presented by y. zhang nov. 18, 2012 2010, 43(1):48-57 if: 21.6(2012)
TRANSCRIPT
Presented by Y. ZhangNov. 18, 2012
2010, 43(1):48-57
IF: 21.6(2012)
Introduction
Aptamers, "chemical antibodies", antibody-like molecules, function primarily in molecular recognition; Single-stranded oligonucleotidesGenerated from SELEX(systematic evolution of ligands by exponetial enrichment)Start with a random libray of 1013-1016 ssDNA or RNAQuick and reproducible synthesisEasy and controllable modification to fulfill different diagnostic and therapeutic purposes Long-term stability as dry powder or in solutionAbility to sustain reversible denaturationNontoxicity and lack of immunogenicityFast tissue penetration
Cell-Based Selection of Aptamers Specific to Cancer Cells
Cancer-related proteins, such as PDGF, VEGF, HER3, NFkB, tenascin-C, or PMSA
Cell-SELEX: proteins may keep their native conformations on cell surface
Unnecessary knowing the number or types of proteins on the cell membrane
A panel of aptamer probes can be selected to profile the molecular characteristics of the target cancer type
(B)Flow cytometry assay to monitor the binding of selected pools withCCRF-CEM cells (target cells) and Ramos cells (negative cells)
(A) Schematics of the cell-based aptamer selection
CCRF-CEM: cultured precursor T cell acute lymphoblastic leukemia (ALL) cell lineRamos: B-cell line from human Burkitt’s lymphoma
Schematics of the working principles of monovalent and bivalent NA ligands. (a) 15Apt, a monovalent ligand, has constant ON and OFF and diffuses into bulk solution immediately after dissociation from thrombin, resulting in low inhibitory function. (b) In contrast, when linked to 27Apt to form a bivalent ligand, 15Apt can rapidly return to the binding site after dissociation because of molecular diffusion confined by 27Apt that is still bound to thrombin. As a result, the equilibrium of the reaction is shifted to the left side.
Kim, Y. et. al Proc. Natl. Acad. Sci. U.S.A. 2008, 105, 5664–5669.
Real-time monitoring of light scattering generated by the coagulation process in the presence of different monovalent or bivalent NA ligands (Bi-xSs). After coagulation is initiated by adding fibrinogen to each sample, the reaction kinetics varied depending on the ligands. The initial reaction rate of each sample was calculated (scattering signal increase divided by time, cps/sec) and then plotted in the Inset. This result is consistent with the clotting test. As the number of spacers increased, the reaction rate went down and then up (Inset). Results show that Bi-8S is the best design of bivalent NA inhibitor.
Secondary structures of a selected aptamer and the truncated one
Aptamer-Based Cancer Cell DetectionMolecular Profiling
sgc8, sgc3, sgd3: T ALLsgc4, sgd2: AML, T ALL, B ALL
Shangguan, D.Clin. Chem. 2007, 53, 1153–1155
Aptamer Nanoparticle Conjugation to Enhance Detection
~80 fluorophore-labeled sgc8 aptamers/nanorod26-fold higher affinity >300-fold higher fluorescence signal
Huang, Y. et al, Anal. Chem. 2008, 80, 567–572.
Absorption spectrum and TEM image of Au Ag NRs
Fluorescence spectrum of fluorescein-labeled aptamers(a) 25 nM aptamer(b) 0.25 nM aptamer(c) NP+0.25 nM aptamer
Flow cytometric assay to monitor the binding of sgc8c (2.5 nM) and NR-sgc8c (0.75 nM) with CCRF-CEM cells (target cells) and Ramos cells (control cells)
Binding assay of KK1HO8 (50 nM) and NR KK1HO8 conjugates (1.88 nM) toward K-562 cells.
Medley, C. Anal. Chem. 2008, 80, 1067–1072
(A-D) TEM images of ACGNPs assembled on different regions of the target cell surface. (E) Image of the control cell surface showing no assembly of the ACGNPs
Images of ACGNPS with increasing amounts of target (top) and control cells (bottom)
(A) Calibration curve illustrating the relationship between the amount of cells and the absorbance intensity at 650 nm for both target cells (black) and control cells (gray). The assay shows a very good dynamic range in addition to excellent sensitivity. (B) Bar graph showing the change in intensity between the target cells and control cells at 650 nm in both cell media (CM) and fetal bovine serum (FBS) for both cell types. The graph also shows the response of anontargeting aptamer sequence to each cell type (random DNA).
(A) Spectra of different sizes of the ACGNPs with target cells to evaluate the red shift based on particle size. (B) The enhancement of the ACGNPs that is a measure of the signal difference between the assay’s response to target cells versus the same amount of control cells.
colorimetric assay for sensitive cancer cell detection limit: 90 cells
Cancer Cell Enrichment and Detection
Two-nanoparticle assay:Aptamer-magnetic nanoparticles for target cell extraction and enrichment
Aptamer-fluorescent dye nanopaticles for cell detection
Detection time <1 h
Joshua Herr. et. al. Anal. Chem. 2006, 78, 2918-2924
Microfluidic poly(dimethylsiloxane) (PDMS)
>80% capture efficiency with 97% purity for the target cells
Image of device attached to syringe pump on confocal microscope (A). The bottom left inlay shows the device, and the top right inlay shows top-down and sideways views with dimensions. Representative images of original mixture of cells before cell capture assay (B) and channel surface after the cell capture assay performed at 154 nL/sec flow rate (C), with target and control cells stained red and green, respectively. Cell-surface density measured over the course of the cell capture experiment showing linear increase in target cells captured over time (D). Target cell capture efficiency decreases with increased fluid flow rate (E).
Joseph A. Phillips, Anal Chem. 2009, 81(3): 1033–1039
Aptamer-Based Target Therapy
Targeted Intracellular Delivery
Liposome vesicles or other delivery vector systems
Xiao, Z. Chemistry, A Eoropean Journal, 2008, 14, 1769–1775
Transferrin-Alexa 633 will both bind to the surface and internalize to the endosomal compartment of CCRF-CEM cells
FIGURE 6. Distribution of sgc8c-Dox conjugates inside CCRF-CEM cells after incubation with cells for (A) 30 min, (B) 1 h, and (C) 2 h,respectively. From left to right, the fluorescence confocal images were monitored for sgc8c-Dox, transferrin-alexa633, overlay of these two channels, and bright field channel, respectively.
Targeted Chemotherapy
Huang, Y. ChemBioChem. 2009, 10, 862–868
FIGURE 7. Cell toxicity assay results for Ramos cells (P < 0.05) after30 min incubation, followed by irradiation of light for 4h and subsequent growth for 36 h.
Targeted Phototherapy
Mallikaratchy, P.ChemMedChem. 2008, 3, 425–428
Phototherapy reagent: Chlorin e6(Ce 6)
Huang, et al. Langmuir, 2008, 24(20):11860-11865
Microscopic images of HeLa cells without NRs (A) and those labeled with sgc8c (50 nM) (B), NR-lib (0.25 nM) (C), and NR-sgc8c (0.25 nM) (D). Cells are irradiated with NIR light (808 nm) at 600 mW for 10 min. Dead cells are stained with PI dye and show red fluorescence. (Left) Fluorescence images of HeLa cells. (Right) Optical images of HeLa cells.
Dead cell percentages of CCRF-CEM cells (target cells) and NB-4 cells (control cells) in all experimental conditions before and after NIR irradiation
Comparison of the relative dead cell percentage between FITC-labeled anti-CD5 CEMcells and NB-4 cells as exposure time increases.
Absorption spectrum and TEM image of Au Ag NRs
Aptamer-Directed Cancer Biomarker Discovery
Biomarker discovery: MS, 2D-GEmembrane proteins (30%, <5%)
1) aptamers bound cell lysate2) membrane proteins separation3) aptamer-protein complex extraction4) SDS-PAGE separation5) MS sequencing6) target protein validation
Shangguan, D. J Proteome Res. 2008, 7(5): 2133–2139
1, markers; 2, membrane extracts;3, protein captured with the nonbinding sequence; 4, magnetic beads only; 5, protein captured with sgc3b; 6, protein captured with sgc8c.
Conclusion and Future Perspective
Cell-SELEX: aptamer probes
Interaction between aptamers and cells
New cancer biomarker
Cancer research: biochemistry and molecular basis
Cancer detection, diagnosis, treatment
Molecular profiling of blood or body fluids
Personalized medicine
Immunity in the tumor microenviroment
Regulatory events/networks in the tumor microenviroment
Inflammation in the tumor microenvironment
Functional genetics of fibroblasts in the tumor microenviro
ment
Cytokine and chemokine networks in the tumor microenvir
oment
Targeting the tumor and the tumor microenviroment VEGF, IL-6, TAK1(TGF-β-activated kinase)