Experiments

1. Exosomal biomarker synthesis

We transfected HEK293T cells with nanoluciferase coding sequence fused with exosomal biomarker membrane protein CD63 (BBa_K2619100) for the subsequent quantification of exosomes.

Figure 1: Negative control was done by transfecting the HEK293T cells with empty pcDNA3.1 plasmid

2. Exosome boosting analysis by luciferase assay

Since the biomarker of exosomes was synthesized successfully in experiment 1. We then conducted experiments to test the exosome production boosting ability of our deigned devices (BBa_K2619014—nSMase and BBa_K2619103—Booster) by co-transfection.

Figure 2: NC: negative control; done by transfecting CD63-nluc and empty pcDNA3.1 plasmid (1:2 ratio). Booster: Steap-SDC4-NadB (BBa_2619103); done by transfecting CD63-nluc, Booster and pcDNA3.1 plasmid (1:1:1 ratio). Booster nSMase: done by transfecting Booster, nSMase and CD63-nluc (1:1:1 ratio)

3. Exosome production boosting analysis by Pierce BCA protein assay

Our team utilized varied methods to confirm the validity of exosomal production enhancement of Booster and nSMase.

Figure 3: NC: negative control; done by transfecting empty pcDNA3.1 plasmid. Booster: Steap-SDC4-NadB; done by transfecting Booster and pcDNA3.1 plasmid (1:1 ratio). Booster nSMase: done by transfecting Booster and nSMase (1:1 ratio)

4. Exosome production boosting analysis by nanoparticle tracking assay (NTA)

The third method of verifying the devices capacity in exosome production enhancement is doing NTA by NanosightTM. After purification of exosomes by Total Exosome Isolation Reagent (Thermo ScientificTM), the exact secreted particle numbers were analyzed.

Figure 4: NC: negative control; done by transfecting empty pcDNA3.1 plasmid. Booster: Steap-SDC4-NadB; done by transfecting Booster and pcDNA3.1 plasmid (1:1 ratio). Booster nSMase: done by transfecting Booster and nSMase (1:1 ratio)

Conclusion

A combination of two exosome production accelerators/enhancing devices is reported to be the most capable of boosting the cells in exosome production.

Lamp2b protein linkage experiment

We fused nanoluciferase gene with exosomal membrane protein Lamp2b to examine its downstream gene expression. (Lamp2b-nluc, BBa_K2619113).

Figure 5: NC: negative control; done by transfecting empty pcDNA3.1 plasmid.