Nanozyme-Based Lateral Flow Immunoassay (LFIA) for Extracellular Vesicle Detection.

Extracellular vesicles (EVs) are biological nanoparticles of great interest as novel sources of biomarkers and as drug delivery systems for personalized therapies. The research in the field and clinical applications require rapid quantification. In this study, we have developed a novel lateral flow immunoassay (LFIA) system based on Fe3O4 nanozymes for extracellular vesicle (EV) detection. Iron oxide superparamagnetic nanoparticles (Fe3O4 MNPs) have been reported as peroxidase-like mimetic systems and competent colorimetric labels. The peroxidase-like capabilities of MNPs coated with fatty acids of different chain lengths (oleic acid, myristic acid, and lauric acid) were evaluated in solution with H2O2 and 3,3,5,5-tetramethylbenzidine (TMB) as well as on strips by biotin-neutravidin affinity assay. As a result, MNPs coated with oleic acid were applied as colorimetric labels and applied to detect plasma-derived EVs in LFIAs via their nanozyme effects. The visual signals of test lines were significantly enhanced, and the limit of detection (LOD) was reduced from 5.73 × 107 EVs/µL to 2.49 × 107 EVs/µL. Our work demonstrated the potential of these MNPs as reporter labels and as nanozyme probes for the development of a simple tool to detect EVs, which have proven to be useful biomarkers in a wide variety of diseases.

Magnetic Lateral Flow Immunoassay for Small Extracellular Vesicles Quantification: Application to Colorectal Cancer Biomarker Detection.

Colorectal cancer (CRC) is the third leading cause of cancer death and the fourth most common cancer in the world. Colonoscopy is the most sensitive test used for detection of CRC; however, their procedure is invasive and expensive for population mass screening. Currently, the fecal occult blood test has been widely used as a screening tool for CRC but displays low specificity. The lack of rapid and simple methods for mass screening makes the early diagnosis and therapy monitoring difficult. Extracellular vesicles (EVs) have emerged as a novel source of biomarkers due to their contents in proteins and miRNAs. Their detection would not require invasive techniques and could be considered as a liquid biopsy. Specifically, it has been demonstrated that the amount of CD147 expressed in circulating EVs is significant higher for CRC cell lines than for normal colon fibroblast cell lines. Moreover, CD147-containing EVs have been used as a biomarker to monitor response to therapy in patients with CRC. Therefore, this antigen could be used as a non-invasive biomarker for the detection and monitoring of CRC in combination with a Point-of-Care platform as, for example, Lateral Flow Immunoassays (LFIAs). Here, we propose the development of a quantitative lateral flow immunoassay test based on the use of magnetic nanoparticles as labels coupled to inductive sensor for the non-invasive detection of CRC by CD147-positive EVs. The results obtained for quantification of CD147 antigen embedded in EVs isolated from plasma sample have demonstrated that this device could be used as a Point-of-Care tool for CRC screening or therapy monitoring thanks to its rapid response and easy operation.

Nano-Encapsulation of Mithramycin in Transfersomes and Polymeric Micelles for the Treatment of Sarcomas.

Sarcomas are aggressive tumors which often show a poor response to current treatments. As a promising therapeutic alternative, we focused on mithramycin (MTM), a natural antibiotic with a promising anti-tumor activity but also a relevant systemic toxicity. Therefore, the encapsulation of MTM in nano-delivery systems may represent a way to increase its therapeutic window. Here, we designed novel transfersomes and PLGA polymeric micelles by combining different membrane components (phosphatidylcholine, Span 60, Tween 20 and cholesterol) to optimize the nanoparticle size, polydispersity index (PDI) and encapsulation efficiency (EE). Using both thin film hydration and the ethanol injection methods we obtained MTM-loaded transferosomes displaying an optimal hydrodynamic diameter of 100-130 nm and EE values higher than 50%. Additionally, we used the emulsion/solvent evaporation method to synthesize polymeric micelles with a mean size of 228 nm and a narrow PDI, capable of encapsulating MTM with EE values up to 87%. These MTM nano-delivery systems mimicked the potent anti-tumor activity of free MTM, both in adherent and cancer stem cell-enriched tumorsphere cultures of myxoid liposarcoma and chondrosarcoma models. Similarly to free MTM, nanocarrier-delivered MTM efficiently inhibits the signaling mediated by the pro-oncogenic factor SP1. In summary, we provide new formulations for the efficient encapsulation of MTM which may constitute a safer delivering alternative to be explored in future clinical uses.

Vitamin D3 Loaded Niosomes and Transfersomes Produced by Ethanol Injection Method: Identification of the Critical Preparation Step for Size Control.

Vesicular nanocarriers have an important role in drug delivery and dietary supplements. Size control and optimization of encapsulation efficiency (EE) should be optimized for those applications. In this work, we report on the identification of the crucial step (injection, evaporation, or sonication) innanovesicles (transfersomes and niosomes) preparation by theethanol injection method (EI). The identification of each production step on the final vesicle size was analyzed in order to optimize further scale-up process. Results indicated that the final size of transfersomeswas clearly influenced by the sonication step while the final size of niosomes was mainly governed by the injection step. Measurements of final surface tension of the different vesicular systems prepared indicate a linear positive tendency with the vesicle size formed. This relation could help to better understand the process and design a vesicular size prediction model for EI. Vitamin D3 (VitD3) was encapsulated in the systems formulated with encapsulation efficiencies larger than 90%. Interaction between the encapsulated compound and the membrane layer components is crucial for vesicle stability. This work has an impact on the scaling-up production of vesicles for further food science applications.

Cu Nanoparticle-Loaded Nanovesicles with Antibiofilm Properties. Part I: Synthesis of New Hybrid Nanostructures.

Copper nanoparticles (CuNPs) stabilized by quaternary ammonium salts are well known as antimicrobial agents. The aim of this work was to study the feasibility of the inclusion of CuNPs in nanovesicular systems. Liposomes are nanovesicles (NVs) made with phospholipids and are traditionally used as delivery vehicles because phospholipids favor cellular uptake. Their capacity for hydrophilic/hydrophobic balance and carrier capacity could be advantageous to prepare novel hybrid nanostructures based on metal NPs (Me-NPs). In this work, NVs were loaded with CuNPs, which have been reported to have a biofilm inhibition effect. These hybrid materials could improve the effect of conventional antibacterial agents. CuNPs were electro-synthesized by the sacrificial anode electrolysis technique in organic media and characterized in terms of morphology through transmission electron microscopy (TEM). The NVs were prepared by the thin film hydration method in aqueous media, using phosphatidylcholine (PC) and cholesterol as a membrane stabilizer. The nanohybrid systems were purified to remove non-encapsulated NPs. The size distribution, morphology and stability of the NV systems were studied. Different quaternary ammonium salts in vesicular systems made of PC were tested as stabilizing surfactants for the synthesis and inclusion of CuNPs. The entrapment of charged metal NPs was demonstrated. NPs attached preferably to the membrane, probably due to the attraction of their hydrophobic shell to the phospholipid bilayers. The high affinity between benzyl-dimethyl-hexadecyl-ammonium chloride (BDHAC) and PC allowed us to obtain stable hybrid NVs c.a. 700 nm in diameter. The stability of liposomes increased with NP loading, suggesting a charge-stabilization effect in a novel antibiofilm nanohybrid material.

Circulating extracellular vesicles as potential biomarkers in chronic fatigue syndrome/myalgic encephalomyelitis: an exploratory pilot study.

Chronic Fatigue Syndrome (CFS), also known as Myalgic Encephalomyelitis (ME) is an acquired, complex and multisystem condition of unknown etiology, no established diagnostic lab tests and no universally FDA-approved drugs for treatment. CFS/ME is characterised by unexplicable disabling fatigue and is often also associated with numerous core symptoms. A growing body of evidence suggests that extracellular vesicles (EVs) play a role in cell-to-cell communication, and are involved in both physiological and pathological processes. To date, no data on EV biology in CFS/ME are as yet available. The aim of this study was to isolate and characterise blood-derived EVs in CFS/ME. Blood samples were collected from 10 Spanish CFS/ME patients and 5 matched healthy controls (HCs), and EVs were isolated from the serum using a polymer-based method. Their protein cargo, size distribution and concentration were measured by Western blot and nanoparticle tracking analysis. Furthermore, EVs were detected using a lateral flow immunoassay based on biomarkers CD9 and CD63. We found that the amount of EV-enriched fraction was significantly higher in CFS/ME subjects than in HCs (p = 0.007) and that EVs were significantly smaller in CFS/ME patients (p = 0.014). Circulating EVs could be an emerging tool for biomedical research in CFS/ME. These findings provide preliminary evidence that blood-derived EVs may distinguish CFS/ME patients from HCs. This will allow offer new opportunities and also may open a new door to identifying novel potential biomarkers and therapeutic approaches for the condition.