A facile and fast method for the functionalization of polymersomes by photoinduced cycloaddition chemistry

Polymersomes are promising platforms for use in biosensing, where their stability may be crucial over that of liposomes. For the introduction of the desired functionality multiple strategies have been reported for functionalization of polymersomes. However, none of them have combined readily available starting materials, facility and in situ quantification. We show a simple 4-step method for functionalization of polymersomes starting from commercially available materials. For the key conjugation step a recently explored light induced cycloaddition was used which is relatively fast (15 min) and allows in situ quantification by the intrinsic fluorescence of the conjugate. The facility of the protocol, the ease of preparation and quantification make this ‘click’-type conjugation method a promising alternative to the established strained cycloadditions

Self-assembled architectures with multiple aqueous compartments

A vital organizational feature of living cells is that of compartmentalization. This allows cells to run concurrently incompatible metabolic processes and to regulate these processes by selective trans-membrane transport. Although strategies that effectively mimic cell function in simple architectures have been researched extensively, soft matter systems with membranes that delineate distinct and multiple aqueous environments have only recently caught attention. We highlight a range of multi-compartmentalized soft matter systems including vesosomes, capsosomes, polymersomes, double emulsions, and their combinations, and demonstrate that the unique properties of the multi-compartmentalized architectures have the potential to add value to application areas such as drug-delivery and multi-enzyme biosynthesis

Hybrid, Nanoscale Phospholipid/Block Copolymer Vesicles

Hybrid phospholipid/block copolymer vesicles, in which the polymeric membrane is blended with phospholipids, display interesting self-assembly behavior, incorporating the robustness and chemical versatility of polymersomes with the softness and biocompatibility of liposomes. Such structures can be conveniently characterized by preparing giant unilamellar vesicles (GUVs) via electroformation. Here, we are interested in exploring the self-assembly and properties of the analogous nanoscale hybrid vesicles (ca. 100 nm in diameter) of the same composition prepared by film-hydration and extrusion. We show that the self-assembly and content-release behavior of nanoscale polybutadiene-b-poly(ethylene oxide) (PB-PEO)/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC) hybrid phospholipid/block copolymer vesicles can be tuned by the mixing ratio of the amphiphiles. In brief, these hybrids may provide alternative tools for drug delivery purposes and molecular imaging/sensing applications and clearly open up new avenues for further investigation

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.Ministry of Education (MOE)Published versionThis research work was funded by the Singapore Ministry of Education Academic Research Fund Tier 2 (MOE2018-T2-1-025) and the NTU-NU Institute for NanoMedicine located at the International Institute for Nanotechnology, Northwestern University, USA and the Nanyang Technological University, Singapore; Agmt10/20/14. KJM was supported by NIH/NCI training grant 5T32CA186897-02. This work made use of the IMSERC at Northwestern University, which has received support from the Soft and Hybrid Nanotechnology Experimental (SHyNE) Resource (NSF NNCI-1542205); the State of Illinois and International Institute for Nanotechnology (IIN). This research was carried out in collaboration with the National Resource for Translational and Developmental Proteomics under Grant P41 GM108569 from the National Institute of General Medical Sciences, National Institutes of Health

Facile mixing of phospholipids promotes self-assembly of low-molecular-weight biodegradable block co-polymers into functional vesicular architectures

In this work, we have used low-molecular-weight (PEG12-b-PCL6, PEG12-b-PCL9 or PEG16-b-PLA38; MW, 1.25–3.45 kDa) biodegradable block co-polymers to construct nano- and micron-scaled hybrid (polymer/lipid) vesicles, by solvent dispersion and electroformation methods, respectively. The hybrid vesicles exhibit physical properties (size, bilayer thickness and small molecule encapsulation) of a vesicular boundary, confirmed by cryogenic transmission electron microscopy, calcein leakage assay and dynamic light scattering. Importantly, we find that these low MW polymers, on their own, do not self-assemble into polymersomes at nano and micron scales. Using giant unilamellar vesicles (GUVs) model, their surface topographies are homogeneous, independent of cholesterol, suggesting more energetically favorable mixing of lipid and polymer. Despite this mixed topography with a bilayer thickness similar to that of a lipid bilayer, variation in surface topology is demonstrated using the interfacial sensitive phospholipase A2 (sPLA2). The biodegradable hybrid vesicles are less sensitive to the phospholipase digestion, reminiscent of PEGylated vesicles, and the degree of sensitivity is polymer-dependent, implying that the nano-scale surface topology can further be tuned by its chemical composition. Our results reveal and emphasize the role of phospholipids in promoting low MW polymers for spontaneous vesicular self-assembly, generating a functional hybrid lipid-polymer interface.Ministry of Education (MOE)Nanyang Technological UniversityPublished versionThe Academic Research Fund (AcRF) Tier 1 Grant supported this work. The authors would like to thank School of Materials Science and Engineering (MSE), Nanyang Technological University for the funding

Biomimetic stratum corneum liposome models: lamellar organization and permeability studies

The stratum corneum (SC), the outer layer of the skin, plays a crucial role as a barrier protecting the underlying cells from external stress. The SC comprises three key components: ceramide (CER), free fatty acid (FFA), and cholesterol, along with small fractions of cholesterol sulfate and cholesterol ester. In order to gain a deeper understanding about the interdependence of the two major components, CER and FFA, on the organizational, structural, and functional properties of the SC layer, a library of SC lipid liposome (SCLL) models was developed by mixing CER (phytosphingosine or sphingosine), FFA (oleic acid, palmitic acid, or stearic acid), cholesterol, and cholesterol sulfate. Self-assembly of the SC lipids into lamellar phases was first confirmed by small-angle X-ray scattering. Short periodicity and long periodicity phases were identified for SCLLs containing phytosphingosines and sphingosine CERs, respectively. Furthermore, unsaturation in the CER acyl and FFA chains reduced the lipid conformational ordering and packing density of the liposomal bilayer, which were measured by differential scanning calorimetry and Fourier transform infrared spectroscopy. The introduction of unsaturation in the CER and/or FFA chains also impacted the lamellar integrity and permeability. This extensive library of SCLL models exhibiting physiologically relevant lamellar phases with defined structural and functional properties may potentially be used as a model system for screening pharmaceuticals or cosmetic agents.Agency for Science, Technology and Research (A*STAR)Published versionThe authors would like to acknowledge the A*STAR BMRC Strategic Positioning Fund (SPF)-A*STAR-P&G Collaboration Grant

Conformational antibody binding to a native, cell-free expressed GPCR in block copolymer membranes.

G-protein coupled receptors (GPCRs) play a key role in physiological processes and are attractive drug targets. Their biophysical characterization is, however, highly challenging because of their innate instability outside a stabilizing membrane and the difficulty of finding a suitable expression system. We here show the cell-free expression of a GPCR, CXCR4, and its direct embedding in diblock copolymer membranes. The polymer-stabilized CXCR4 is readily immobilized onto biosensor chips for label-free binding analysis. Kinetic characterization using a conformationally sensitive antibody shows the receptor to exist in the correctly folded conformation, showing binding behaviour that is commensurate with heterologously expressed CXCR4

A Nanocompartment system (Synthosome) Designed for Biotechnical Applications

A nanocompartment system based on two deletion mutants of the large channel protein FhuA (FhuA Delta 1-129; FhuA Delta 1-160) and an ABA triblock copolymer (PMOXA-PDMS-PMOXA) has been developed for putative biotechnological applications. FhuA is ideally suited for applications in biotechnology due to its monomeric structure, large pore diameter (39-46 angstrom elliptical cross-section) that ensures rapid compound flux, and solved crystallographic structure. Two areas of application were targeted as proof of principle: (A) selective product recovery in nanocompartments and (B) enzymatic conversion in nanocompartments. Selective recovery of negatively charged compounds has been achieved on the example of sulforhodamine B by using positively charged polylysine molecules as trap inside the nanocompartment. Conversion in nanocompartments has been achieved by 3,3 ',5,5 '-tetrainethylbenzidine oxidation employing horseradish peroxidase (HRP)