Sonication Aided Exosomes Drug Loading

Exosomes are natural, intrinsic, cell-derived inter-cellular communication phospholipid membrane nanovesicles in the size range of 40-100 nm. Besides carrying several cell-derived growth factor, they can serve as a great tool for intracellular drug delivery.

These biologically natural nanocarriers possess many advantages such as:

1- They are known to bypass biological barriers, such as the blood-brain barrier.

2- Exosomes have the property of homing, making them helpful in tissue targeting.

3- High biocompatibility.

4- Cargo protection from degradation.

Due to these promising properties exosomes can be used as drug delivery carrier.

Procedure:

1-    measuring exosome concentration using BCA assay kit or Bradford assay.

2-    Exosomes are diluted in PBS to a concentration 0.15 mg/mL total protein.

3-    Determination of the drug solubility to determine the used solvent (PBS or DMSO).

4-    Post loading strategy; the drug solution in PBS (0.5 mg/mL) was added to 250 µl of exosomes to the final concentration 0.1 mg/mL total protein.

Illustration:

125 ul (drug) added to 250 ul EXO

MV=MV

150×250=100V2

V2=375 µl

Therefore, 125 µl Drug+250 µl EXO =375 total volume

5-    Drug-EXO mixture is sonicated using probe sonicator (750 v, 20% power, 6 cycles by 4 sec pulse /2 sec pause (total time 24 sec), cooled down on ice for 2 min, and then sonicated again.

Note: 1-it is preferred to put the EXO-DRUG mixture in falcon tube during sonication to avoid loss of volume during sonication.

6-    Incubation in 37 °C for 1 hr without shaking (The membrane integrity of the exosomes has been found to be restored within an hour when the exosomes are incubated at 37 °C).

7-    Put 300 ul of the EXO-DRUG mixture in Nanosep. (or in dialysis sac)

8-    Centrifugation (cooling) 7500 rpm for 15 min for 2 runs (twice).

9-    The amount of Drug loaded into exosomes was measured by a high-performance liquid chromatography (HPLC) or any suitable device (GC-mass – ICP) method according of the drug.

10- To ensure the exosomes integrity and structure we can confirm using TEM.

Refrences:

Haney, M. J., Klyachko, N. L., Zhao, Y., Gupta, R., Plotnikova, E. G., He, Z., … & Batrakova, E. V. (2015). Exosomes as drug delivery vehicles for Parkinson’s disease therapy. Journal of controlled release, 207, 18-30.

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