Size-dependent cellular uptake of exosomes

doi:10.1016/j.nano.2016.12.009

Nanomedicine: Nanotechnology, Biology and Medicine

Volume 13, Issue 3, April 2017, Pages 1011-1020

https://doi.org/10.1016/j.nano.2016.12.009 Get rights and content

Abstract

The ability of exosomes to elicit specific cellular responses suggests that they may be increasingly used as therapeutics. Their vesicular nature makes them suitable as potential nanocarriers for drugs or nucleic acids delivery. Here we address the question whether the method of preparation of enriched exosomal fractions can affect their uptake by cells and their ability to trigger a response. We compared ultracentrifugation and polymer-based precipitation methods on supernatants of glioma-associated stem cells isolated from a high-grade glioma patient. We determined particle size distributions after purification and their correlation with uptake, proliferation and migration in glioblastoma cell cultures. Our findings indicate that polymer-based precipitation leads to smaller particle size distributions, faster uptake by target cells and increased cellular motility. The different effect that isolation method-dependent populations of particles have on cell motility suggests their size distribution could also profoundly affect exosomes therapeutic potential.

Graphical Abstract

Exosomes from glioma-associated stem cells (GASC) were purified using either ExoQuick (EQ) or ultracentrifugation (UC). The two purification methods generated different particle size distributions and atomic force microscope images showed that EQ preparations were richer in smaller vesicles compared to UC. Also, cellular uptake from A172 glioblastoma cells showed that small EQ exosomes were preferentially internalized compared to UC and this increased the motility of EQ-exposed cells compared to UC and a negative control (NC) not exposed to GASC exosomes. We concluded that smaller exosomes are preferentially internalized and they elicit a cellular response more effectively.

Section snippets

Cell cultures

Human glioma samples were collected at the Department of Neuroscience, Santa Maria della Misericordia University Hospital in Udine, after a written informed consent from the patient was obtained, in accordance with the declaration of Helsinki, and with approval by the independent ethics committee of the hospital. Glioma associated stem cells (GASC) and A172 human glioblastoma cells were cultured as described in26, 27 and detailed in the Supplementary methods.

Exosome purification, size and density measurements

A172 and GASC supernatants were

Effect of the extraction method on the size distribution of exosomal fractions

EQ and UC isolated exosomes from three GASC cultures supernatants derived from the same patient were adsorbed on mica coverslips and imaged using AFM to estimate the size distribution of the particles in the exosome fractions. Figure 1, A and B are representative 5 × 5 μm images of EQ and UC purified exosomes respectively, obtained from the same supernatant. Although both preparations produced individually separated particles with a near-spherical morphology, the EQ method generated particles

Discussion

In this work, we emphasized the importance of looking at the physical properties of exosomal preparations rather than limiting the characterization to molecular attributes. Our rationale is that exosomes play an important role in cellular communication and, regardless of the specific molecules they carry, the nature of the “envelope” might be of great importance for the delivery of the message. For example, in the case of nanoengineered nanoparticles it has been shown that size per se can

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: Funding: Timea Palmai-Pallag was funded by the BBSRC grant BB/K019597/1.

: Conflict of interest: No competing interests are present.

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Original ArticleSize-dependent cellular uptake of exosomes

Abstract

Graphical Abstract

Section snippets

Cell cultures

Exosome purification, size and density measurements

Effect of the extraction method on the size distribution of exosomal fractions

Discussion

J Proteomics

Curr Opin Cell Biol

Methods

J Thromb Haemost

Colloids Surf B Biointerfaces

Adv Drug Deliv Rev

J Immunol Methods

Blood

Am J Pathol

J Thromb Haemost

Exosomes: current knowledge of their composition, biological functions, and diagnostic and therapeutic potentials

Biochim Biophys Acta Gen Subj

Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells

Nat Cell Biol

ExoCarta 2012: database of exosomal proteins, RNA and lipids

Nucleic Acids Res

The complete exosome workflow solution: from isolation to characterization of RNA cargo

Biomed Res Int

Therapeutic Uses of exosomes

J Circ Biomark

Extracellular vesicles: exosomes, microvesicles, and friends

J Cell Biol

The impact of disparate isolation methods for extracellular vesicles on downstream RNA profiling

J Extracell Vesicles

Engineered nanoparticles interacting with cells: size matters

J Nanobiotechnol

Isolation and characterization of exosomes from cell culture supernatants and biological fluids

Curr Protoc Cell Biol

Original Article
Size-dependent cellular uptake of exosomes