Extracellular Vesicles in Cardiovascular Diseases: Alternative Biomarker Sources, Therapeutic Agents, and Drug Delivery Carriers.

Cardiovascular diseases (CVD) represent the leading cause of morbidity and mortality globally. The emerging role of extracellular vesicles (EVs) in intercellular communication has stimulated renewed interest in exploring the potential application of EVs as tools for diagnosis, prognosis, and therapy in CVD. The ubiquitous nature of EVs in biological fluids presents a technological advantage compared to current diagnostic tools by virtue of their notable stability. EV contents, such as proteins and microRNAs, represent specific signatures of cellular activation or injury. This feature positions EVs as an alternative source of biomarkers. Furthermore, their intrinsic activity and immunomodulatory properties offer EVs unique opportunities to act as therapeutic agents per se or to serve as drug delivery carriers by acting as miniaturized vehicles incorporating bioactive molecules. In this article, we aim to review the recent advances and applications of EV-based biomarkers and therapeutics. In addition, the potential of EVs as a drug delivery and theranostic platform for CVD will also be discussed.

EXOPLEXs: Chimeric Drug Delivery Platform from the Fusion of Cell-Derived Nanovesicles and Liposomes.

Cell-derived nanovesicles (CDNs) have been recently investigated as novel drug delivery systems (DDSs), due to the preservation of key features from the cell membrane of their precursor cells, which are responsible for an efficient cellular uptake by target cells. However, CDNs suffer from low drug loading efficiencies as well as challenges in functionalization compared to conventional DDS like liposomes. Here, we describe the first study proposing the fusion of CDNs with liposomes to form EXOPLEXs. We report the preservation of cell membranes from precursor cells similarly to CDNs, as well as high loading efficiencies of more than 65% with doxorubicin hydrochloride, a model chemotherapeutic drug. The doxorubicin-loaded EXOPLEXs (DOX-EXO) also demonstrated a higher in vitro cell killing effect than liposomes, while EXOPLEXs alone did not show any remarkable cytotoxicity. Taken together, these results illustrate the potential of EXOPLEXs as a novel DDS for targeted delivery of chemotherapeutics.

Superhydrophobic-oleophobic Ag nanowire platform: an analyte-concentrating and quantitative aqueous and organic toxin surface-enhanced Raman scattering sensor.

The ultratrace detection and quantification of toxins in both water and organic liquids remains a challenge due to the random spreading and dilution of liquids on substrate-based sensors, especially for organic liquids with low surface tension. Herein, we fabricate a superhydrophobic-oleophobic (SHP-OP) 3D Ag nanowire mesh-like surface-enhanced Raman scattering (SERS) platform to overcome the random spreading issue, demonstrating ultratrace toxin sensing in both water and organic liquid. Our SHP-OP SERS platform is able to concentrate analyte solutions in water and toluene to 100-fold and 8-fold smaller areas, respectively, as compared to its omniphilic counterparts. The synergy of analyte-concentrating ability and intense SERS-enhancing properties on our SHP-OP SERS platform enables quantitative and ultratrace detection of melamine and Sudan I down to 0.1 fmol in water and toluene, respectively, using just 1 µL of analyte solution. These detection limits are 10(3)-fold lower than the regulatory limits, clearly indicating our SHP-OP SERS platform as an appealing universal ultratrace toxin sensor. The ultratrace detection of spiked melamine in liquid milk down to 100 fmol also highlights the suitability of our SHP-OP SERS platform for the sensing of food toxins in real samples.

Hierarchical 3D SERS substrates fabricated by integrating photolithographic microstructures and self-assembly of silver nanoparticles.

Most of the surface-enhanced Raman scattering (SERS) substrates are 2D planar systems, which limits the SERS active area to a single Cartesian plane. Here, we fabricate 3D SERS substrates with the aim to break the traditional 2D SERS active area limitation, and to extend the SERS hotspots into the third dimension along the z-axis. Our 3D SERS substrates are tailored with increased SERS hotspots in the z-direction from tens of nanometers to tens of micrometers, increasing the hotspots in the z-direction by at least an order of magnitude larger than the confocal volume (~1 µm) of most Raman spectrometers. Various hierarchical 3D SERS-active microstructures are fabricated by combining 3D laser photolithography with Langmuir-Blodgett nanoparticle assembly. 3D laser photolithography creates microstructured platforms required to extend the SERS-active area into 3D, and the self-assembly of Ag nanoparticles ensures homogeneous coating of SERS-active Ag nanoparticles over the entire microstructured platforms. Large-area 3D Raman imaging demonstrates that homogeneous signals can be collected throughout the entire 3D SERS substrates. We vary the morphology, height, and inclination angles of the 3D microstructures, where the inclination angle is found to exhibit strong influence on the SERS signals. We also demonstrate a potential application of this hierarchical 3D SERS substrate in information tagging, storage and encryption as SERS micro-barcodes, where multiple micro-barcodes can be created within a single set of microstructures.

Cell-derived nanovesicles from mesenchymal stem cells as extracellular vesicle-mimetics in wound healing.

Wound healing is a dynamic process that involves a series of molecular and cellular events aimed at replacing devitalized and missing cellular components and/or tissue layers. Recently, extracellular vesicles (EVs), naturally cell-secreted lipid membrane-bound vesicles laden with biological cargos including proteins, lipids, and nucleic acids, have drawn wide attention due to their ability to promote wound healing and tissue regeneration. However, current exploitation of EVs as therapeutic agents is limited by their low isolation yields and tedious isolation processes. To circumvent these challenges, bioinspired cell-derived nanovesicles (CDNs) that mimic EVs were obtained by shearing mesenchymal stem cells (MSCs) through membranes with different pore sizes. Physical characterisations and high-throughput proteomics confirmed that MSC-CDNs mimicked MSC-EVs. Moreover, these MSC-CDNs were efficiently uptaken by human dermal fibroblasts and demonstrated a dose-dependent activation of MAPK signalling pathway, resulting in enhancement of cell proliferation, cell migration, secretion of growth factors and extracellular matrix proteins, which all promoted tissue regeneration. Of note, MSC-CDNs enhanced angiogenesis in human dermal microvascular endothelial cells in a 3D PEG-fibrin scaffold and animal model, accelerating wound healing in vitro and in vivo. These findings suggest that MSC-CDNs could replace both whole cells and EVs in promoting wound healing and tissue regeneration.

Extracellular Vesicles and Their Mimetics: A Comparative Study of Their Pharmacological Activities and Immunogenicity Profiles.

Extracellular vesicles (EVs), which are miniaturised carriers loaded with functional proteins, lipids, and nucleic acid material, are naturally secreted by cells and show intrinsic pharmacological effects in several conditions. As such, they have the potential to be used for the treatment of various human diseases. However, the low isolation yield and laborious purification process are obstacles to their translation for clinical use. To overcome this problem, our lab developed cell-derived nanovesicles (CDNs), which are EV mimetics produced by shearing cells through membrane-fitted spin cups. To evaluate the similarities between EVs and CDNs, we compare the physical properties and biochemical composition of monocytic U937 EVs and U937 CDNs. Besides having similar hydrodynamic diameters, the produced CDNs had proteomic, lipidomic, and miRNA profiles with key communalities compared to those of natural EVs. Further characterisation was conducted to examine if CDNs could exhibit similar pharmacological activities and immunogenicity when administered in vivo. Consistently, CDNs and EVs modulated inflammation and displayed antioxidant activities. EVs and CDNs both did not exert immunogenicity when administered in vivo. Overall, CDNs could serve as a scalable and efficient alternative to EVs for further translation into clinical use.

Enhanced skin penetration of berberine from proniosome gel attenuates pain and inflammation in a mouse model of osteoarthritis.

Dermal delivery of bioactive molecules remains an attractive route of administration in osteoarthritis (OA) due to the local accumulation of drugs while avoiding their systemic side effects. In this study we propose a proniosome gel comprising non-ionic surfactants that self-assemble into de-hydrated vesicles for the delivery of the natural anti-inflammatory compound berberine. By modulating the hydrating ability of the proniosome gel, berberine can be efficiently released with minimal mechanical force. A combination of sorbitan oleate (S80) and polyethlene glycol sorbitan monolaurate (T20) in a sorbitan stearate (S60)-based proniosome enables a readily hydrated gel to deliver berberine into the skin, as confirmed by ex vivo skin permeation studies. Concurrently, an in vitro model of OA using primary mouse chondrocytes demonstrated that the release of berberine at a concentration as low as 1 µg mL-1 is sufficient to restore the production of sulphated glycosaminoglycans (sGAG) to levels comparable to healthy chondrocytes while avoiding the cytotoxic concentrations (IC50 = 33 µg mL-1) on skin keratinocytes. In a mouse model of OA, the optimized formulation is able to attenuate inflammation and pain and minimize cartilage degeneration. Taken together, these data demonstrate the feasibility of adopting proniosome gels as a suitable platform to deliver active molecules for the management of osteoarthritis.

Plant-derived extracellular vesicles: Recent advancements and current challenges on their use for biomedical applications.

Extracellular vesicles (EVs) represent a diverse class of lipid bilayer membrane vesicles released by both animal and plant cells. These ubiquitous vesicles are involved in intercellular communication and transport of various biological cargos, including proteins, lipids, and nucleic acids. In recent years, interest in plant-derived EVs has increased tremendously, as they serve as a scalable and sustainable alternative to EVs derived from mammalian sources. In vitro and in vivo findings have demonstrated that these plant-derived vesicles (PDVs) possess intrinsic therapeutic activities that can potentially treat diseases and improve human health. In addition, PDVs can also act as efficient and biocompatible drug carriers. While preclinical studies have shown promising results, there are still several challenges and knowledge gaps that have to be addressed for the successful translation of PDVs into clinical applications, especially in view of the lack of standardised protocols for material handling and PDV isolation from various plant sources. This review provides the readers with a quick overview of the current understanding and research on PDVs, critically analysing the current challenges and highlighting the immense potential of PDVs as a novel class of therapeutics to treat human diseases. It is expected that this work will guide scientists to address the knowledge gaps currently associated with PDVs and promote new advances in plant-based therapeutic solutions.

Investigations on Cellular Uptake Mechanisms and Immunogenicity Profile of Novel Bio-Hybrid Nanovesicles.

In drug delivery, the development of nanovesicles that combine both synthetic and cellular components provides added biocompatibility and targeting specificity in comparison to conventional synthetic carriers such as liposomes. Produced through the fusion of U937 monocytes' membranes and synthetic lipids, our nano-cell vesicle technology systems (nCVTs) showed promising results as targeted cancer treatment. However, no investigation has been conducted yet on the immunogenic profile and the uptake mechanisms of nCVTs. Hence, this study was aimed at exploring the potential cytotoxicity and immune cells' activation by nCVTs, as well as the routes through which cells internalize these biohybrid systems. The endocytic pathways were selectively inhibited to establish if the presence of cellular components in nCVTs affected the internalization route in comparison to both liposomes (made up of synthetic lipids only) and nano-cellular membranes (made up of biological material only). As a result, nCVTs showed an 8-to-40-fold higher cellular internalization than liposomes within the first hour, mainly through receptor-mediated processes (i.e., clathrin- and caveolae-mediated endocytosis), and low immunostimulatory potential (as indicated by the level of IL-1α, IL-6, and TNF-α cytokines) both in vitro and in vivo. These data confirmed that nCVTs preserved surface cues from their parent U937 cells and can be rationally engineered to incorporate ligands that enhance the selective uptake and delivery toward target cells and tissues.

Doxorubicin-loaded cell-derived nanovesicles: an alternative targeted approach for anti-tumor therapy.

Cell-derived nanovesicles (CDNs) are an emerging class of biological drug delivery systems (DDS) that retain the characteristics of the cells they were derived from, without the need for further surface functionalization. CDNs are also biocompatible, being derived from natural sources and also take advantage of the enhanced permeability and retention effect due to their nanodimensions. Furthermore, CDNs derived from monocytes were shown to have an in vivo targeting effect, accumulating at the tumor site in a previous study conducted in a mouse tumor model. Here, we report a systematic approach pertaining to various loading methods of the chemotherapeutic drug doxorubicin into our CDNs and examine the differential cellular uptake of drug-loaded CDNs in cancerous (HeLa) and healthy (HEK293) cell lines. Lastly, we proved that the addition of doxorubicin-loaded CDNs to the HeLa and HEK293 co-cultures showed a clear discrimination toward cancer cells at the cellular level. Our results further reinforce the intriguing potential of CDNs as an alternative targeted strategy for anticancer therapy.

ZnO Nano-Rod Devices for Intradermal Delivery and Immunization.

Intradermal delivery of antigens for vaccination is a very attractive approach since the skin provides a rich network of antigen presenting cells, which aid in stimulating an immune response. Numerous intradermal techniques have been developed to enhance penetration across the skin. However, these methods are invasive and/or affect the skin integrity. Hence, our group has devised zinc oxide (ZnO) nano-rods for non-destructive drug delivery. Chemical vapour deposition was used to fabricate aligned nano-rods on ZnO pre-coated silicon chips. The nano-rods' length and diameter were found to depend on the temperature, time, quality of sputtered silicon chips, etc. Vertically aligned ZnO nano-rods with lengths of 30-35 µm and diameters of 200-300 nm were selected for in vitro human skin permeation studies using Franz cells with Albumin-fluorescein isothiocyanate (FITC) absorbed on the nano-rods. Fluorescence and confocal studies on the skin samples showed FITC penetration through the skin along the channels formed by the nano-rods. Bradford protein assay on the collected fluid samples indicated a significant quantity of Albumin-FITC in the first 12 h. Low antibody titres were observed with immunisation on Balb/c mice with ovalbumin (OVA) antigen coated on the nano-rod chips. Nonetheless, due to the reduced dimensions of the nano-rods, our device offers the additional advantage of excluding the simultaneous entrance of microbial pathogens. Taken together, these results showed that ZnO nano-rods hold the potential for a safe, non-invasive, and painless intradermal drug delivery.

Using the Langmuir-Schaefer technique to fabricate large-area dense SERS-active Au nanoprism monolayer films.

Interfacial self-assembly of nanoparticles is capable of creating large-area close-packed structures for a variety of applications. However, monolayers of hydrophilic cetyltrimethylammonium bromide (CTAB)-coated Au nanoparticles are challenging to assemble via interfacial self-assembly. This report presents a facile and scalable process to fabricate large-area monolayer films of ultrathin CTAB-coated Au nanoprisms at the air-water interface using the Langmuir-Schaefer technique. This is first achieved by a one-step functionalization of Au nanoprisms with poly(vinylpyrrolidone) (PVP). PVP functionalization is completed within a short time without loss of nanoprisms due to aggregation. Uniform and near close-packed monolayers of the Au nanoprisms formed over large areas (∼1 cm(2)) at the air-water interface can be transferred to substrates with different wettabilities. The inter-prism gaps are tuned qualitatively through the introduction of dodecanethiol and oleylamine. The morphological integrity of the nanoprisms is maintained throughout the entire assembly process, without truncation of the nanoprism tips. The near close-packed arrangement of the nanoprism monolayers generates large numbers of hot spots in the 2D arrays in the tip-to-tip and edge-to-edge inter-particle regions, giving rise to strong surface-enhanced Raman scattering (SERS) signals. When deposited on an Au mirror film, additional hotspots are created in the 3(rd) dimension in the gaps between the 2D nanoprism monolayers and the Au film. SERS enhancement factors reaching 10(4) for non-resonant probe molecules are achieved.