By NanoFCM
Taking advantage of the Flow NanoAnalyzer multiparameter analysis of single EVs as small as 40 nm, six commonly used drug-loading strategies (coincubation, electroporation, extrusion, freeze-thawing, sonication, and surfactant treatment) were explored by employing doxorubicin (Dox) as the model drug. Encapsulation ratio, EV concentration, drug content, and membrane proteins of Dox-loaded EVs were measured at the single-particle level. Data indicated that coincubation and electroporation outperformed other methods with an encapsulation ratio of approximately 45% and a higher Dox content in single EVs. Interestingly, the labeling ratios of membrane proteins indicated that varying degrees of damage to the surface proteins of EVs occurred upon extrusion, freeze-thawing, sonication, and surfactant treatment. Figure 1. Encapsulation ratio of Dox-loaded EVs in different drug-loading strategies Figure 2. The expression of membrane proteins of Dox-loaded EVs in differemt drug-loading strategies Confocal fluorescence microscopy and nano-flow cytometry analysis revealed that Dox-loaded EVs prepared by electroporation induced the strongest apoptosis followed by coincubation. These results correlated well with their cellular uptake rate and fundamentally with the Dox encapsulation efficiency of single EVs. Flow The NanoAnalyzer provides a rapid and sensitive platform for single-particle assessment of drug-loading strategies for incorporating drugs into EVs. Single-particle analysis by the Flow NanoAnalyzer was further employed to reveal the heterogeneous encapsulation behaviour of Dox-mEVs which would otherwise be overlooked by bulk-based approaches. Correlation analysis between doxorubicin auto-fluorescence and the fluorescence of a lipophilic dye DiD suggested that only the lipid-enclosed particles were actively loadable. Meanwhile, immunofluorescence analysis revealed that more than 85% of the casein positive particles was doxorubicin free. These findings further inspired the development of the lipid-probe- and immuno-mediated magnetic isolation techniques to selectively remove the contaminants of non-lipid enclosed particles and casein assemblies, respectively. Finally, the intracellular assessments confirmed the superior performance of SEAL-prepared mEV formulations, and demonstrated the impact of encapsulation heterogeneity on therapeutic outcome. Figure 1.Single particle analysis of the drug loading capacity of SEAL-prepared Dox-mEVs by nano-flow cytometry. Figure 2. Multi-parameter analysis of the correlation between drug loading capacity and lipid labelling pattern of Dox-mEVs. The developed cargo-loading approach and the Flow NanoAnalyzer-based characterization method provides instructive insight into the development of EV-based delivery systems. In this study, the authors harness RBCEVs and engineer them via exogenous drug loading and surface functionalization to develop an efficient drug delivery system for AML and characterise the single particles using the Flow NanoAnalyzer. Figure 1. EVs surface antibody modification strategies Figure 2. The Flow NanoAnalyzer provides an estimation of the modification efficiency To estimate the antibody conjugation efficiency at single-RBCEV level, the Flow NanoAnalyzer was used to detect RBCEVs coated with biotinylated anti-CD33 antibody and isotype monoclonal antibody (IgG-EV). The nano-flow cytometric analysis indicated that ~74% of RBCEVs were successfully conjugated with the CD33 monoclonal antibody. Subsequent in vivo experiments demonstrated that RBCEVs loaded with ASO significantly suppresses AML progression. Nano-flow cytometry plays a crucial role in the validation of exosome modification results and is expected to advance the development of engineered EVs drugs. Using the Flow NanoAnalzyer to identify scaffold proteins, and analyze the expression of several scaffold proteins at single EVs particle level, the researchers showed that PTGFRN protein was expressed in 97% of exosomes. In contrast to the semi-quantitative Western Blot and the ELISA method which can only provide an average value, the Flow NanoAnalzyer can determine the proportion of positive subpopulations. Furthermore, the fluorescence intensity can be used to further reflect information on the amount of protein expression on single EVs particle. Figure 1. The expression of PTGFRN proteins and IL2 Figure 2. Proteins’s different proportion of expression on the Flow NanoAnalyzer The application of the Flow NanoAnalzyer is the first single particle assay for genetically modified PTGFRN exosomes at single particle level. Traditional ELISA can only quantify the protein on a large number of exosomes, while the Flow NanoAnalzyer can identify and quantify the protein expression at the individual exosome level, and found that PTGFRN is an ideal scaffold protein with expression percentage of 97%. In this study, a laboratory-built nano-flow cytometer (nFCM) that can detect single EVs as small as 40 nm in diameter and single DNA fragments of 200 bp upon SYTO™ 16 staining was used to study EV-DNA at the single-vesicle level. Through simultaneous side-scatter and fluorescence (FL) detection of single particles and with the combination of enzymatic treatment, present study revealed that: (1) naked DNA or DNA associated with non-vesicular entities is abundantly presented in EV samples prepared from cell culture medium by ultracentrifugation; (2) the quantity of EV-DNA in individual EVs exhibits large heterogeneity and the population of DNA positive (DNA+ ) EVs varies from 30% to 80% depending on the cell type; (3) external EV-DNA is mainly localized on relatively small size EVs (e.g. <100 nm for HCT-15 cell line) and the secretion of external DNA+ EVs can be significantly reduced by exosome secretion pathway inhibition; (4) internal EV-DNA is mainly packaged inside the lumen of relatively large EVs (e.g. 80-200 nm for HCT-15 cell line); (5) double-stranded DNA (dsDNA) is the predominant form of both the external and internal EV-DNA; (6) histones (H3) are not found in EVs, and EV-DNA is not associated with histone proteins and (7) genotoxic drug induces an enhanced release of DNA+ EVs, and the number of both external DNA+ EVs and internal DNA+ EVs as well as the DNA content in single EVs increase significantly. The Flow NanoAnalyzer was employed to analyze the liposomes, exosomes and gETL NPs at single-particle level, which played important roles in the optimization and screening of conditions for genetically engineered exosomes and the production of gETL NPs.
