Purification Analysis, Intracellular Tracking, and Colocalization of Extracellular Vesicles Using Atomic Force and 3D Single-Molecule Localization Microscopy

Puthukodan, Sujitha; Hofmann, Martina; Mairhofer, Mario; Janout, Hannah; Schurr, Jonas; Hauser, Fabian; Naderer, Christoph; Preiner, Johannes; Winkler, Stephan; Sivun, Dmitry; Jacak, Jaroslaw

doi:10.1021/acs.analchem.3c00144

Cited by 1 publication

(1 citation statement)

References 68 publications

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“…This holds promise for the development of more biocompatible and scalable intracellular drug delivery nanocarriers with a reduced immunogenicity. These results align well with previous studies utilizing Nanoflow cytometry , and single molecule localization microscopy, which have also observed a similar range of cargo loading in engineered EV subpopulations, with a similar percentage of cargo-loaded extracellular vesicles at the single-molecule and single-vesicle level. Furthermore, the additional cleaning procedures in this study provided stable results for Ab-loading and insights into the relationship between nanovesicle physical properties and cargo nature, offering opportunities to enhance the purity of production yields and customize these nanovesicles for specific therapeutic agent for Ab delivery with substantially lower side effects.…”

Section: Discussionmentioning

confidence: 99%

Antibody-Loading of Biological Nanocarrier Vesicles Derived from Red-Blood-Cell Membranes

Sanaee,

Ronquist,

Sandberg

et al. 2024

ACS Omega

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Antibodies, disruptive potent therapeutic agents against pharmacological targets, face a barrier in crossing immune systems and cellular membranes. To overcome these, various strategies have been explored including shuttling via liposomes or biocamouflaged nanoparticles. Here, we demonstrate the feasibility of loading antibodies into exosome-mimetic nanovesicles derived from human red-blood-cell membranes, which can act as nanocarriers for intracellular delivery. Goat-antichicken antibodies are loaded into erythrocyte-derived nanovesicles, and their loading yields are characterized and compared with smaller dUTP-cargo molecules. Applying dual-color coincident fluorescence burst analyses, the loading yield of nanocarriers is rigorously profiled at the single-vesicle level, overcoming challenges due to size-heterogeneity and demonstrating a maximum antibody-loading yield of 38–41% at the optimal vesicle radius of 52 nm. The achieved average loading yields, amounting to 14% across the entire nanovesicle population, with more than two antibodies per loaded vesicle, are fully comparable to those obtained for the much smaller dUTP molecules loaded in the nanovesicles after additional exosome-spin-column purification. The results suggest a promising new avenue for therapeutic delivery of antibodies, potentially encompassing also intracellular targets and suitable for large-scale pharmacological applications, which relies on the exosome-mimetic properties, biocompatibility, and low-immunogenicity of bioengineered nanocarriers synthesized from human erythrocyte membranes.

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Section: Discussionmentioning

confidence: 99%