A survey to evaluate parameters governing the selection and application of extracellular vesicle isolation methods.

Extracellular vesicles (EVs) continue to gain interest across the scientific community for diagnostic and therapeutic applications. As EV applications diversify, it is essential that researchers are aware of challenges, in particular the compatibility of EV isolation methods with downstream applications and their clinical translation. We report outcomes of the first cross-comparison study looking to determine parameters (EV source, starting volume, operator experience, application and implementation parameters such as cost and scalability) governing the selection of popular EV isolation methods across disciplines. Our findings highlighted an increased clinical focus, with 36% of respondents applying EVs in therapeutics and diagnostics. Data indicated preferential selection of ultracentrifugation for therapeutic applications, precipitation reagents in clinical settings and size exclusion chromatography for diagnostic applications utilising biofluids. Method selection was influenced by operator experience, with increased method diversity when EV research was not the respondents primary focus. Application and implementation criteria were indicated to be major influencers in method selection, with UC and SEC chosen for their abilities to process large and small volumes, respectively. Overall, we identified parameters influencing method selection across the breadth of EV science, providing a valuable overview of practical considerations for the effective translation of research outcomes.

Defining the influence of size-exclusion chromatography fraction window and ultrafiltration column choice on extracellular vesicle recovery in a skeletal muscle model.

Extracellular vesicles (EVs) have the potential to provide new insights into skeletal muscle (SM) physiology and pathophysiology. However, current isolation protocols often do not eliminate co-isolated components such as lipoproteins and RNA binding proteins that could confound outcomes and hinder downstream clinical translation. In this study, we validated an EV isolation protocol that combined size-exclusion chromatography (SEC) with ultrafiltration (UF) to increase sample throughput, scalability and purity, while providing the very first analysis of the effects of UF column choice and fraction window on EV recovery. C2C12 myotube conditioned medium was pre-concentrated using either Amicon® Ultra 15 or Vivaspin®20 100 KDa UF columns and processed by SEC (IZON, qEV 70 nm). The resulting thirty fractions obtained were individually analysed to identify an optimal fraction window for EV recovery. The EV marker TSG101 could be detected from fractions 5 to 14, while CD9 and Annexin A2 only up to fraction 6. ApoA1+ lipoprotein co-isolates were detected from fraction 6 onwards for both protocols. Strikingly, Amicon and Vivaspin UF concentration protocols led to qualitative and quantitative variations in EV marker profiles and purity. Eliminating lipoprotein co-isolation by reducing the SEC fraction window resulted in a net loss of particles, but increased measures of sample purity and had only a negligible impact on the presence of EV marker proteins. In conclusion, our study developed an effective UF+SEC protocol for the isolation of EVs based on sample purity (fractions 1-5) and total EV abundance (fractions 2-10). We provide evidence to demonstrate that the choice of UF column can affect the composition of the resulting EV preparation and needs to be considered when being applied in EV isolation studies in SM. The resulting protocols will be valuable in isolating highly pure EV preparations for applications in a range of therapeutic and diagnostic studies.

Direct contact-mediated non-viral gene therapy using thermo-sensitive hydrogel-coated dressings.

Nanotechnologies are being increasingly applied as systems for peptide and nucleic acid macromolecule drug delivery. However systemic targeting of these, or efficient topical and localized delivery remains an issue. A controlled release system that can be patterned and locally administered such as topically to accessible tissue (skin, eye, intestine) would therefore be transformative in realizing the potential of such strategies. We previously developed a technology termed GAG-binding enhanced transduction (GET) to efficiently deliver a variety of cargoes intracellularly, using GAG-binding peptides to mediate cell targeting, and cell penetrating peptides (CPPs) to promote uptake. Herein we demonstrate that the GET transfection system can be used with the moisturizing thermo-reversible hydrogel Pluronic-F127 (PF127) and methyl cellulose (MC) to mediate site specific and effective intracellular transduction and gene delivery through GET nanoparticles (NPs). We investigated hydrogel formulation and the temperature dependence of delivery, optimizing the delivery system. GET-NPs retain their activity to enhance gene transfer within our formulations, with uptake transferred to cells in direct contact with the therapy-laden hydrogel. By using Azowipe™ material in a bandage approach, we were able to show for the first-time localized gene transfer in vitro on cell monolayers. The ability to simply control localization of gene delivery on millimetre scales using contact-mediated transfer from moisture-providing thermo-reversible hydrogels will facilitate new drug delivery methods. Importantly our technology to site-specifically deliver the activity of novel nanotechnologies and gene therapeutics could be transformative for future regenerative medicine.

Efficient Delivery of Transducing Polymer Nanoparticles for Gene-Mediated Induction of Osteogenesis for Bone Regeneration.

Developing non-viral gene therapy vectors that both protect and functionally deliver nucleic acid cargoes will be vital if gene augmentation and editing strategies are to be effectively combined with advanced regenerative medicine approaches. Currently such methodologies utilize high concentrations of recombinant growth factors, which result in toxicity and off-target effects. Herein we demonstrate the use of modified cell penetrating peptides (CPPs), termed Glycosaminoglycan (GAG)-binding Enhanced Transduction (GET) peptides with plasmid DNA (pDNA) encapsulated poly (lactic-co-glycolic acid) PLGA nanoparticles (pDNA-encapsulated PLGA NPs). In order to encapsulate the pDNA, it was first condensed with a cationic low molecular weight Poly L-Lysine (PLL) into 30-60 nm NPs followed by encapsulation in PLGA NPs by double emulsion; yielding encapsulation efficiencies (EE) of ∼30%. PLGA NPs complexed with GET peptides show enhanced intracellular delivery (up to sevenfold) and transfection efficiencies (up to five orders of magnitude). Moreover, the pDNA cargo has enhanced protection from nucleases (such as DNase I) promoting their translatability. As an example, we show these NPs efficiently deliver pBMP2 which can promote osteogenic differentiation in vitro. Gene delivery to human Mesenchymal Stromal Cells (hMSCs) inducing their osteogenic programming was confirmed by Alizarin red calcium staining and bone lineage specific gene expression (Q RT-PCR). By combining simplistic and FDA-approved PLGA polymer nanotechnology with the GET delivery system, therapeutic non-viral vectors could have significant impact in future cellular therapy and regenerative medicine applications.