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. 2012 Feb 13;51(7):1563-7.
doi: 10.1002/anie.201105432. Epub 2011 Dec 1.

Switching the targeting pathways of a therapeutic antibody by nanodesign

Sanjib Bhattacharyya  1 , Raman Deep Singh, Richard Pagano, J David Robertson, Resham Bhattacharya, Priyabrata Mukherjee
Affiliations

Switching the targeting pathways of a therapeutic antibody by nanodesign

Sanjib Bhattacharyya et al. Angew Chem Int Ed Engl. .
No abstract available

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Figures

Figure 1
Figure 1. Effect on dynamin-2 requirement upon nano surface coverage and mechanism 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 μm). (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 βMCD (methyl-β-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 μm). (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 μm). (f) Quantification of antibody uptake as affected by the pharmacological inhibitors treatments.
Figure 2
Figure 2. Role of Cdc42 GTPase on the internalization of C225, Au-C225-P and Au-C225-C and structural Elucidation of CI Pathways
(a) PANC-1 cell transfected with GFP-Cdc42F17 dominant negative mutants were treated with Cy3 labeled C225, Au-C225-P and Au-C225-C for 1 h at 37 °C. Fluorescence images shows the inhibition of uptake of AuNPs partially covered with C225 (Au-C225-P) while the uptake of C225, Au-C225-C remains unaffected. Lower panel represent the images for Dex-Red particles (positive control) treated transfected cell (Scale bar 10 μm). (b) Quantification of nanoconjugates internalization in the presence of dominant negative mutant, GFP-Cdc42F17 transfected PANC-1 cell. (c) TEM images represent the arrest of the caveolar invagination, triggered by the treatment with C225 and Au-C225-C to PANC-1 cell at 4 °C, incubated for 2 h (Scale bar 100 nm).
Figure 3
Figure 3. Lipid micro domain involved during cetuximab-nanoconjugates uptake
(a) PANC-1 cell was preincubated Fumonisin Synthase B1 (FB1) for 48 h followed by treatment with Cy3 labeled C225, Au-C225-P and Au-C225-C for 1h at 37 °C. Fluorescence images show the inhibition of internalization (> 90 %, upper panel) of C225and its gold conjugate (Au-C225-P and Au-C225-C). Uptake of the C225 and Au-C225-C was restored upon addition of exogenous monosialoganglioside (GM3) (200 μg/ml) for 1 h at 37 °C. Lower panel images show the restoration of uptake of C225 and Au-C225-C when incubated with GM3, whereas inhibition of Au-C225-P cannot be restored by GM3 (Scale bar 20 μm). (b) Quantification of uptake of C225 and its nanoconjugates rescued by the addition of GM3 to FB-1 treated PANC-1 cell. (c) Proposed mechanism of pathway switch as triggered by the nanoconjugated antibody (Au-C225-P and Au-C225-C).
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