Volume 14, Issue 2, Pages 192-201 (August 2006) Labeling and Intracellular Tracking of Functionally Active Plasmid DNA with Semiconductor Quantum Dots  Charudharshini Srinivasan, Jeunghoon Lee, Fotios Papadimitrakopoulos, Lawrence K. Silbart, Minhua Zhao, Diane J. Burgess  Molecular Therapy  Volume 14, Issue 2, Pages 192-201 (August 2006) DOI: 10.1016/j.ymthe.2006.03.010 Copyright © 2006 The American Society of Gene Therapy Terms and Conditions

FIG. 1 Schematic illustration of conjugation of QDs to plasmid DNA. (A) Attachment of PNA-decorated QDs onto plasmid DNA (gWIZ) using pGeneGrip motif [36]. (B) Water solubilization and maleimide functionalization of CdSe/ZnS core/shell QDs and (C) synthetic route for covalently attaching PNA–SPDP linkages onto the maleimide functionalities of QDs (B). Molecular Therapy 2006 14, 192-201DOI: (10.1016/j.ymthe.2006.03.010) Copyright © 2006 The American Society of Gene Therapy Terms and Conditions

FIG. 2 Characterization of QD–DNA conjugates. (A and B) Gel electrophoresis (A) before and (B) after staining with SYBR gold: (lane 1) plasmid DNA alone, (lane 2) physical mixture of QDs and plasmid DNA, (lane 3) QDs alone, (lane 4) purified QD–DNA conjugates, (lane 5) supernatant after ultracentrifugation of purified QD–DNA conjugate, (lane 6) first wash filtrate, (lane 7) second wash filtrate, and (lane 8) third wash filtrate. (C and D) Topographical atomic force micrographs: (C) DNA alone and (D) QD–DNA conjugates on a mica substrate. Arrowheads in (D) indicate the QD clusters on the ring structure of two plasmid DNAs. The arrows indicate the different segments, I, II, and III, on the DNA strands of the conjugate. Based on average height analysis, the segments indicate a relaxed plasmid strand at I, a supercoiled plasmid strand at II (twice the average height of I), and the overlap of the relaxed and supercoiled plasmid strands at III (three times the average height of I). Molecular Therapy 2006 14, 192-201DOI: (10.1016/j.ymthe.2006.03.010) Copyright © 2006 The American Society of Gene Therapy Terms and Conditions

FIG. 3 Comparison of QD and rhodamine long-term photostability. Consecutive images of (A, B, and C) QD–DNA conjugates and (D, E, and F) rhodamine complexes (Rh–PE/DNA) in the presence of Lipofectamine2000 taken at different time points after 3 h transfection in CHO-K1 cells. (G) Graph representing the change in fluorescence intensity of QDs and rhodamine complexes for a total period of 100 min, during which both the fluorophores were exposed to continuous excitation at 543 nm (Kr/Ne laser) (mean ± SD, n = 3). QD–DNA conjugates were added as 1.469 × 1013 particles per 2.5 × 106 cells per well. Bars, 25 μm. Molecular Therapy 2006 14, 192-201DOI: (10.1016/j.ymthe.2006.03.010) Copyright © 2006 The American Society of Gene Therapy Terms and Conditions

FIG. 4 Cellular uptake studies of conjugated and unconjugated QDs. Overlapped epifluorescence images of: the nuclear stain, Hoechst 33342 (DAPI excitation and Fura-2 emission filters), and red QDs (Texas red excitation filter and Quad emission filters) after 3 h incubation of materials (described below) in live CHO-K1 cells, followed by washing to remove unbound material. (A) QDs alone, (B) QDs in the presence of Lipofectamine2000 mixture, (C) physical mixture of QDs and plasmid DNA in the presence of Lipofectamine2000, and (D) QD–DNA conjugates in the presence of Lipofectamine2000. The arrowheads (A–D) indicate red dots that are in the cytoplasm or outside the cell membrane and arrows in D indicate colocalization of red dots with the blue nuclear stain (shown as pink dots). QD–DNA conjugates were added as 1.469 × 1013 particles per 2.5 × 106 cells per well. Bar, 25 μm. Molecular Therapy 2006 14, 192-201DOI: (10.1016/j.ymthe.2006.03.010) Copyright © 2006 The American Society of Gene Therapy Terms and Conditions

FIG. 5 In vitro expression and long-term imaging using QD–DNA conjugates. Time-lapse imaging experiments performed using confocal microscopy to track uptake of QD–DNA conjugates (in the presence of Lipofectamine2000) and subsequent EGFP expression in CHO-K1 cells (0–11 h). The arrows indicate an increase in the intensity of EGFP expression associated with the uptake of red QD–DNA conjugates within a selected cell. QD–DNA conjugates were added as 1.469 × 1013 particles per 2.5 × 106 cells per well. Bars, 25 μm. Molecular Therapy 2006 14, 192-201DOI: (10.1016/j.ymthe.2006.03.010) Copyright © 2006 The American Society of Gene Therapy Terms and Conditions

FIG. 6 Tracking of QD–DNA conjugates. Confocal images following incubation with QD–DNA conjugates at different time points in CHO-K1 cells in the presence of Lipofectamine2000 are shown. Cells were stained with SYTO-16 nuclear stain. Nuclear uptake of QD–DNA conjugates is demonstrated by red QD–DNA conjugates imaged using a 633 nm He–Ne laser line excitation source, which does not excite the SYTO-16 green nuclear stain (ex 488/em 518). (A, C, and E) Representative images using the red channel (633 nm) at 6, 10, and 24 h, respectively. (B, D, and F) Overlapped images of red and green channel at 6, 10, and 24 h, respectively. Arrows indicate the presence of QD–DNA conjugates (as yellow/orange dots) at the perinuclear/nuclear region at 6-, 10-, and 24-h time points. QD–DNA conjugates were added as 1.469 × 1013 particles per 2.5 × 106 cells per well. Bar, 25 μm. Molecular Therapy 2006 14, 192-201DOI: (10.1016/j.ymthe.2006.03.010) Copyright © 2006 The American Society of Gene Therapy Terms and Conditions

FIG. 7 MTT toxicity assay showing percentage cell survival following incubation with QDs and QD–DNA conjugates in CHO-K1 cells after 24 h transfection. QD–DNA conjugates were added as 2.35 × 1012 particles per 0.4 × 106 cells per well. The values represent percentage cell survival (means ± SD), n = 4. Molecular Therapy 2006 14, 192-201DOI: (10.1016/j.ymthe.2006.03.010) Copyright © 2006 The American Society of Gene Therapy Terms and Conditions