Over the past few decades, a range of vesicle-based drug delivery systems have entered clinical practice and several others are in various stages of clinical translation. While most of these vesicle constructs are lipid-based (liposomes), or polymer-based (polymersomes), recently new classes of vesicles have emerged that defy easy classification. Examples include assemblies with small molecule amphiphiles, biologically derived membranes, hybrid vesicles with two or more classes of amphiphiles, or more complex hierarchical structures such as vesicles incorporating gas bubbles or nanoparticulates in the lumen or membrane. In this review, we explore these recent advances and emerging trends at the edge and just beyond the research fields of conventional liposomes and polymersomes. A focus of this review is the distinct behaviors observed for these classes of vesicles when exposed to physical stimuli -such as ultrasound, heat, light and mechanical triggers -and we discuss the resulting potential for new types of drug delivery, with a special emphasis on current challenges and opportunities.

Liposomes are well-established systems for drug delivery and biosensing applications. The design of a liposomal carrier requires careful choice of lipid composition and formulation method. These determine many vesicle properties including lamellarity, which can have a strong effect on both encapsulation efficiency and the efflux rate of encapsulated active compounds. Despite this, a comprehensive study on how the lipid composition and formulation method affect vesicle lamellarity is still lacking. Here, we combine small-angle neutron scattering and cryogenic transmission electron microscopy to study the effect of three different well-established formulation methods followed by extrusion through 100 nm polycarbonate membranes on the resulting vesicle membrane structure. Specifically, we examine vesicles formulated from the commonly used phospholipids 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) via film hydration followed by (i) agitation on a shaker or (ii) freeze–thawing, or (iii) the reverse-phase evaporation vesicle method. After extrusion, up to half of the total lipid content is still assembled into multilamellar structures. However, we achieved unilamellar vesicle populations when as little as 0.1 mol % PEG-modified lipid was included in the vesicle formulation. Interestingly, DPPC with 5 mol % PEGylated lipid produces a combination of cylindrical micelles and vesicles. In conclusion, our results provide important insights into the effect of the formulation method and lipid composition on producing liposomes with a defined membrane structure.

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It has been demonstrated that some commonly used Extracellular Vesicle (EV) isolation techniques can lead to substantial contamination with non-EV factors. Whilst it has been established that this impacts the identification of biomarkers, the impact on apparent EV bioactivity has not been explored. Extracellular vesicles have been implicated as critical mediators of therapeutic human mesenchymal stem cell (hMSC) paracrine signalling. Isolated hMSC-EVs have been used to treat multiple in vitro and in vivo models of tissue damage. However, the relative contributions of EVs and non-EV factors have not been directly compared. The dependence of hMSC paracrine signalling on EVs was first established by ultrafiltration of hMSC-conditioned medium to deplete EVs, which led to a loss of signalling activity. Here, we show that this method also causes depletion of non-EV factors, and that when this is prevented proangiogenic signalling activity is fully restored in vitro. Subsequently, we used size-exclusion chromatography (SEC) to separate EVs and soluble proteins to directly and quantitatively compare their relative contributions to signalling. Non-EV factors were found to be necessary and sufficient for the stimulation of angiogenesis and wound healing in vitro. EVs in isolation were found to be capable of potentiating signalling only when isolated by a low-purity method, or when used at comparatively high concentrations. These results indicate a potential for contaminating soluble factors to artefactually increase the apparent bioactivity of EV isolates and could have implications for future studies on the biological roles of EVs.

Enabling concurrent, high throughput analysis of single nanoparticles would greatly increase the capacity to study size, composition and inter and intra particle population variance with applications in a wide range of fields from polymer science to drug delivery. Here, we present a comprehensive platform for Single Particle Automated Raman Trapping Analysis (SPARTA) able to integrally analyse nanoparticles ranging from synthetic polymer particles to liposomes without any modification. With the developed highly controlled automated trapping process, single nanoparticles are analysed with high throughput and sensitivity to resolve particle mixtures, obtain detailed compositional spectra of complex particles, track sequential functionalisations, derive particle sizes and monitor the dynamics of click reactions occurring on the nanoparticle surface. The SPARTA platform opens up a wide range of new avenues for nanoparticle research through label-free integral high-throughput single particle analysis, overcoming key limitations in sensitivity and specificity of existing bulk analysis methods.

The enzyme sphingomyelinase (SMase) is an important biomarker for several diseases such as Niemann Pick’s, atherosclerosis, multiple sclerosis, and HIV. We present a two-component colorimetric SMase activity assay that is more sensitive and much faster than currently available commercial assays. Herein, SMase-triggered release of cysteine from a sphingomyelin (SM)-based liposome formulation with 60 mol % cholesterol causes gold nanoparticle (AuNP) aggregation, enabling colorimetric detection of SMase activities as low as 0.02 mU/mL, corresponding to 1.4 pM concentration. While the lipid composition offers a stable, nonleaky liposome platform with minimal background signal, high specificity toward SMase avoids cross-reactivity of other similar phospholipases. Notably, use of an SM-based liposome formulation accurately mimics the natural in vivo substrate: the cell membrane. We studied the physical rearrangement process of the lipid membrane during SMase-mediated hydrolysis of SM to ceramide using small- and wide-angle X-ray scattering. A change in lipid phase from a liquid to gel state bilayer with increasing concentration of ceramide accounts for the observed increase in membrane permeability and consequent release of encapsulated cysteine. We further demonstrated the effectiveness of the sensor in colorimetric screening of small-molecule drug candidates, paving the way for the identification of novel SMase inhibitors in minutes. Taken together, the simplicity, speed, sensitivity, and naked-eye readout of this assay offer huge potential in point-of-care diagnostics and high-throughput drug screening.

Vesicles are dynamic supramolecular structures with a bilayer membrane consisting of lipids or synthetic amphiphiles enclosing an aqueous compartment. Lipid vesicles have often been considered as mimics for biological cells. In this paper, we present a novel strategy for the preparation of three-dimensional multilayered structures in which vesicles containing amphiphilic β-cyclodextrin are interconnected by proteins using cyclodextrin guests as bifunctional linker molecules. We compared two pairs of adhesion molecules for the immobilization of vesicles: mannose-concanavalin A and biotin-streptavidin. Microcontact printing and thiol-ene click chemistry were used to prepare suitable substrates for the vesicles. Successful immobilization of intact vesicles through the mannose-concanavalin A and biotin-streptavidin motifs was verified by fluorescence microscopy imaging and dynamic light scattering, while the vesicle adlayer was characterized by quartz crystal microbalance with dissipation monitoring. In the case of the biotin-streptavidin motif, up to six layers of intact vesicles could be immobilized in a layer-by-layer fashion using supramolecular interactions. The construction of vesicle multilayers guided by noncovalent vesicle-vesicle junctions can be taken as a minimal model for artificial biological tissue.

The host-guest complexation of a tailored Si(IV) phthalocyanine with supramolecular β-cyclodextrin vesicles (CDV) was studied, revealing a reduced aggregation of the photoactive center upon binding to the CDV, even in aqueous environments. For this purpose, a photosensitizing unit axially decorated with one adamantyl group and one pyridinium moiety on the other side was obtained by two successive click reactions on a bis-azido-functionalized derivative of Si(IV) phthalocyanine. To evaluate its potential as a photosensitizer against antibiotic-resistant bacteria, comparative studies of the photophysical properties including absorption and emission spectroscopy, lifetimes as well as fluorescence and singlet oxygen quantum yields were determined for the Si(IV) phthalocyanine alone and upon self-assembly on the CDV surface. In vitro phototoxicity against the methicillin-resistant Staphylococcus aureus (MRSA) USA300 was evaluated, showing an almost complete inactivation.