A reconstitution method for integral membrane proteins in hybrid lipid-polymer vesicles for enhanced functional durability

Seneviratne, Rashmi; Khan, Sanobar; Moscrop, Ellen; Rappolt, Michael; Muench, Stephen P.; Jeuken, Lars J. C.; Beales, Paul A.

doi:10.1016/j.ymeth.2018.01.021

Cited by 41 publications

(55 citation statements)

References 42 publications

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“…It was expected that the polymeric vesicles would be more resistant to heat exposure as polymer membranes are believed to be more robust than those formed by lipids alone. Polymersomes with a large membrane thickness (PEE 37 -PEO 40 and PBd 46 -PEO 26 ) have previously been shown to have both encapsulation and colloidal stability to autoclave conditions, although there is a slight shift to a smaller vesicle size distribution [28]. To enhance the stability of the vesicles during heat sterilisation, it has been suggested that sugars or polyols could be used to stabilise the vesicles [52] and the drug could be encapsulated after vesicles have been autoclaved [38].…”

Section: Discussionmentioning

confidence: 99%

“…Liposomes, composed of natural lipids, form bilayer membranes that closely mimic the structural matrix of native biomembranes. This makes them highly biocompatible and provides a native-like environment if integral membrane proteins are desired to add functionality to the membrane [23][24][25][26][27]. However, deformations, but weak under stretching deformations, with a lysis strain of less than 5% [28,29].…”

Section: Introductionmentioning

confidence: 99%

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Hybrid Vesicle Stability under Sterilisation and Preservation Processes Used in the Manufacture of Medicinal Formulations

et al. 2020

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Sterilisation and preservation of vesicle formulations are important considerations for their viable manufacture for industry applications, particular those intended for medicinal use. Here, we undertake an initial investigation of the stability of hybrid lipid-block copolymer vesicles to common sterilisation and preservation processes, with particular interest in how the block copolymer component might tune vesicle stability.We investigate two sizes of polybutadiene-block-poly(ethylene oxide) polymers (PBd 12 -PEO 11 and PBd 22 -PEO 14 ) mixed with the phospholipid 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) considering the encapsulation stability of a fluorescent cargo and the colloidal stability of vesicle size distributions. We find that autoclaving and lyophilisation cause complete loss of encapsulation stability under the conditions studied here. Filtering through 200 nm pores appears to be viable for sterilisation for all vesicle compositions with comparatively low release of encapsulated cargo, even for vesicle size distributions which extend beyond the 200 nm filter pore size. Freeze-thaw of vesicles also shows promise for the preservation of hybrid vesicles with high block copolymer content. We discuss the process stability of hybrid vesicles in terms of the complex mechanical interplay between bending resistance, stretching elasticity and lysis strain of these membranes and propose strategies for future work to further enhance the process stability of these vesicle formulations.Polymers 2020, 12, 914 2 of 17 liposomes can have inherent instabilities. Their membranes are highly flexible under bending deformations, but weak under stretching deformations, with a lysis strain of less than 5% [28,29]. The labile fluidity of the membrane can lead to transient membrane defects that frustrates long term encapsulation stability and lipid peroxidation can cause chemical instabilities in these structures.Polymersomes, due to their synthetic nature, are often less biocompatible than their lipid counterparts but offer greater mechanical stability and a broader chemical parameter space [30]. Amphiphilic copolymers that form vesicles can have several different architectures, the most common being AB diblock (A = hydrophilic, B = hydrophobic) [6], ABA [31] and ABC tri-block polymers [32] where A and C are chemically different hydrophilic blocks and finally graft copolymers [33]. Formation of polymersomes depends on the amphiphilic co-polymers molecular weight, polydispersity and hydrophilic/hydrophobic block lengths ratio, which is ideally less than 1:3 to form vesicular structures [34]. Their membranes are often thicker than liposome membranes, which provides greater bending resistance and their enhanced elasticity under stretching makes them tough and durable [22]. The polymer chemistry can be designed to minimise chemical instability but also to incorporate novel functionality. In blending liposomes and polymersomes to create hybrid vesicles, the ambition is to combine advantageous properties of these m...

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Hybrid Vesicle Stability under Sterilisation and Preservation Processes Used in the Manufacture of Medicinal Formulations

et al. 2020

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“…A future study could investigate this phenomenon. It will be also interesting to apply our methodology to lipid/polymer hybrid systems and their use as platform for membrane proteins as the combined properties in terms of stability and biocompatibility would be beneficial [50][51][52][53] . Most importantly, the potential optimization has been shown to yield functional proteovesicles composed of the lipid DOPC or an ABA block copolymer.…”

Section: Discussionmentioning

confidence: 99%

Optimized reconstitution of membrane proteins into synthetic membranes

et al. 2018

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Light-driven proton pumps, such as proteorhodopsin, have been proposed as an energy source in the field of synthetic biology. Energy is required to power biochemical reactions within artificially created reaction compartments like proto-or nanocells, which are typically based on either lipid or polymer membranes. The insertion of membrane proteins into these membranes is delicate and quantitative studies comparing these two systems are needed. Here we present a detailed analysis of the formation of proteoliposomes and proteopolymersomes and the requirements for a successful reconstitution of the membrane protein proteorhodopsin. To this end, we apply design of experiments to provide a mathematical framework for the reconstitution process. Mathematical optimization identifies suitable reconstitution conditions for lipid and polymer membranes and the obtained data fits well to the predictions. Altogether, our approach provides experimental and modeling evidence for different reconstitution mechanisms depending on the membrane type which resulted in a surprisingly similar performance.

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“…Fatty acid-phospholipid blended membranes display increased stabilityb ut still maintain permeability for small (charged) solutes. [50] To day's biological membranes are mostlyc omposed of lipids, in which proteins are embedded. [47] Well-sealed, stable membranes can also be formed from block copolymers, [48] but the functional incorporation of integral membranep roteins is challenging, especially when the proteins requirespecific lipids as cofactors.…”

Section: Building Blocks For Membranesmentioning

confidence: 99%

Cell Fuelling and Metabolic Energy Conservation in Synthetic Cells

et al. 2019

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We are aiming for a blue print for synthesizing (moderately complex) subcellular systems from molecular components and ultimately for constructing life. However, without comprehensive instructions and design principles, we rely on simple reaction routes to operate the essential functions of life. The first forms of synthetic life will not make every building block for polymers de novo according to complex pathways, rather they will be fed with amino acids, fatty acids and nucleotides. Controlled energy supply is crucial for any synthetic cell, no matter how complex. Herein, we describe the simplest pathways for the efficient generation of ATP and electrochemical ion gradients. We have estimated the demand for ATP by polymer synthesis and maintenance processes in small cell‐like systems, and we describe circuits to control the need for ATP. We also present fluorescence‐based sensors for pH, ionic strength, excluded volume, ATP/ADP, and viscosity, which allow the major physicochemical conditions inside cells to be monitored and tuned.

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A reconstitution method for integral membrane proteins in hybrid lipid-polymer vesicles for enhanced functional durability

Cited by 41 publications

References 42 publications

Hybrid Vesicle Stability under Sterilisation and Preservation Processes Used in the Manufacture of Medicinal Formulations

Hybrid Vesicle Stability under Sterilisation and Preservation Processes Used in the Manufacture of Medicinal Formulations

Optimized reconstitution of membrane proteins into synthetic membranes

Cell Fuelling and Metabolic Energy Conservation in Synthetic Cells

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