These results suggest that, possibly RhoA/Cdc42 dependent pinocytosis and actin polymerization are required for Au-C225-P uptake. To further confirm the involvement of RhoA/Cdc42 during Au-C225-P internalization, we overexpressed dominant negative RhoA (RhoAT19N) and Cdc42 (Cdc42T17N) mutants in PANC-1 cells and studied internalization of C225, Au-C225-C and Au-C225-P. GSL (Glycosphingolipid) domains at the plasma membrane followed by dynamin-2 (dyn-2) dependent caveolar endocytosis. However, partially covered nanoconjugate (Au-C225-P; 1 C225/particle) is internalized via dyn-2 independent Cdc42 dependent pinocytosis/phagocytosis that requires actin polymerization. Regulating the endocytosis of cetuximab between caveolar and pinocytic internalization by hCIT529I10 appropriate nanodesign may be useful to target specific intracellular pathways for better therapeutic intervention with reduced side effects. To determine the mechanism and molecular machinery involved in the uptake of C225 and its nanoconjugates we designed two nanoconjugates as we have described recently; a) AuNP surface partially covered by C225, termed as Au-C225-P and b) AuNP surface fully covered by cetuximab, termed as Au-C225-C (Figure 1a). The C225 to AuNP Syringic acid ratio in Au-C225-P and Au-C225-C is 1 and 3, respectively[5]. To study the interaction between C225 and its nanoconjugates with EGFR by confocal microscopy, we transfected PANC-1 cells with EGFR-GFP (see methods) and treated with C225, Au-C225-P or Au-C225-C for 1h where C225 was Cy3 labeled. Both C225 and Au-C225-C demonstrated similar co-localization with EGFR at the cell membrane indicating that the receptor-binding motif of C225 remained unaffected upon assembly on AuNPs. However, relatively lower extent of co-localization of Au-C225-P with EGFR at the plasma membrane suggests other modes of interaction with the cell membrane (Supplementary Figure 2). == Figure 1. Effect on dynamin-2 requirement upon nano surface coverage and mechanism Syringic acid of C225-nanoconjugate uptake; role of CME vs. CI. == (a)Cartoon presentation of the reaction scheme and structure of the antibody-nanoconjugates (Au-C225-P and Au-C225-C).(b)To study the dependency on dynamin-2, PANC-1 cells infected with dyn-2 WT and dominant negative mutant (K44A) adenovirus, show switching of endocytosis pathway upon increasing AuNP surface coverage (Au-C225-C) with C225 compared to Au-C225-P (showing dyn-2 independent internalization, Scale bar 20 m).(c)Quantification of antibody uptake by Au-C225-C treatment with Dyn-2 WT and DynK44A infected cell.(d)PANC-1 cells were preincubated with 10 mM MCD (methyl–cyclodextrin) for 30 min in a chamber slides and processed for confocal microscopy. Fluorescence images show the inhibition of internalization of Cy3-labeled C225 and its different nanoconjugates (Au-C225-P and Au-C225-C) for 1 h at 37 C (Scale bar 20 m).(e)To study the clathrin independent (CI) endocytosis, PANC-1 cell transfected with GFP-EGFR constructs were preincubated with clostridium difficile toxin B (660 ng/mL) for 1 h and then treated with Cy3 labeled C225, Au-C225-P and Au-C225-C for 1h at 37 C. Fluorescence images show that uptake was only inhibited for Au-C225-P by toxin B treatment (Scale bar 20 m).(f)Quantification of antibody uptake as affected by the pharmacological inhibitors treatments. We earlier demonstrated that C225 internalization in PANC-1 cell is dyn-2 dependent[6]. Here, we wanted to investigate whether the dyn-2 dependent internalization of C225 could possibly be modified by nanodesign. We contaminated the PANC-1 cells with crazy type dyn-2 (Dyn-2-WT) and mutant Syringic acid dyn-2 (Dyn-2-K44A) adenovirus and supervised the Syringic acid internalization of Cy3 tagged C225 and its own different nanoconjugates (Au-C225-P and Au-C225-C) by confocal microscopy. Considerable inhibition of Au-C225-C internalization (Shape 1b, c) was seen in PANC-1 cells expressing mutant dyn-2-K44A when compared with dyn2-WT expressing cells, whereas the same cells didn’t inhibit the uptake of Au-C225-P (Shape 1b). Therefore, dyn-2 reliant endocytosis of C225 could be modified by suitable nanodesign. Because dyn-2 can be involved with both CME (Clathrin Mediated Endocytosis) and CI (Clathrin 3rd party) pathways, we wished to delineate additional particular endocytic pathways included during internalization of C225-nanoconjugates[7]. Chlorpromazine, a CME inhibitor, does not have any apparent influence on the internalization of either C225 or its different nanoconjugates (Au-C225-P and Au-C225-C uptake) (Supplementary Shape 3)[8]. However, like a positive control, it inhibited the internalization of transferrin (Supplementary Shape 4). These data obviously claim that C225 and its own different nanoconjugates usually do not need the CME pathway as their major path of internalization. Since C225 and its own nanoconjugates usually do not internalize by CME, we investigated CI pathways then. To look for the participation of lipid raft we utilized -methyl cyclodextrin (BmCD), a reagent that components cholesterol.