Self-assembling complexes of quantum dots and scFv antibodies for cancer cell targeting and imaging

Abstract

Semiconductor quantum dots represent a novel class of fluorophores with unique physical and chemical properties which could enable a remarkable broadening of the current applications of fluorescent imaging and optical diagnostics. Complexes of quantum dots and antibodies are promising visualising agents for fluorescent detection of selective biomarkers overexpressed in tumor tissues. Here we describe the construction of self-assembling fluorescent complexes of quantum dots and anti-HER1 or anti-HER2/neu scFv antibodies and their interactions with cultured tumor cells. A binding strategy based on a very specific non-covalent interaction between two proteins, barnase and barstar, was used to connect quantum dots and the targeting antibodies. Such a strategy allows combining the targeting and visualization functions simply by varying the corresponding modules of the fluorescent complex.

Figure 1. Gene construct encoding the 425scFv-Bs recombinant protein.

The 425scFv-Bs-His₆ construct starts with an N-terminal short FLAG tag (F, dark blue) followed by 425scFv in V_H-linker-V_L orientation (V_H, cyan; L, gray; V_L, turquoise), 16-amino-acid hinge linker (green), barstar (purple). The construct terminates with a His₆-tag (dark blue) attached to the C-terminus of 425scFv-barstar fusion protein. The fusion gene is under control of the lac promoter. OmpA – the signal peptide for directed secretion of the recombinant protein to the E. coli periplasm.

Figure 2. The design of fluorescent probes on the basis of QDs and 425scFv (a, green antibody) or 4D5scFv (b, blue antibody) for specific cancer cell imaging.

Binding of QDs to scFv antibodies via barnase-barstar molecular adaptors (A) and BBS-based molecular constructor comprising of a set of variable fluorescing and targeting modules (B) are shown.

Figure 3. Purification and characterization of fusion proteins.

(A) 12% SDS-PAGE confirming the purification of (4D5scFv)₂-Bn (a, 71 kDa) and 425scFv-Bs (a', 40 kDa), Coomassie Brillian Blue R-25 stained gel; standard protein marker (M) and fusion protein lanes (P) are shown. (B) RNAse activity of (4D5scFv)₂-Bn (solid line) compared with activity of free barnase (dashed line) and evaluated with acid-insoluble RNA precipitation assay. (C) Inhibition of free barnase by 425scFv-Bs (solid line) compared with inhibition by free barstar (dashed line).

Figure 4. Characterization of QD-BBS protein conjugates.

(A) Electrophoretic mobility of QDs and QD-conjugates in 1% agarose gel, in Tris-acetate-EDTA buffer (pH 7.4). QDs run from cathode (–) to anode (+). Lanes: 1 – QD₆₀₅, 2 – mixture of QD₆₀₅ and barstar (without EDC), 3 - QD₆₀₅-Bs conjugate, 4- QD₅₆₅, 5 –mixture of QD₅₆₅ and barnase (without EDC), 6 - QD₅₆₅-Bn conjugate. (B) Normalized fluorescence spectra of QD₅₆₅ (blue line), QD₆₀₅ (violet line) and their conjugates, QD₅₆₅-Bn (green line) and QD₆₀₅-Bs (red line). (C) The ribonuclease activity of QD₆₀₅-Bn (red line and circles) and free barnase RNAse activity inhibition of QD₆₀₅-Bs (blue line and square). QD₆₀₅ (green line) do not affect ribonuclease activity of barnase.

Figure 5. Live cell imaging.

(A) Optical microscopy of HER1overexpressing A431 cells that were preincubated with QD₆₀₅ (a), QD₆₀₅-Bn (b), 425scFv-Bs and QD₆₀₅-Bn (c). HER1-negative CHO cells were used as controls for staining with 425-Bs and QD₆₀₅-Bn (d). As additional control competitive binding test of free 425scFv and anti-HER1 425scFv-Bs/QD₆₀₅-Bn complex was carried out (e). Left row, bright-field image; right row, fluorescence image with 488 nm excitation and 605 nm emission peaks. (B) Flow cytometry of SKOV-3, A431 <$>\raster(70%)="rg1"<$> CHO cells incubated with QD₆₀₅ (green line), QD₆₀₅-Bn (yellow line), QD₆₀₅-Bs (orange line), (4D5scFv)₂-Bn and QD₆₀₅-Bs (red line), 425scFv-Bsand QD₆₀₅-Bn (blue line).

Figure 6.Two. approaches for tumor cells imaging using QD-scFv antibody complexes based on barnase-barstar.

Legends as in figure 1.

Figure 7. In vitro cytotoxicity analysis of used QD₅₆₅ (A) and QD₆₀₅ (B) probes.

Relatively cell viability of SKOV-3 cells after treatment with initial QDs (red line), their conjugates with barstar (blue line) and their complex with 4D5scFv (orange line) are shown.