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. 2014 Mar 1;448:41-9.
doi: 10.1016/j.ab.2013.12.001. Epub 2013 Dec 9.

Maximizing Exosome Colloidal Stability Following Electroporation

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Free PMC article

Maximizing Exosome Colloidal Stability Following Electroporation

Joshua L Hood et al. Anal Biochem. .
Free PMC article

Abstract

Development of exosome-based semisynthetic nanovesicles for diagnostic and therapeutic purposes requires novel approaches to load exosomes with cargo. Electroporation has previously been used to load exosomes with RNA. However, investigations into exosome colloidal stability following electroporation have not been considered. Herein, we report the development of a unique trehalose pulse media (TPM) that minimizes exosome aggregation following electroporation. Dynamic light scattering (DLS) and RNA absorbance were employed to determine the extent of exosome aggregation and electroextraction post electroporation in TPM compared to common PBS pulse media or sucrose pulse media (SPM). Use of TPM to disaggregate melanoma exosomes post electroporation was dependent on both exosome concentration and electric field strength. TPM maximized exosome dispersal post electroporation for both homogenous B16 melanoma and heterogeneous human serum-derived populations of exosomes. Moreover, TPM enabled heavy cargo loading of melanoma exosomes with 5nm superparamagnetic iron oxide nanoparticles (SPION5) while maintaining original exosome size and minimizing exosome aggregation as evidenced by transmission electron microscopy. Loading exosomes with SPION5 increased exosome density on sucrose gradients. This provides a simple, label-free means of enriching exogenously modified exosomes and introduces the potential for MRI-driven theranostic exosome investigations in vivo.

Keywords: Electroporation; Exosomes; Iron oxide; Superparamagnetic; Trehalose.

Figures

Figure 1
Figure 1
Exosome electroporation in the presence of trehalose pulse media. Post storage at −80° C, B16 melanoma exosomes were subjected to electroporation at 4°C using selected electric field strengths. (a) The amount of RNA electroextracted from 50 μg of exosomes was determined by RNA absorbance and reported as percent electroextraction. Background RNA absorbance in the absence of electroporation (0 kV/cm) was set to 0%; (n = 15 for 3 experiments and 5 measurements per experiment); error bars = s.d. (b) B16 melanoma exosome (50 μg) size was measured by DLS as a function of varying electric field strengths; (n = 27 for 3 experiments and 9 measurements per experiment); error bars = s.e.m. (c) B16 melanoma exosome size was measured using a constant electric field strength of 0.75 kV/cm while varying exosome concentration (micrograms of protein per 0.75 ml of trehalose pulse media); (n = 10 for 3 pooled exosome isolations); error bars = s.d. Best fit dotted lines denote linear regression analyses indicated by R2 values. Connecting bars denote statistically significant differences; p values < 0.05 were considered significant.
Figure 2
Figure 2
Comparing the effects of PBS and trehalose PBS pulse media on exosome size and electroextraction. B16 melanoma exosomes were electroporated in the presence of PBS or trehalose PBS pulse media using electric field strengths of 0, 0.75 and 1.5 kV/cm. (a) Exosome size (n = 30 for 3 experiments and 10 measurements per experiment, error bars = s.e.m.) and (b) RNA electroextraction (n = 15 for 3 experiments and 10 measurements per experiment, error bars = s.d.) were determined by DLS and RNA absorbance post electroporation. For background RNA absorbance in the absence of electroporation, electroextraction in PBS at 0 kV/cm was normalized to 100%. Connecting bars denote statistically significant differences; p values < 0.05 were considered significant.
Figure 3
Figure 3
Comparing the effects of sucrose and trehalose pulse media on exosome size and zeta potential. B16 melanoma exosomes (50 μg) were electroporated in the presence of different concentrations of sucrose PBS (25, 50, 100 mM) or trehalose PBS (25, 50, 100 mM) pulse media using an electric field strength of 0.75 kV/cm. (a) Exosome size (n = 30 for 3 experiments and 10 measurements per experiment, error bars = s.e.m.) was determined by DLS. S = sucrose; T = trehalose (b) Exosome electrokinetic mobility was determined using a ZetaPlus Zeta Potential analyzer in the presence of PBS, 50 mM sucrose PBS, or 50 mM trehalose PBS pulse media (n = 50 for 4 experiments, error bars = s.e.m.). Connecting bars denote statistically significant differences; p values < 0.05 were considered significant.
Figure 4
Figure 4
Comparing the effects of PBS and trehalose pulse media on human serum exosome size. After storage at −80°C, exosomes isolated from pooled human serum (Innovative Research Inc.) were subjected to electroporation in the presence of PBS or trehalose PBS pulse media at 4°C at selected electric field strengths. Exosome size was measured by DLS (n = 10 measurements; error bars = s.d.) Best fit dotted lines denote linear regression analyses indicated by R2 values. Significant differences between PBS and trehalose pulse media at selected electric field strengths are denoted by p values; p values < 0.05 were considered significant.
Figure 5
Figure 5
Loading exosomes with SPION5. B16 melanoma exosomes were loaded with SPION5 passively or by electroporation in the presence of trehalose pulse media at 0.75 kV/cm. (a) B16 melanoma exosomes visualized by TEM in the absence of electroporation or post electroporation with SPION5; the black lower left corner scale bar = 100 nm (b) Selected stages of SPION5 loading or unloading of exosome contents are observed by TEM after electroporation. (c) SPION5 loaded exosome size was assessed with DLS (n = 6; error bars = s.d.) (d) DiI labeled SPION5 loaded exosomes were isolated on a representative continuous sucrose gradient, fractions corresponding to peak exosome densities collected, densities determined and exosomes detected using Xenogen IVIS. (e) SPION5 loaded exosome iron content was assessed for exosome peak fractions using ICP-OES (n =3; error bars = s.d.). EP = electroporated; F = fraction

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