| Autor: |
Morrison G; Department of Chemical Engineering, Imperial College London, South Kensington Campus, London, SW7 2AZ, UK. rongjun.chen@imperial.ac.uk., Henry N; Department of Chemical Engineering, Imperial College London, South Kensington Campus, London, SW7 2AZ, UK. rongjun.chen@imperial.ac.uk., Kopytynski M; Department of Chemical Engineering, Imperial College London, South Kensington Campus, London, SW7 2AZ, UK. rongjun.chen@imperial.ac.uk., Chen R; Department of Chemical Engineering, Imperial College London, South Kensington Campus, London, SW7 2AZ, UK. rongjun.chen@imperial.ac.uk. |
| Abstrakt: |
Saporin is a 28 621 Da protein and plant toxin possessing rRNA N -glycosidase activity. Due to its potent ribosome-inactivating ability, saporin is commonly studied as an anticancer agent. However, its enzymatic activity is greatly hindered by its poor plasma membrane permeability. To overcome this barrier, we used a bioinspired intracellular delivery platform based on the pH-responsive pseudopeptide, poly(L-lysine isophthalamide) grafted with L-phenylalanine at a stoichiometric molar percentage of 50% (PP50). PP50 was co-incubated with saporin (PP50/saporin) in a mildly acidic pH environment to aid intracellular delivery and increase saporin's therapeutic potential. We demonstrated that PP50 greatly enhanced the cytotoxicity of saporin in the 2D monolayer of A549 cells and 3D A549 multicellular spheroids whilst remaining non-toxic when administered alone. To elucidate the mechanism of cell death, we assessed the activation of caspases, the inhibition of protein synthesis, the onset of apoptosis and the mechanism of PP50/saporin entry. Inhibition of protein synthesis and activation of caspases 3/7, 8 and 9 were found to occur before the onset of apoptosis and cell death. PP50/saporin was also shown to rely on micropinocytosis and caveolae-mediated endocytosis for cell entry. In addition, fluorescein isothiocyanate-labelled saporin (FITC-saporin) was localized within the cytoplasm and nuclei when delivered with Cyanine5-labelled PP50 (Cy5-PP50). Taken together, this suggests that multiple pathways are triggered to initiate apoptosis and cell death in cells treated with PP50/saporin. Therefore, these results make PP50 a potential intracellular delivery platform for the internalization of protein therapeutics. |
