In this project, we are determined to create potential oxygen carriers, transporting hemoglobin using the cargo loading of exosomes, for the development of artificial blood. PNCV plasmids have been transfected into HEK 293T cell line, and the successful translation of Vhb (Vitreoscilla hemoglobin) was proved by western blot results. Exosomes were extracted from the medium. SDS-PAGE results and TEM observation both yielded expected results, demonstrating the feasibility of this extraction. Attaching Vhb to exosomes didn’t affect either protein, which was again shown by TEM results. COD testing proved that the heme group was functioning successfully: better in the case of membrane anchored protein CD63 than that of WWtag3. Repeated testing, as well as comparison between control groups and fusion protein groups demonstrated the success of experiment: by concluding all experimental evidence, we see that an efficient oxygen carrier, composed of Vhb and exosome, has been produced. Although more experiments still have to be performed to demonstrate its behaviors in vivo, and more considerations for its practice need to be taken, we consider it an essential step in the whole process of developing artificial blood.
Western blot results indicated production of Vhb proteins.
SDS-PAGE yielded correct protein bonds for exosomes. TEM visualized the correct shape and size.
TEM demonstrated that the fusion of Vhb didn’t affect the shape or concentration of exosomes. COD proved the potential function of fusion proteins to transport oxygen efficiently. The loading of Vhb into exosome has been successful.1: Successful extraction lysis, and TEM imaging of exosomes:
We’ve used total exosome isolation kit (from medium) to extract the exosomes. The exosomes is visible in the bottom of the tube after centrifuge. After ripa lysis, SDS-PAGE results shown correct protein bands for exosomes. TEM result of paraformaldehyde fixed exosomes proves that exosomes extracted using this method has the correct shape and size.
2: CD63-Vhb Fusion Protein can transport Vhb into exosomes and the function of Vhb is not disturbed.
Cell content western blotting first proved successful translation of PNCV plasmids inside HEK 293T cells. Exosome content shown successful result of fusion protein band around 40 kda. TEM result of sample shown that the protein doesn’t effect the shape or the concentration of exosomes. COD result compared to normal exosomes shown that the fusion protein contains heme group that increases the chemical demand of exosomes and thus proving the function of Vhb is not disturbed. Also, color difference can be seen between normal and fusion-protein containing exosomes.
3: WW3-Vhb can be actively transported into exosomes, but more experiment is needed.
Cell content western blotting first proved successful translation of PNW3V plasmids inside HEK 293T cells. Exosome content shown pale fusion protein band around 30kda. TEM result of sample shown that the protein doesn’t effect the shape or the concentration of exosomes. COD result compared to normal exosomes shown that the COD value increased but the difference is not as huge as PNCV transfected once.
4:COD value can be used to test loading of Vhb into exosomes.
Repeat test on COD value of normal exosome shown that normal exosomes extracted from different plates have approximately the same base COD value. A difference can be seen between regular, PNV transfected and PNCV transfected exosomes. WW3 transfected does not show a significant difference.