant systems require the use of organic solvents. By contrast, nature has optimized strategies to compartmentalize and protect proteins inside the cell-a purely aqueous environment. Although the mechanisms whereby aspects of the cytosol is able to stabilize proteins are unknown, the crowded nature of many newly discovered, liquid phase separated "membraneless organelles" that achieve protein compartmentalization suggests that the material environment surrounding the protein may be critical in determining stability. Here, encapsulation strategies based on liquid-liquid phase separation, and complex coacervation in particular, which has many of the key features of the cytoplasm as a material, are reviewed. The literature on protein encapsulation via coacervation is also reviewed and the parameters relevant to creating protein-containing coacervate formulations are discussed. Additionally, potential opportunities associated with the creation of tailored materials to better facilitate protein encapsulation and stabilization are highlighted.

Recent progress in the science of complex coacervation.

Complex coacervation is an associative, liquid-liquid phase separation that can occur in solutions of oppositely-charged macromolecular species, such as proteins, polymers, and colloids. This process results in a coacervate phase, which is a dense mix of the oppositely-charged components, and a supernatant phase, which is primarily devoid of these same species. First observed almost a century ago, coacervates have since found relevance in a wide range of applications; they are used in personal care and food products, cutting edge biotechnology, and as a motif for materials design and self-assembly. There has recently been a renaissance in our understanding of this important class of material phenomena, bringing the science of coacervation to the forefront of polymer and colloid science, biophysics, and industrial materials design. In this review, we describe the emergence of a number of these new research directions, specifically in the context of polymer-polymer complex coacervates, which are inspired by a number of key physical and chemical insights and driven by a diverse range of experimental, theoretical, and computational approaches.

The benefit of poor mixing: kinetics of coacervation.

Complex coacervation has become a prominent area of research in the fields of food science, personal care, drug stabilization, and more. However, little has been reported on the kinetics of assembly of coacervation itself. Here, we describe a simple, low-cost way of looking at the kinetics of coacervation by creating poorly mixed samples. In particular, we examine how polymer chain length, the patterning and symmetry of charges on the oppositely charged polyelectrolytes, and the presence of salt and a zwitterionic buffer affect the kinetics of complex coacervation. Our results suggest an interesting relationship between the time for equilibration and the order of addition of polymers with asymmetric patterns of charge. Furthermore, we demonstrated that increasing polymer chain length resulted in a non-monotonic trend in the sample equilibration times as a result of opposing factors such as excluded volume and diffusion. We also observed differences in the rate of sample equilibration based on the presence of a neutral, zwitterionic buffer, as well as the presence and identity of added salt, consistent with previous reports of salt-specific effects on the rheology of complex coacervates. While not a replacement for more advanced characterization strategies, this turbidity-based method could serve as a screening tool to identify interesting and unique phenomena for further study.

Recent Advances in Encapsulation, Protection, and Oral Delivery of Bioactive Proteins and Peptides using Colloidal Systems.

There are many areas in medicine and industry where it would be advantageous to orally deliver bioactive proteins and peptides (BPPs), including ACE inhibitors, antimicrobials, antioxidants, hormones, enzymes, and vaccines. A major challenge in this area is that many BPPs degrade during storage of the product or during passage through the human gut, thereby losing their activity. Moreover, many BPPs have undesirable taste profiles (such as bitterness or astringency), which makes them unpleasant to consume. These challenges can often be overcome by encapsulating them within colloidal particles that protect them from any adverse conditions in their environment, but then release them at the desired site-of-action, which may be inside the gut or body. This article begins with a discussion of BPP characteristics and the hurdles involved in their delivery. It then highlights the characteristics of colloidal particles that can be manipulated to create effective BPP-delivery systems, including particle composition, size, and interfacial properties. The factors impacting the functional performance of colloidal delivery systems are then highlighted, including their loading capacity, encapsulation efficiency, protective properties, retention/release properties, and stability. Different kinds of colloidal delivery systems suitable for encapsulation of BPPs are then reviewed, such as microemulsions, emulsions, solid lipid particles, liposomes, and microgels. Finally, some examples of the use of colloidal delivery systems for delivery of specific BPPs are given, including hormones, enzymes, vaccines, antimicrobials, and ACE inhibitors. An emphasis is on the development of food-grade colloidal delivery systems, which could be used in functional or medical food applications. The knowledge presented should facilitate the design of more effective vehicles for the oral delivery of bioactive proteins and peptides.