Preparation And Authentication Of Cpps-β-Glucosidase-Cetuximab Triple Conjugates

ObjectiveDiscusses application of Sulfo-SMCC to prepare CPPs-β-glucosidase-cetuximab conjugates feasibilty and identify its CPPs activity, P-glucosidase activity and cetuximab activity, for the further study of its anti-tumor efficacy laid the foundation.Methods1. Cell-penetrating peptides (CPPs)and the amino water-soluble quantum dots were crosslinked by Sulfo-SMCC. Incubation CPPs-quantum dots with human bladder cancer EJ cells together, identifying its CPPs activity.2. Application of Sulfo-SMCC coupled Cetuximab with β-glucosidase. Cetuximab-β-glucosidase conjugates and its P-glucosidase and Cetuximab activity were detected by SDS-PAGE,colorimetry assay, labeling NHS-Alexa Fluor 488.3.CPPs and β-glucosidase and cetuximab were crosslinked by Sulfo-SMCC. Using non-reduced SDS-PAGE, colorimetry assay identify its connections and components, enzymatic activity. CPPs-p-glucosidase-cetuximab conjugates were labeled by NHS-Alexa Fluor 488 and incubated it with EJ cells together, identifying its CPPs activity and antibody activity.Results1. The results of penetrating activity:Incubation CPPs-quantum dots with EJ cells for one hour and two hours, with the extension of time under the Olympus BX51 upright fluorescence microscope can be observed, the red fluorescence of human bladder cancer EJ cells increasingly more obvious, while the control group did not see the fluorescence, indicating that the CCPs remains good penetrating activity.2. The results of Cetuximab-p-glucosidase Conjugates:SDS-PAGE results are shown in the 3 lane shows a clear polymer side bands, and in the P-glucosidase、cetuximab corresponding location no bands. Colorimetry assay showed that at the same of concentration conjugates its enzymatic activity was lower than β-glucosidase (U/L:669.56 ± 39.23 vs 870.60 ± 61.34, t-7.863, P <0.05), conjugates still maintain good enzymatic activity. Labeled with the green fluorescent cetuximab-p-glucosidase conjugates incubated with EJ cells for one hour, under the Olympus BX51 upright fluorescence microscope can be seen the green fluorescent around cells significantly, indicating that the conjugate still remains good antibody activity.3. The results of CPPs-β-glucosidase-Cetuximab triple Conjugates:we can see the clear polymer side bands in the 4 lane and in the β-glucosidase, CPPs-β-glucosidase, cetuximab corresponding location no bands through the electropherogram. Colorimetry assay showed that at the same of concentration triple conjugates its enzymatic activity was lower than β-glucosidase (U/L: 612.75±10.58 vs 917.03±22.72, t=21.02, P<0.05), but still maintain good enzymatic activity. Labeled with the green fluorescent dye CPPs-β-glucosidase-Cetuximab Conjugates incubated with EJ cells for one hour, under the Olympus BX51 upright fluorescence microscope we can see within cells and surrounding cells apparent green fluorescence,indicating that the conjugates still remain good penetrating activity and antibody activity.ConclusionCell Penetrating Peptides(CPPs), β-glucosidase and Cetuximab were crosslinked successfully by Sulfo-SMCC, and the conjugates still maintain good CPPs activity, enzymatic activity, antibody activity. The establishment of the system which laid a foundation for further study on its anti-tumor effect and providing a new way of thinking to the antibody directed enzyme prodrug therapy(ADEPT), and has great significance for the treatment of invasive tumors.