Amphiphilic Block Copolymer Nanostructures as a Tunable Delivery Platform: Perspective and Framework for the Future Drug Product Development.

Nanoformulation of active payloads or pharmaceutical ingredients (APIs) has always been an area of interest to achieve targeted, sustained, and efficacious delivery. Various delivery platforms have been explored, but loading and delivery of APIs have been challenging because of the chemical and structural properties of these molecules. Polymersomes made from amphiphilic block copolymers (ABCPs) have shown enormous promise as a tunable API delivery platform and confer multifold advantages over lipid-based systems. For example, a COVID booster vaccine comprising polymersomes encapsulating spike protein (ACM-001) has recently completed a Phase I clinical trial and provides a case for developing safe drug products based on ABCP delivery platforms. However, several limitations need to be resolved before they can reach their full potential. In this Perspective, we would like to highlight such aspects requiring further development for translating an ABCP-based delivery platform from a proof of concept to a viable commercial product.

Outer-Membrane Protease (OmpT) Based E. coli Sensing with Anionic Polythiophene and Unlabeled Peptide Substrate.

E. coli and Salmonella are two of the most common bacterial pathogens involved in foodborne and waterborne related deaths. Hence, it is critical to develop rapid and sensitive detection strategies for near-outbreak applications. Reported is a simple and specific assay to detect as low as 1 CFU mL-1 of E. coli in water within 6 hours by targeting the bacteria's surface protease activity. The assay relies on polythiophene acetic acid (PTAA) as an optical reporter and a short unlabeled peptide (LL37FRRV ) previously optimized as a substrate for OmpT, an outer-membrane protease on E. coli. LL37FRRV interacts with PTAA to enhance its fluorescence while also inducing the formation of a helical PTAA-LL37FRRV construct, as confirmed by circular dichroism. However, in the presence of E. coli LL37FRRV is cleaved and can no longer affect the conformations and optical properties of PTAA. This ability to distinguish between an intact and cleaved peptide was investigated in detail using LL37FRRV sequence variants.

A Bottom-Up Proteomic Approach to Identify Substrate Specificity of Outer-Membrane Protease OmpT.

Identifying peptide substrates that are efficiently cleaved by proteases gives insights into substrate recognition and specificity, guides development of inhibitors, and improves assay sensitivity. Peptide arrays and SAMDI mass spectrometry were used to identify a tetrapeptide substrate exhibiting high activity for the bacterial outer-membrane protease (OmpT). Analysis of protease activity for the preferred residues at the cleavage site (P1, P1') and nearest-neighbor positions (P2, P2') and their positional interdependence revealed FRRV as the optimal peptide with the highest OmpT activity. Substituting FRRV into a fragment of LL37, a natural substrate of OmpT, led to a greater than 400-fold improvement in OmpT catalytic efficiency, with a kcat /Km value of 6.1×106  L mol-1 s-1 . Wild-type and mutant OmpT displayed significant differences in their substrate specificities, demonstrating that even modest mutants may not be suitable substitutes for the native enzyme.

Rapid Detection of Escherichia coli: Optimized Peptide-Polythiophene Interactions Help Reduce Assay Time and Improve Naked-Eye Detection.

Recent improvements in methods for rapid detection of microbial contamination in food and water samples have aided in the development of on-site and point-of-care testing. Early detection, made possible via on-site testing, can help limit the spread of food and waterborne illnesses. Recently, we reported a novel fluorescence-based Omptin-Polythiophene assay (the assay) to detect Escherichia coli in contaminated water samples. The assay targets OmpT─an E. coli outer membrane protease─and exploits the protease's ability to cleave at dibasic sites within a peptide. By combining a peptide substrate optimized for OmpT with a conjugated polythiophene reporter molecule whose optical properties vary upon interaction with the intact or cleaved peptide, we demonstrated the detection of 1-10 CFU/mL and 105 CFU/mL E. coli in 5.5 and 1 h, respectively. In comparison, most microbial detection methods that rely on culturing and plating techniques take anywhere between 8 and 24 h to report their results. Herein we report significant improvements in the assay which include reducing the assay time from an already short 1 h to a mere 10 min for detecting E. coli in highly contaminated samples and augmenting the assay with colorimetric sensing capability for naked eye discernment under normal visible light or under UV-A light. These improvements were made possible by characterizing the optical changes resulting from the interaction of the peptide with five carboxylate-functionalized polythiophene variants carrying different 3- side chain carboxylic acids and by identifying preferential peptide substrates via the screening of ten peptide sequence variants for OmpT activity.

A Perspective on Polythiophenes as Conformation Dependent Optical Reporters for Label-Free Bioanalytics.

Poly(3-alkylthiophene) (PT)-based conjugated polyelectrolytes (CPEs) constitute an important class of responsive polymers with excellent optical properties. The electrostatic interactions between PTs and target analytes trigger complexation and concomitant conformational changes of the PT backbones that produce distinct optical responses. These conformation-induced optical responses of the PTs enable them to be utilized as reporters for detection of various analytes by employing simple UV-vis spectrophotometry or the naked eye. Numerous PTs with unique pendant groups have been synthesized to tailor their interactions with analytes such as nucleotides, ions, surfactants, proteins, and bacterial and viral pathogens. In this perspective, we discuss PT-target analyte complexation for bioanalytical applications and highlight recent advancements in point-of-care and field deployable assays. Subsequently, we highlight a few areas of critical importance for future applications of PTs as reporters, including (i) design and synthesis of specific PTs to advance the understanding of the mechanisms of interaction with target analytes, (ii) using arrays of PTs and linear discriminant analysis for selective and specific detection of target analytes, (iii) translation of conventional homogeneous solution-based assays into heterogeneous membrane-based assay formats, and finally (iv) the potential of using PT as an alternative to conjugated polymer nanoparticles and dots in bioimaging.

Artificial Cell Membrane Polymersome-Based Intranasal Beta Spike Formulation as a Second Generation Covid-19 Vaccine.

Current parenteral coronavirus disease 2019 (Covid-19) vaccines inadequately protect against infection of the upper respiratory tract. Additionally, antibodies generated by wild type (WT) spike-based vaccines poorly neutralize severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants. To address the need for a second-generation vaccine, we have initiated a preclinical program to produce and evaluate a potential candidate. Our vaccine consists of recombinant Beta spike protein coadministered with synthetic CpG adjuvant. Both components are encapsulated within artificial cell membrane (ACM) polymersomes, synthetic nanovesicles efficiently internalized by antigen presenting cells, including dendritic cells, enabling targeted delivery of cargo for enhanced immune responses. ACM vaccine is immunogenic in C57BL/6 mice and Golden Syrian hamsters, evoking high serum IgG and neutralizing responses. Compared to an ACM-WT spike vaccine that generates predominantly WT-neutralizing antibodies, the ACM-Beta spike vaccine induces antibodies that neutralize WT and Beta viruses equally. Intramuscular (IM)-immunized hamsters are strongly protected from weight loss and other clinical symptoms after the Beta challenge but show delayed viral clearance in the upper airway. With intranasal (IN) immunization, however, neutralizing antibodies are generated in the upper airway concomitant with rapid and potent reduction of viral load. Moreover, antibodies are cross-neutralizing and show good activity against Omicron. Safety is evaluated in New Zealand white rabbits in a repeated dose toxicological study under Good Laboratory Practice (GLP) conditions. Three doses, IM or IN, at two-week intervals do not induce an adverse effect or systemic toxicity. Cumulatively, these results support the application for a Phase 1 clinical trial of ACM-polymersome-based Covid-19 vaccine (ClinicalTrials.gov identifier: NCT05385991).

Role of Lipopolysaccharide in Protecting OmpT from Autoproteolysis during In Vitro Refolding.

Outer membrane protease (OmpT) is a 33.5 kDa aspartyl protease that cleaves at dibasic sites and is thought to function as a defense mechanism for E. coli against cationic antimicrobial peptides secreted by the host immune system. Despite carrying three dibasic sites in its own sequence, there is no report of OmpT autoproteolysis in vivo. However, recombinant OmpT expressed in vitro as inclusion bodies has been reported to undergo autoproteolysis during the refolding step, thus resulting in an inactive protease. In this study, we monitor and compare levels of in vitro autoproteolysis of folded and unfolded OmpT and examine the role of lipopolysaccharide (LPS) in autoproteolysis. SDS-PAGE data indicate that it is only the unfolded OmpT that undergoes autoproteolysis while the folded OmpT remains protected and resistant to autoproteolysis. This selective susceptibility to autoproteolysis is intriguing. Previous studies suggest that LPS, a co-factor necessary for OmpT activity, may play a protective role in preventing autoproteolysis. However, data presented here confirm that LPS plays no such protective role in the case of unfolded OmpT. Furthermore, OmpT mutants designed to prevent LPS from binding to its putative LPS-binding motif still exhibited excellent protease activity, suggesting that the putative LPS-binding motif is of less importance for OmpT's activity than previously proposed.

S-Layer Protein for Resistive Switching and Flexible Nonvolatile Memory Device.

In this work, a flexible resistive switching memory device consisting of S-layer protein (Slp) is demonstrated for the first time. This novel device (Al/Slp/indium tin oxide/polyethylene terephthalte) based on a simple and easy fabrication method is capable of bistable switching to low resistive state (LRS) and high resistive state (HRS). This device exhibits bistable memory behavior with stability and a long retention time (>4 × 103 s), being stable up to a 500 cycle endurance test and with significant HRS/LRS ratio. The device possesses consistent switching performance for more than 100 times bending, corresponding to desired applicability for biocompatible wearable electronics. The memory mechanism is attributed to a trapping/de-trapping process in S-layer protein. These promising results of the flexible memory device could find a way in the wearable storage applications like smart bands and sports equipments' sensors.