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. 2012 Jan 16;17(1):796-808.
doi: 10.3390/molecules17010796.

Quantum dot-conjugated anti-GRP78 scFv inhibits cancer growth in mice

Weiming Xu  1 , Lizhi Liu, Nicola J Brown, Sven Christian, David Hornby
Affiliations

Quantum dot-conjugated anti-GRP78 scFv inhibits cancer growth in mice

Weiming Xu et al. Molecules. .

Abstract

Semiconductor quantum dots (Qdots) have recently been shown to offer significant advantages over conventional fluorescent probes to image and study biological processes. The stability and low toxicity of QDs are well suited for biological applications. Despite this, the potential of Qdots remains limited owing to the inefficiency of existing delivery methods. By conjugating Qdots with small antibody fragments _targeting membrane-bound proteins, such as GRP78, we demonstrate here that the Quantum dot- Anti-GRP78 scFv (Qdot-GRP78) retains its immunospecificity and its distribution can be monitored by visualization of multi-color fluorescence imaging both in vitro and in vivo. Moreover we demonstrate here for the first time that Qdot-GRP78 scFv bioconjugates can be efficiently internalized by cancer cells, thus upregulate phophosphate-AKT-ser473 and possess biological anti-tumour activity as shown by inhibition of breast cancer growth in a xenograft model. This suggests that nanocarrier-conjugated scFvs can be used as a therapeutic antibody for cancer treatment.

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Figures

Figure 1
Figure 1
scFv-Grp78-H19 antibody was conjugated to a Qdot-625. (A) Coomassie blue-stained SDS-PAGE gel showing the Qdot labeled scFv; (B) Dot blot analysis of GRP78 protein binding by the Qdot625 labeled scFv; (C) Western blot analysis of GRP78 protein binding with Qdot625-GRP78 antibody with recombinant protein or whole cell extract (WCE) (upper panel). The same blot was hybridized with a goat-anti-GRP78 polyclonal antibody [N20, detected by horseradish peroxidase chemiluminescence (lower panel)]; (D) GRP78 protein was diluted in electrophoresis buffer and serial dilutions were then prepared and the western blot was probed with either anti-GRP78-Qdot625 scFv antibody (detected by UV illumination) or anti-GRP78-C20 polyclonal antibody (detected by horseradish peroxidase chemiluminescence).
Figure 2
Figure 2
Immunohistochemistry of Qdot-GRP78 antibody in MDA-MB-231/GFP breast cells and LNCaP prostate cancer cells. (A) and (B) Fluorescence images of cells staining with Qdot625-GRP78 antibody. (A) control cells staining with unlabeled nanobeads; (B) MDA-MB-231/GFP cells staining with Qdot-GRP78 antibody; (C) Scanned confocal microscopy imaging in LNCaP prostate cancer cell, stained with anti-GRP78 mouse monoclonal antibody with secondary Goat anti-mouse IgG labeled with Alexa488(green)and Qdot-GRP78(red dots) and overlapped with two probes (yellow); (D) and (E) Internalization of Qdot-GRP78 antibody by MDA-MB-231/GFP cells. Cells were incubated with unlabelled nanobeads. (D) or Qdot-GRP78 antibody; (E) at 37 °C for 16 h. Cells were then washed with PBS and analyzed by fluorescence microscopy; (F) Scanned confocal microscopy imaging in MDA-MB-231/GFP cell (green), treated with control unlabelled beads; (G) Qdot-GRP78(red dots) was detected inside MDA-MB-231/GFP cell; (H) 3D reconstruction of confocal Z stack with 0.8-μM, GFP cell showing in green channel, while Qdot-GRP78 showing in red channel. Scale bar represents 20 μm; (I) Western blot analysis of GRP78 protein in Qdot-GRP78 antibody treated cells. Cells either treated with control (unlabelled Qdot) or Qdot-labeled antibody. Phosphorylated Akt-ser473 protein was detected by an anti-anti-Akt-se473 antibody and Pan-Akt antibody was used as a loading control. The western blot represents three independent experiments. (J) MDA-MB-231/GFP cells were incubated with various concentrations of Qot-GRP78 antibody for 24h. Apoptotic nuclei or nuclear DNA strand breaks were visualized using Hoechst DNA dye H33342 (10). A minimum of 200 cells were counted in each sample and condensed or fragmented nuclei were expressed as a percentage of the total number of nuclei. Values are presented as mean ± S.D. of three determinations. * indicates significant difference (p < 0.05) between none treatment cells and antibody treatment cells.
Figure 3
Figure 3
Qdot-GRP78 antibody-conjugates inhibits breast cancer growth. Two-color fluorescence imaging of effect of the injected antibody(red) and GFP tumor(green). (A) before injection; (B) two days after injection; (C) five days after injection; (D) two weeks after injection; (E) five weeks after injection; (F) five weeks after unlabeled nanobeads only injection; (G) Total 2 × 106 tumor cells were injected subcutaneously into each Balb/c nu/nu mouse. Each experiment used five female mice. In the treatment group, the Qdot-GRP78 antibody was intratumorally injected into pre-established tumors (on the second week) (white square). Then injected at weekly interval for three weeks. In control groups, mice received unlabelled nanobeads alone (black circle. * p < 0.05, two-tailed Student’s t test. Scale bar represents 5 mm.
Figure 4
Figure 4
Qdot-GRP78 antibody detected in the tumor samples. The red fluorescence of Qdot-GRP78 antibody was detected in some of the cells inside tumor. Scale bar represents 20 μm.
Figure 5
Figure 5
Schematic illustration of the Qdot-GRP78-scFv and GRP78 complex. The complex enters the intracellular space through GRP78 movement from cell into cytoplasm. The Qdot-GRP78-scFv:GRP78 complex prevents GRP78 binds to AKT, freeing AKT to be phosphorylated at Ser374, thereby mediating its downstream _target processing including cancer cell invasion and growth.
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