Advancement of Measurement Techniques for Tunable Resistive Pulse Sensing

<p>Tunable resistive pulse sensing (TRPS) is a particle-by-particle analysis technique combining the Coulter principle with size-tunable pores. TRPS can be used to characterize biological and synthetic particles 50 nm - 20 µm in diameter. Information is obtained from the resistive pulse signal, a transient change in ionic current observed when a particle passes through the pore. TRPS has been shown to provide excellent resolution and accuracy for measuring particle size and concentration as well as providing information about particle charge. TRPS is therefore applicable to many industrial and fundamental research areas involving aptamers, drug delivery particles, extracellular vesicles and other biological particle types. Advancement of this technology requires a better understanding of the technique, particularly in the area of particle surface charge measurement and this Thesis helps to provide that understanding. In this work, firstly particle ζ-potential measurement using TRPS was investigated. A number of different measurement methods are presented and the uncertainties associated with each method are outlined. The ζ-potential for a variety of particles with different surface charges were measured in a range of electrolytes. Particle ζ-potential measurements were then improved upon with the addition of streaming potential measurements to measure the pore surface charge. The ζ-potential of the pore surface, which makes a significant contribution to particle ζ-potential calculations, was measured using a set up which works alongside the qNano. Streaming potential measurements were also used to investigate changes in the pore surface charge following application of number of different chemical coatings. The volume of data collected and detail of analysis in this work (including uncertainties) is unprecedented in TRPS ζ potential measurements. Biphasic pulses arising from the charge on the particles were investigated. The pulse is conventionally resistive, but biphasic pulses which include both resistive and conductive components are significant for less than 50 mM salt concentrations when measuring 200 nm particles. The experimental variables investigated include the concentration of the electrolyte, particle charge, pore size, applied voltage, and the direction of particlemotion. Conductive pulse size was seen to decrease with increasing electrolyte concentration and pore size and increase with applied voltage. A linear relationship was found between conductive pulse magnitude and particle surface group density. The influence of direction of motion on conductive pulses was consistent with concentration polarization of an ion selective pore. Biphasic pulses were also seen to affect conventional TRPS particle size measurements. Finally, size distribution broadening due to varying particle trajectories was investigated. Pulse size distributions for monodisperse particles became broader when the pore size was increased and featured two distinct peaks. Relatively large pulses are produced by particles with trajectories passing near to the edge of the pore. Other experiments determined that pulse size distributions are independent of applied voltage but broaden with increasing pressure applied across the membrane.</p>

Advancement of Measurement Techniques for Tunable Resistive Pulse Sensing

<p>Tunable resistive pulse sensing (TRPS) is a particle-by-particle analysis technique combining the Coulter principle with size-tunable pores. TRPS can be used to characterize biological and synthetic particles 50 nm - 20 µm in diameter. Information is obtained from the resistive pulse signal, a transient change in ionic current observed when a particle passes through the pore. TRPS has been shown to provide excellent resolution and accuracy for measuring particle size and concentration as well as providing information about particle charge. TRPS is therefore applicable to many industrial and fundamental research areas involving aptamers, drug delivery particles, extracellular vesicles and other biological particle types. Advancement of this technology requires a better understanding of the technique, particularly in the area of particle surface charge measurement and this Thesis helps to provide that understanding. In this work, firstly particle ζ-potential measurement using TRPS was investigated. A number of different measurement methods are presented and the uncertainties associated with each method are outlined. The ζ-potential for a variety of particles with different surface charges were measured in a range of electrolytes. Particle ζ-potential measurements were then improved upon with the addition of streaming potential measurements to measure the pore surface charge. The ζ-potential of the pore surface, which makes a significant contribution to particle ζ-potential calculations, was measured using a set up which works alongside the qNano. Streaming potential measurements were also used to investigate changes in the pore surface charge following application of number of different chemical coatings. The volume of data collected and detail of analysis in this work (including uncertainties) is unprecedented in TRPS ζ potential measurements. Biphasic pulses arising from the charge on the particles were investigated. The pulse is conventionally resistive, but biphasic pulses which include both resistive and conductive components are significant for less than 50 mM salt concentrations when measuring 200 nm particles. The experimental variables investigated include the concentration of the electrolyte, particle charge, pore size, applied voltage, and the direction of particlemotion. Conductive pulse size was seen to decrease with increasing electrolyte concentration and pore size and increase with applied voltage. A linear relationship was found between conductive pulse magnitude and particle surface group density. The influence of direction of motion on conductive pulses was consistent with concentration polarization of an ion selective pore. Biphasic pulses were also seen to affect conventional TRPS particle size measurements. Finally, size distribution broadening due to varying particle trajectories was investigated. Pulse size distributions for monodisperse particles became broader when the pore size was increased and featured two distinct peaks. Relatively large pulses are produced by particles with trajectories passing near to the edge of the pore. Other experiments determined that pulse size distributions are independent of applied voltage but broaden with increasing pressure applied across the membrane.</p>

Determining Extracellular Vesicles Properties and miRNA Cargo Variability in Bovine Milk from Healthy Cows and Cows Undergoing Subclinical Mastitis

Background: Subclinical mastitis, the inflammation of the mammary gland lacking clinical symptoms, is one of the most prevalent and costly diseases in dairy farming worldwide. Milk microRNAs (miRNAs) encapsulated in extracellular vesicles (EVs) have been proposed as potential biomarkers of different mammary gland conditions, including subclinical mastitis. However, little is known about the robustness of EVs analysis regarding sampling time-point or natural infections. To estimate the reliability of EVs measurements in raw bovine milk, we first evaluated changes in EVs size and concentration using Tunable Resistive Pulse Sensing (TRPS) during three consecutive days. Then, we analysed daily differences in miRNA cargo using small RNA-seq. Finally, we compared milk EVs differences from naturally infected udder quarters with their healthy adjacent quarters and quarters from uninfected udders.Results: We found that the milk EV miRNA cargo is very stable over the course of three days regardless of the health status of the quarter, and that infected quarters do not induce relevant changes in milk EVs of adjacent healthy quarters. Chronic subclinical mastitis induced changes in milk EV miRNA cargo, but neither in EVs size nor concentration. We observed that the changes in immunoregulatory miRNAs in quarters with chronic subclinical mastitis are cow-individual, however, the most upregulated miRNA was bta-miR-223-3p across all individuals.Conclusions: Our results showed that the miRNA profile and particle size characteristics remained constant throughout consecutive days, suggesting that miRNAs packed in EVs are physiological state-specific. In addition, since infected quarters are solely affected while adjacent healthy quarters remain unaffected. Finally, the cow-individual miRNA changes pointed towards infection-specific alterations.

Neuron-derived extracellular vesicles extracted from plasma show altered size and miRNA cargo as a function of antidepressant drug response

Previous work has demonstrated that microRNAs (miRNAs) change as a function of antidepressant treatment (ADT) response. However, it is unclear how representative these peripherally detected miRNA changes are to those occurring in the brain. This study aimed to use peripherally extracted neuron-derived extracellular vesicles (NDEVs) to circumvent these limitations and investigate neuronal miRNA changes associated with antidepressant response. Samples were collected at two time points (baseline and after 8 weeks of follow-up) from depressed patients who responded (N=20) and did not respond (N=20) to escitalopram treatment, as well as controls (N=20). Total extracellular vesicles (EVs) were extracted from plasma, and then further enriched for NDEVs by immunoprecipitation with L1CAM. EV size was measured using tunable resistive pulse sensing, and NDEV miRNA cargo was extracted and sequenced. Subsequently, studies in cell lines and postmortem tissue were conducted. Characterization of NDEVs revealed they were smaller than other EVs isolated from plasma (p<0.0001), had brain-specific neuronal markers, and contained miRNAs enriched for brain functions (p<0.0001) Furthermore, NDEVs from depressed patients were smaller than controls (p<0.05), and NDEV size increased with ADT response (p<0.01). Finally, changes in NDEV cargo, specifically changes in miR-21-5p, miR-30d-5p and miR-486-5p together (p<0.01), were associated with ADT response. Targets of these three miRNAs were altered in brain tissue from depressed individuals (p<0.05). Together, this study indicates that changes in peripherally isolated NDEVs can act as both a clinically accessible and informative biomarker of ADT response specifically through size and cargo.

Elucidating Methods for Isolation and Quantification of Exosomes: A Review

Exosomes are the smallest extracellular vesicles present in most of the biological fluids. They are found to play an important role in cell signaling, immune response, tumor metastasis, etc. Studies have shown that these vesicles also have diagnostic and therapeutic roles for which their accurate detection and quantification is essential. Due to the complexity in size and structure of exosomes, even the gold standard methods face challenges. This comprehensive review discusses the various standard methods such as ultracentrifugation, ultrafiltration, size-exclusion chromatography, precipitation, immunoaffinity, and microfluidic technologies for the isolation of exosomes. The principle of isolation of each method is described, as well as their specific advantages and disadvantages. Quantification of exosomes by nanoparticle tracking analysis, flow cytometry, tunable resistive pulse sensing, electron microscopy, dynamic light scattering, and microfluidic devices are also described, along with the applications of exosomes in various biomedical domains.

uPAR-expressing melanoma exosomes promote angiogenesis by VE-Cadherin, EGFR and uPAR overexpression and rise of ERK1,2 signaling in endothelial cells

Exosomes (Exos) have been reported to promote pre-metastatic niche formation, proliferation, angiogenesis and metastasis. We have investigated the role of uPAR in melanoma cell lines-derived Exos and their pro-angiogenic effects on human microvascular endothelial cells (HMVECs) and endothelial colony-forming cells (ECFCs). Melanoma Exos were isolated from conditioned media of A375 and M6 cells by differential centrifugation and filtration. Tunable Resistive Pulse Sensing (TRPS) and Nanoparticle tracking analysis were performed to analyze dimension and concentration of Exos. The CRISPR–Cas 9 technology was exploited to obtain a robust uPAR knockout. uPAR is expressed in melanoma Exos that are internalized by HMVECs and ECFCs, enhancing VE-Cadherin, EGFR and uPAR expression in endothelial cells that undergo a complete angiogenic program, including proliferation, migration and tube formation. uPAR loss reduced the pro-angiogenic effects of melanoma Exos in vitro and in vivo by inhibition of VE-Cadherin, EGFR and uPAR expression and of ERK1,2 signaling in endothelial cells. A similar effect was obtained with a peptide that inhibits uPAR–EGFR interaction and with the EGFR inhibitor Gefitinib, which also inhibited melanoma Exos-dependent EGFR phosphorylation. This study suggests that uPAR is required for the pro-angiogenic activity of melanoma Exos. We propose the identification of uPAR-expressing Exos as a potentially useful biomarker for assessing pro-angiogenic propensity and eventually monitoring the response to treatment in metastatic melanoma patients.

Particle Detection and Characterization for Biopharmaceutical Applications: Current Principles of Established and Alternative Techniques

Detection and characterization of particles in the visible and subvisible size range is critical in many fields of industrial research. Commercial particle analysis systems have proliferated over the last decade. Despite that growth, most systems continue to be based on well-established principles, and only a handful of new approaches have emerged. Identifying the right particle-analysis approach remains a challenge in research and development. The choice depends on each individual application, the sample, and the information the operator needs to obtain. In biopharmaceutical applications, particle analysis decisions must take product safety, product quality, and regulatory requirements into account. Biopharmaceutical process samples and formulations are dynamic, polydisperse, and very susceptible to chemical and physical degradation: improperly handled product can degrade, becoming inactive or in specific cases immunogenic. This article reviews current methods for detecting, analyzing, and characterizing particles in the biopharmaceutical context. The first part of our article represents an overview about current particle detection and characterization principles, which are in part the base of the emerging techniques. It is very important to understand the measuring principle, in order to be adequately able to judge the outcome of the used assay. Typical principles used in all application fields, including particle–light interactions, the Coulter principle, suspended microchannel resonators, sedimentation processes, and further separation principles, are summarized to illustrate their potentials and limitations considering the investigated samples. In the second part, we describe potential technical approaches for biopharmaceutical particle analysis as some promising techniques, such as nanoparticle tracking analysis (NTA), micro flow imaging (MFI), tunable resistive pulse sensing (TRPS), flow cytometry, and the space- and time-resolved extinction profile (STEP®) technology.

Development of Artificial Plasma Membranes Derived Nanovesicles Suitable for Drugs Encapsulation

Extracellular vesicles (EVs) are considered as promising nanoparticle theranostic tools in many pathological contexts. The increasing clinical employment of therapeutic nanoparticles is contributing to the development of a new research area related to the design of artificial EVs. To this aim, different approaches have been described to develop mimetic biologically functional nanovescicles. In this paper, we suggest a simplified procedure to generate plasma membrane-derived nanovesicles with the possibility to efficiently encapsulate different drugs during their spontaneously assembly. After physical and molecular characterization by Tunable Resistive Pulse Sensing (TRPS) technology, transmission electron microscopy, and flow cytometry, as a proof of principle, we have loaded into mimetic EVs the isoquinoline alkaloid Berberine chloride and the chemotherapy compounds Temozolomide or Givinostat. We demonstrated the fully functionality of these nanoparticles in drug encapsulation and cell delivery, showing, in particular, a similar cytotoxic effect of direct cell culture administration of the anticancer drugs. In conclusion, we have documented the possibility to easily generate scalable nanovesicles with specific therapeutic cargo modifications useful in different drug delivery contexts.