Molecular Engineering of pH-Responsive Anchoring Systems onto Poly(ethylene glycol) Corona.

An adaptive surface that can sense and respond to environmental stimuli is integral to smart functional materials. Here, we report pH-responsive anchoring systems onto the poly(ethylene glycol) (PEG) corona of polymer vesicles. The hydrophobic anchor, pyrene, is reversibly inserted into the PEG corona through the reversible protonation of its covalently linked pH-sensing group. Depending on the pKa of the sensor, the pH-responsive region is engineered from acidic to neutral and basic conditions. The switchable electrostatic repulsion between the sensors contributes to the responsive anchoring behavior. Our findings provide a new responsive binding chemistry for the creation of smart nanomedicine and a nanoreactor.

Porous Polymersomes as Carriers for Silver Nanoparticles and Nanoclusters: Advantages of Compartmentalization for Antimicrobial Usage.

The global threat to public health posed by antibiotic-resistant bacterial infections requires the exploration of innovative approaches. Nanomaterials, particularly silver nanoparticles (AgNPs) and nanoclusters (AgNCs), have emerged as potential solutions to address the pressing issue of a bacterial healthcare crisis. However, the high cytotoxicity levels and low stability associated with AgNPs and AgNCs limit their applicability. To overcome these challenges, AgNCs and AgNPs were synthesized in the presence of porous polymersomes, resulting in a compartmentalized system that enhances stability, reduces cytotoxicity, and maintains high antimicrobial activity. The encapsulated particles exhibit a distribution of silver components on both the surface and the core, which is confirmed through the analysis of surface charge and center of mass. Moreover, our investigation demonstrates improved stability of the nanoparticles and nanoclusters upon entrapment in the porous system, as evidenced by the ion release assay. The antimicrobial effectiveness of porous polymersomes containing AgNPs and AgNCs was demonstrated by visualizing the biofilms and quantifying the penetration depth. Furthermore, cytotoxicity studies showed that compartmentalization increases cell compatibility for AgNC-based systems, showcasing the many advantages this system holds.

Surface-Area Determination of Anisotropic Polymersomes by Amphiphilic Molecular Probe Loading.

The surface area of anisotropic polymeric assemblies is a critical parameter concerning their properties. However, it is still a grand challenge for traditional techniques to determine the surface area. Here, a molecular probe loading (MPL) method is developed to measure the surface area of anisotropic polymersomes in the shape of tube, disc, and stomatocyte. This method uses an amphiphilic molecular probe, comprising hydrophobic pyrene as the anchor and hydrophilic tetraethylene glycol (EG4 ) as the float. The surface area of spherical polymersomes determined by dynamic light scattering is quantitatively correlated with the loading amount of probes, allowing the calculation of the average separation distance between the loaded probes. With the separation distance, we successfully determine the surface area of anisotropic polymersomes by measuring the loading amount. We envision that the MPL method will assist in the real-time surface area characterization, enabling the customization of functions.

Modular Approach to the Functionalization of Polymersomes.

Functionalizing polymersomes remains a challenge due to the limitation in reaction conditions applicable to the chemistry on the surface, hindering their application for selective targeting. In order to overcome this limitation, functionalization can be introduced right before the self-assembly. Here, we have synthesized a library (32 examples) of PEG-b-PS and PEG-b-PDLLA with various functional groups derived from the amine-functionalized polymers, leading to functionally active polymersomes. We show that polymersome formation is possible via the general method with all functionalized groups and that these handles are present on the surface and are able to undergo reactions. Additionally, this methodology provides a general synthetic tool to tailor the functional group of the polymersome right before self-assembly, without limitation on the reaction conditions.

Evaluation of a standardised Vi poly-l-lysine ELISA for serology of Vi capsular polysaccharide antibodies.

Typhoid vaccines based on protein-conjugated capsular Vi polysaccharide (TCVs) prevent typhoid in infants and young children. Analysis of the serum anti-Vi IgG response following immunisation against typhoid confirms the immunogenicity of TCVs and forms an important part of the pathway to licensing. Comparative studies could expedite the licencing process, and the availability of a standardised ELISA method alongside the 1st International Standard (IS) 16/138 for anti-typhoid capsular Vi polysaccharide IgG (human) will facilitate this process. To this end, a non-commercial ELISA based on a coat of Vi and poly-l-lysine (Vi-PLL ELISA) was evaluated by 10 laboratories. Eight serum samples, including IS 16/138, were tested in the standardised Vi-PLL ELISA (n = 10), a commercial Vi ELISA (n = 3) and a biotinylated Vi ELISA (n = 1). Valid estimates of potencies relative to IS 16/138 were obtained for all samples in the Vi-PLL ELISA and the commercial ELISA, with good repeatability and reproducibility evident from the study results and concordant estimates obtained by the two ELISA methods. The study demonstrates that the Vi-PLL ELISA can be used in clinical trial studies to determine the immunogenicity of TCVs.

Consensus Report on Shigella Controlled Human Infection Model: Immunological Assays.

Moderate to severe diarrhea caused by Shigella is a global health concern due to its substantial contribution to morbidity and mortality in children aged <5 years in low- and middle-income countries. Although antibiotic treatment can be effective, emerging antimicrobial resistance, limited access, and cost affirm the role of vaccines as the most attractive countermeasure. Controlled human infection models (CHIMs) represent a valuable tool for assessing vaccine efficacy and potentially accelerating licensure. Currently, immunological analysis during CHIM studies is customized based on vaccine type, regimen, and administration route. Additionally, differences in type of immunoassays and procedures used limit comparisons across studies. In November 2017, an expert working group reviewed Shigella CHIM studies performed to date and developed consensus guidelines on prioritization of immunoassays, specimens, and collection time points. Immunoassays were ranked into 3 tiers, with antibodies to Shigella lipopolysaccharide (LPS) being the highest priority. To facilitate comparisons across clinical studies, a second workshop was conducted in December 2017, which focused on the pathway toward a recognized enzyme-linked immunosorbent assay (ELISA) to determine serum immunoglobulin G titers against Shigella LPS. The consensus of the meeting was to establish a consortium of international institutions with expertise in Shigella immunology that would work with the National Institute for Biological Standards and Control to establish a harmonized ELISA, produce a reference sera, and identify a reliable source of Shigella LPS for global utilization. Herein we describe efforts toward establishing common procedures to advance Shigella vaccine development, support licensure, and ultimately facilitate vaccine deployment and uptake.

Interference of the T Cell and Antigen-Presenting Cell Costimulatory Pathway Using CTLA4-Ig (Abatacept) Prevents Staphylococcal Enterotoxin B Pathology.

Staphylococcal enterotoxin B (SEB) is a bacterial superantigen that binds the receptors in the APC/T cell synapse and causes increased proliferation of T cells and a cytokine storm syndrome in vivo. Exposure to the toxin can be lethal and cause significant pathology in humans. The lack of effective therapies for SEB exposure remains an area of concern, particularly in scenarios of acute mass casualties. We hypothesized that blockade of the T cell costimulatory signal by the CTLA4-Ig synthetic protein (abatacept) could prevent SEB-dependent pathology. In this article, we demonstrate mice treated with a single dose of abatacept 8 h post SEB exposure had reduced pathology compared with control SEB-exposed mice. SEB-exposed mice showed significant reductions in body weight between days 4 and 9, whereas mice exposed to SEB and also treated with abatacept showed no weight loss for the duration of the study, suggesting therapeutic mitigation of SEB-induced morbidity. Histopathology and magnetic resonance imaging demonstrated that SEB mediated lung damage and edema, which were absent after treatment with abatacept. Analysis of plasma and lung tissues from SEB-exposed mice treated with abatacept demonstrated significantly lower levels of IL-6 and IFN-γ (p < 0.0001), which is likely to have resulted in less pathology. In addition, exposure of human and mouse PBMCs to SEB in vitro showed a significant reduction in levels of IL-2 (p < 0.0001) after treatment with abatacept, indicating that T cell proliferation is the main target for intervention. Our findings demonstrate that abatacept is a robust and potentially credible drug to prevent toxic effects from SEB exposure.

Evaluation of two WHO First International Standards for Vi polysaccharide from Citrobacter freundii and Salmonella enterica subspecies enterica serovar Typhi.

Numerous Vi capsular polysaccharide (Vi PS) conjugate vaccines to protect young children and infants from Typhoid are either licensed or under development. These vaccines are evaluated by laboratory methods to ensure their potency and that quality requirement are met. International Standard (IS) preparations of Vi PS are needed to calibrate and harmonise these assays. Twenty laboratories from 12 countries participated in a collaborative study to evaluate two candidate ISs: Citrobacter freundii Vi PS (NIBSC code 12/244) and Salmonella enterica serovar Typhi Vi PS (16/126). On the basis of returned results and stability profiles, these standards were established by the WHO Expert Committee on Biological Standardization in Oct 2017 as the First WHO IS for C. freundii Vi PS with a content of 1.94 ±â€¯0.12 mg Vi PS per ampoule (expanded uncertainty with coverage factor of k = 2.11 corresponding to a 95% level of confidence) and the First WHO IS for S. Typhi Vi PS with a content of 2.03 ±â€¯0.10 mg Vi PS per ampoule (expanded uncertainty with coverage factor of k = 2.11), as determined by quantitative NMR. The study also showed the ISs are suitable for physicochemical and immuno assays used for the quantitation of the Vi PS component in Vi PS and conjugate vaccines.

Establishment of the first International Standard for human anti-typhoid capsular Vi polysaccharide IgG.

Vi capsular polysaccharide (Vi) conjugate vaccines, which can prevent typhoid in infants and young children, are being developed. Comparative immunogenicity studies are facilitated by an International Standard (IS) for human anti-Vi IgG. 16/138, a pool of sera from volunteers which received either Vi conjugate vaccine or plain Vi vaccine, was assessed as an IS alongside U.S. reference reagent Vi-IgGR1, 2011. Samples were tested in a commercial ELISA (n = 7), a standardised ELISA based on biotinylated Vi (n = 7) and in-house ELISAs (n = 7). Valid estimates were obtained for the potency of all samples in the commercial ELISA, and the commutability of 16/138 and Vi-IgGR1, 2011 was evident for the commercial ELISA and in-house ELISAs based on a coating of Vi and protein. The WHO Expert Committee on Biological Standardization established 16/138 as the first IS for anti-Vi IgG with 100 IU per ampoule and assigned 163 IU per vial of Vi-IgGR1, 2011.

Establishment of replacement International Standard 13/132 for human antibodies to Toxoplasma gondii.

Sixteen laboratories carried out a collaborative study to validate 13/132 as a replacement International Standard (IS) for TOXM (3rd IS for anti-Toxoplasma Serum, Human, 1000 IU). 13/132 is a freeze dried preparation of pooled human plasma from six donors who experienced a recent Toxoplasma gondii infection. The potency of 13/132 was compared to TOXM and 01/600 (1st IS for anti-Toxoplasma IgG, Human, 20 IU). Samples were tested for IgA, IgG, IgG avidity and IgM in agglutination assays; enzyme linked immunosorbent assays (ELISA), enzyme linked fluorescent assays, immunoblots, immunofluorescence assays and the Sabin-Feldman dye test for Ig. 13/132 was strongly positive for Ig, IgA, IgG and IgM and the reproducibility was very good. 13/132 contains high levels of anti-Toxoplasma Ig, IgG and IgM and its potency falls between TOXM and 01/600. The avidity of IgG was found to be low, similar to the avidity of IgG from TOXM. 13/132 was established by the Expert Committee on Biological Standardization as the 4th IS for Antibodies, Human, to T. gondii with an assigned unitage of 160 IU per ampoule for Ig by dye test and 263 U per ampoule for IgG by ELISA.

Biofilm Disruption from within: Light-Activated Molecular Drill-Functionalized Polymersomes Bridge the Gap between Membrane Damage and Quorum Sensing-Mediated Cell Death.

Bacterial biofilms represent an escalating global health concern with the proliferation of drug resistance and hospital-acquired infections annually. Numerous strategies are under exploration to combat biofilms and preempt the development of antibacterial resistance. Among these, mechanical disruption of biofilms and enclosed bacteria presents a promising avenue, aiming to induce membrane permeabilization and consequent lethal damage. Herein, we introduce a hemithioindigo (HTI) motor activated by visible light, capable of disrupting sessile bacteria when integrated into a polymeric vesicle carrier. Under visible light, bacteria exhibited a notable outer membrane permeability, reduced membrane fluidity, and diminished viability following mechanical drilling. Moreover, various genetic responses pertaining to the cell envelope were examined via qRT-PCR, alongside the activation of a self-lysis mechanism associated with phage stress, which was coupled with increases in quorum sensing, demonstrating a potential self-lysis cascade from within. The multifaceted mechanisms of action, coupled with the energy efficiency of mechanical damage, underscore the potential of this system in addressing the challenges posed by pathogenic biofilms.

Selective photodimerization of acenaphthylene in polymersome nanoreactors.

PEG-b-PLA polymersomes are used as nanoreactors for the photodimerization of acenaphthylene (ACE), increasing reaction rate significantly. The reaction steered towards almost exclusive formation of anti product (94 : 6). This selectivity is remarkable, as other known systems commonly mediate formation of the syn product.

Adaptive insertion of a hydrophobic anchor into a poly(ethylene glycol) host for programmable surface functionalization.

Covalent and non-covalent molecular binding are two strategies to tailor surface properties and functions. However, the lack of responsiveness and requirement for specific binding groups makes spatiotemporal control challenging. Here, we report the adaptive insertion of a hydrophobic anchor into a poly(ethylene glycol) (PEG) host as a non-covalent binding strategy for surface functionalization. By using polycyclic aromatic hydrocarbons as the hydrophobic anchor, hydrophilic charged and non-charged functional modules were spontaneously loaded onto PEG corona in 2 min without the assistance of any catalysts and binding groups. The thermodynamically favourable insertion of the hydrophobic anchor can be reversed by pulling the functional module, enabling programmable surface functionalization. We anticipate that the adaptive molecular recognition between the hydrophobic anchor and the PEG host will challenge the hydrophilic understanding of PEG and enhance the progress in nanomedicine, advanced materials and nanotechnology.

Photo-Cross-Linking Polymersome Nanoreactors with Size-Selective Permeability.

The design of stable, inert, and permeable nanoreactors remains a challenge due to the additives required to create a cross-linked network, limiting their potential for catalysis. Polymersomes are nanovesicles self-assembled from amphiphilic block copolymers that can act as nanoreactors by encapsulating catalysts. A major restriction toward their use is their stability and reduced permeability. In order to overcome this, polymersome membranes can be cross-linked to retain their shape and function. Here, we report the synthesis of a PEG-b-P(S-co-4-VBA) polymer, which can self-assemble into polymersomes and subsequently be cross-linked using UV light. We demonstrate that these polymersomes are stable over a long period of time in various organic solvents, that incorporation of functional handles on their surface is possible, and that they are able to undergo reactions. Additionally, we show that co-assembly with up to 40% PEG-b-PS present results in the formation of pores in the membrane structure, which allows for the structure to be used as a nanoreactor. By encapsulating a platinum nanocatalyst, we are able to catalyze the depropargylation of a small coumarin substrate, which was able to enter and leave the porous nanoreactor.

Reversible speed control of one-stimulus-double-response, temperature-sensitive asymmetric hydrogel micromotors.

Soft, one-stimulus-double-response, thermo-sensitive, PNIPAm-based microgels are designed for controlled autonomous motion under stimuli. At higher temperature, the motors with physically encapsulated catalase move faster, while motors in which catalase is chemically linked to PNIPAm ceased moving. The phenomenon is reversible over multiple cycles of temperature.

Recent Developments in Enteric Bacterial Vaccines.

In this issue, we present promising developments in the field of bacterial enteric vaccines [...].

Generating biomembrane-like local curvature in polymersomes via dynamic polymer insertion.

Biomembrane curvature formation has long been observed to be essential in the change of membrane morphology and intracellular processes. The significant importance of curvature formation has attracted scientists from different backgrounds to study it. Although magnificent progress has been achieved using liposome models, the instability of these models restrict further exploration. Here, we report a new approach to mimic biomembrane curvature formation using polymersomes as a model, and poly(N-isopropylacrylamide) to induce the local curvature based on its co-nonsolvency phenomenon. Curvatures form when poly(N-isopropylacrylamide) becomes hydrophobic and inserts into the membrane through solvent addition. The insertion area can be fine-tuned by adjusting the poly(N-isopropylacrylamide) concentration, accompanied by the formation of new polymersome-based non-axisymmetric shapes. Moreover, a systematic view of curvature formation is provided through investigation of the segregation, local distribution and dissociation of inserted poly(N-isopropylacrylamide). This strategy successfully mimicks biomembrane curvature formation in polymersomes and a detailed observation of the insertion can be beneficial for a further understanding of the curvature formation process. Furthermore, polymer insertion induced shape changing could open up new routes for the design of non-axisymmetric nanocarriers and nanomachines to enrich the boundless possibilities of nanotechnology.

Development of a Monocyte Activation Test as an Alternative to the Rabbit Pyrogen Test for Mono- and Multi-Component Shigella GMMA-Based Vaccines.

Generalised modules for membrane antigens (GMMA)-based vaccines comprise the outer membrane from genetically modified Gram-negative bacteria containing membrane proteins, phospholipids and lipopolysaccharides. Some lipoproteins and lipopolysaccharides are pyrogens; thus, GMMA-based vaccines are intrinsically pyrogenic. It is important to control the pyrogenic content of biological medicines, including vaccines, to prevent adverse reactions such as febrile responses. The rabbit pyrogen test (RPT) and bacterial endotoxin test (BET) are the most commonly employed safety assays used to detect pyrogens. However, both tests are tailored for detecting pyrogenic contaminants and have considerable limitations when measuring the pyrogen content of inherently pyrogenic products. We report the adaptation of the monocyte activation test (MAT) as an alternative to the RPT for monitoring the pyrogenicity of Shigella GMMA-based vaccines. The European Pharmacopoeia endorses three MAT methods (A-C). Of these, method C, the reference lot comparison test, was identified as the most suitable. This method was evaluated with different reference materials to ensure parallelism and consistency for a mono- and multi-component Shigella GMMA vaccine. We demonstrate the drug substance as a promising reference material for safety testing of the matched drug product. Our results support the implementation of MAT as an alternative to the RPT and use of the defined parameters can be extended to GMMA-based vaccines currently in development, aiding vaccine batch release.

Evidence of Extended Thermo-Stability of Typhoid Polysaccharide Conjugate Vaccines.

Typhoid conjugate vaccines (TCV) are effective in preventing enteric fever caused by Salmonella enterica serovar Typhi in Southeast Asia and Africa. To facilitate vaccination with the Vi capsular polysaccharide-tetanus toxoid conjugate vaccine, Typbar TCV, and allow it to be transported and stored outside a cold chain just prior to administration, an extended controlled-temperature conditions (ECTC) study was performed to confirm the quality of the vaccine at 40 °C for 3 days at the end of its shelf-life (36 months at 2-8 °C). Studies performed in parallel by the vaccine manufacturer, Bharat Biotech International Limited, and an independent national control laboratory (NIBSC) monitored its stability-indicating parameters: O-acetylation of the Vi polysaccharide, integrity of the polysaccharide-protein conjugate, and its molecular size and pH. ECTC samples stored at 40 °C and 45 °C in comparison with control samples stored at 4 °C and 55 or 56 °C, were shown to have stable O-acetylation and pH; only very slight increases in the percentage of free saccharide and corresponding decreases in molecular size were observed. The deoxycholate method for precipitating conjugated polysaccharide was very sensitive to small incremental increases in percentage of free saccharide, in line with storage temperature and duration. This extended ECTC study demonstrated minimal structural changes to the Vi polysaccharide and conjugate vaccine and a stable formulation following extended exposure to elevated temperatures for the desired durations. This outcome supports the manufacturer's ECTC claim for the vaccine to be allowed to be taken outside the cold chain before its administration.