Difference between revisions of "Team:NJU-China/Results/Conclusions"
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Revision as of 12:53, 1 November 2017
Nanoparticle tracking analysis to characterize exosome
We performed a nanoparticle tracking analysis (NTA) for further evaluation of the quantity and size of secreted exosomes. The use of Nanosight enabled quantification and size determination of the extracellular vesicles, as nanoparticles can be automatically tracked and sized based on Brownian motion and the diffusion coefficient. The size of exosomes attained ranged around 172nm, and particle concentration is about 9.74e8 particles/ml, according to the dilute ratio(1) and protein concentration(715ng/ul), then the relationship between protein and exosome is 1ng protein equals 1400 exosome particles, All the data collected helped us decide the transfection dosage of siRNA and dosing of treatment prepared for animal experiment.
(a)
(b)
(c)
(d)
(e)
Nanoparticle tracing analysis(NTA) for exosome characterization. (a) Intensity / Size graph for Experiment.(b) FTLA Concentration / Size graph for three Experiments, most exosomes’ diameters are around 170nm. (c) Averaged FTLA Concentration / Size for Experiment.(d) Experiment settings. (e)Results.
Targeting valuation in vivo
To ensure the targeting of our tpep modified exosome, we obtained a qualitative description of in vivo drug distribution after systematic administration. Egfr( epidermal growth factor receptor) siRNA was used in the place of Bcl2 siRNA in this validation. The mice in control group were injected with egfr siRNA exosomes and experimental group were injected with egfr siRNA-tpep exosomes. 12hours after the injection, the mice were dissected and their heart, liver, kidney and fat tissue were separated, then we isolated tissue RNA and ran a Qpcr. Comparing to the control group, egfr siRNA expression level in kidney and heart is marginally increased, and there is a downward trend in liver, while a significant rise of egfr siRNA expression level in fat tissue is indicated, demonstrating that our tpep module has a specific affinity to adipose tissue.
Tpep exosome is targeted to fat tissue. Qpcr analysis of egfr siRNA level in different tissues
Characterization of Bcl2 siRNA-tpep exosome by TEM imaging.
To characterize the exosome based drug delivery system in a more intuitive way, we performed a transmission electron microscopy (TEM). The TEM image showed that the exosomes presented normal morphological characteristics after outside modification and siRNA loading, with a diameter of approximately 130 nanometer and a double-layer membrane.
TEM image of tpep-modified exosomes packaging bcl2 siRNA
