Physical stimuli-responsive vesicles in drug delivery: Beyond liposomes and polymersomes

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

A robust liposomal platform for direct colorimetric detection of sphingomyelinase enzyme and inhibitors

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

Highly purified extracellular vesicles from human cardiomyocytes demonstrate preferential uptake by human endothelial cells

Extracellular vesicles (EVs) represent a promising cell-free alternative for treatment of cardiovascular diseases. Nevertheless, the lack of standardised and reproducible isolation methods capable of recovering pure, intact EVs presents a significant obstacle. Additionally, there is significant interest in investigating the interactions of EVs with different cardiac cell types. Here we established a robust technique for the production and isolation of EVs harvested from an enriched (>97% purity) population of human induced pluripotent stem cell (iPSC)-derived cardiomyocytes (CMs) with size exclusion chromatography. Utilizing an advanced fluorescence labelling strategy, we then investigated the interplay of the CM-EVs with the three major cellular components of the myocardium (fibroblasts, cardiomyocytes and endothelial cells) and identified that cardiac endothelial cells show preferential uptake of these EVs. Overall, our findings provide a great opportunity to overcome the translational hurdles associated with the isolation of intact, non-aggregated human iPSC-CM EVs at high purity. Furthermore, understanding in detail the interaction of the secreted EVs with their surrounding cells in the heart may open promising new avenues in the field of EV engineering for targeted delivery in cardiac regeneration

Molecular imaging of extracellular vesicles in vitro via Raman metabolic labelling

Extracellular vesicles (EVs) are biologically-derived nanovectors important for intercellular communication and trafficking. As such, EVs show great promise as disease biomarkers and therapeutic drug delivery vehicles. However, despite the rapidly growing interest in EVs, understanding of the biological mechanisms that govern their biogenesis, secretion, and uptake remains poor. Advances in this field have been hampered by both the complex biological origins of EVs, which make them difficult to isolate and identify, and a lack of suitable imaging techniques to properly study their diverse biological roles. Here, we present a new strategy for simultaneous quantitative in vitro imaging and molecular characterisation of EVs in 2D and 3D based on Raman spectroscopy and metabolic labelling. Deuterium, in the form of deuterium oxide (D2O), deuterated choline chloride (d-Chol), or deuterated D-glucose (d-Gluc), is metabolically incorporated into EVs through the growth of parent cells on medium containing one of these compounds. Isolated EVs are thus labelled with deuterium, which acts as a bio-orthogonal Raman-active tag for direct Raman identification of EVs when introduced to unlabelled cell cultures. Metabolic deuterium incorporation demonstrates no apparent adverse effects on EV secretion, marker expression, morphology, or global composition, indicating its capacity for minimally obstructive EV labelling. As such, our metabolic labelling strategy could provide integral insights into EV biocomposition and trafficking. This approach has the potential to enable a deeper understanding of many of the biological mechanisms underpinning EVs, with profound implications for the design of EVs as therapeutic delivery vectors and applications as disease biomarkers

Integrated photodynamic Raman theranostic system for cancer diagnosis, treatment, and post-treatment molecular monitoring

Theranostics, the combination of diagnosis and therapy, has long held promise as a means to achieving personalised precision cancer treatments. However, despite its potential, theranostics has yet to realise significant clinical translation, largely due the complexity and overriding toxicity concerns of existing theranostic nanoparticle strategies. / Methods: Here, we present an alternative nanoparticle-free theranostic approach based on simultaneous Raman spectroscopy and photodynamic therapy (PDT) in an integrated clinical platform for cancer theranostics. / Results: We detail the compatibility of Raman spectroscopy and PDT for cancer theranostics, whereby Raman spectroscopic diagnosis can be performed on PDT photosensitiser-positive cells and tissues without inadvertent photosensitiser activation/photobleaching or impaired diagnostic capacity. We further demonstrate that our theranostic platform enables in vivo tumour diagnosis, treatment, and post-treatment molecular monitoring in real-time. / Conclusion: This system thus achieves effective theranostic performance, providing a promising new avenue towards the clinical realisation of theranostics

Highly purified extracellular vesicles from human cardiomyocytes demonstrate preferential uptake by human endothelial cells

Extracellular vesicles (EVs) represent a promising cell-free alternative for treatment of cardiovascular diseases. Nevertheless, the lack of standardised and reproducible isolation methods capable of recovering pure, intact EVs presents a significant obstacle. Additionally, there is significant interest in investigating the interactions of EVs with different cardiac cell types. Here we established a robust technique for the production and isolation of EVs harvested from an enriched (>97% purity) population of human induced pluripotent stem cell (iPSC)-derived cardiomyocytes (CMs) with size exclusion chromatography. Utilizing an advanced fluorescence labelling strategy, we then investigated the interplay of the CM-EVs with the three major cellular components of the myocardium (fibroblasts, cardiomyocytes and endothelial cells) and identified that cardiac endothelial cells show preferential uptake of these EVs. Overall, our findings provide a great opportunity to overcome the translational hurdles associated with the isolation of intact, non-aggregated human iPSC-CM EVs at high purity. Furthermore, understanding in detail the interaction of the secreted EVs with their surrounding cells in the heart may open promising new avenues in the field of EV engineering for targeted delivery in cardiac regeneration