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Lecture 18 BIOE 498/598 DP 04/09/2014

Lecture 18 BIOE 498/598 DP 04/09/2014. Subcutaneous and Orthotopic Xenograft Models. Comparison of Different Implantation Methods in Preclinical Targeted Imaging. Real-time in vivo imaging of invasive- and biomaterial-associated bacterial infections using fluorescently labeled  vancomycin.

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Lecture 18 BIOE 498/598 DP 04/09/2014

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  1. Lecture 18BIOE 498/598 DP04/09/2014

  2. Subcutaneous and OrthotopicXenograft Models

  3. Comparison of Different Implantation Methods in Preclinical Targeted Imaging

  4. Real-time in vivo imaging of invasive- and biomaterial-associated bacterial infections using fluorescently labeled vancomycin

  5. (a) Imaging of a mouse with E. coli (Xen16)-induced myositis in the left hind limb and S. aureus (Xen36)-induced myositis in the right hind limb was performed with the IVIS SpectrumCT Imaging System at 8 and 24 h after intravenous administration of 1.8 mg kg−1 vanco-800CW. Left side: bioluminescence imaging (open filter, field of view (FOV) 12.8 cm, F-stop 1, 30 s acquisition time); right side: fluorescence imaging (excitation 745 nm, emission 840 nm, field of view (FOV) 12.8 cm, F-stop 2, 0.5 s acquisition time). (b) Excised muscle tissue of the E. coli-infected left leg (left) and the S. aureus-infected right leg (right) of the mouse shown in a. (c) Micro-computed tomography (CT) imaging of the mouse shown in a with bioluminescence (BLI; rainbow scale) and fluorescence (FLI; red–yellow scale) coregistration. A fluorescent signal from the bladder is detectable behind the spine. (d) Fluorescence microscopy of infected muscle tissue. A cluster of vanco-800CW-labelled Gram-positive cocci (that is, S. aureus) is indicated in the right panel (red) and a chain of Gram-negative rods (that is, E. coli) is indicated in the left panel (green). DAPI (4',6-diamidino-2-phenylindole)-stained cell nuclei are labelled green. Scale bar, 10 μm. Vanco-800CW imaging: excitation 710 nm, emission >785 nm; DAPI imaging: excitation 360 nm and emission >458 nm. Nature Communications 4, Article number: 2584

  6. uPAR-targeted Contrasts as Theranostic Agents for Tumor Margin Detection Urokinaseplasminogen activator (uPA) and its receptor (uPAR) are the ligand/cell surface target pair for the development of targeted optical imaging probes for enhancing imaging contrasts in the tumor border. Recombinant peptides of the amino terminal fragment (ATF) of the receptor binding domain of uPA were labeled with near infrared fluorescence (NIR) dye molecules either as peptide-imaging or peptide-conjugated nanoparticle imaging probes. Theranostics2014; 4(1):106-118.

  7. Simultaneous imaging of uPAR and EGFR expression in a human breast cancer xenograft using NIR-dye labeled ATF and ScFvEGFR peptide probes. 

  8. Time course optical imaging of mouse mammary tumors using NIR-830-mouse ATF imaging probes- Peptide

  9. Time course optical imaging of mouse mammary tumors using NIR-830-mouse ATF imaging probes.

  10. A variety of extra- and intracellular biomarkers serve as potential targets for contrast agents

  11. Nanoparticle-based Optical Contrast Agents Carbon-based Material

  12. A) Schematic illustration of QD structure. B) QDs display distinctive colors under UV excitation.

  13. Quantum Dots (Q-Dots) • Fluorescent Qdots are ultra-small (2-8 nm diameter) semiconductor nanocrystals, having broad absorption band with narrow and symmetric emission band • (full width at half-maximum :25-40 nm) • emit in the visible to NIR spectral range • Qdots absorption is associated with the promotion of electrons from the conduction band to the valence band when the excitation energy exceeds that band gap energy between two electron bands (semiconductor gap), resulting in the formation of an electron-hole pair, called an exciton.

  14. What Happens at The Quantum Level? (Fermi level: the top of filled level) • When the Qdot is smaller than the Bohr exciton radius (which is typically a few nanometers) the quantum confinement effect is observed in such nanocrystals. • In this situation, Qdot energy levels are quantized, with values directly related to the Qdotsize (Qdotsize-dependent band gap energy) • Qdot emission is due to a radiative recombination of an exciton, which is characterized by a long lifetime (>10 ns), leading to the emission of a photon in a narrow and symmetric energy band: no different vibrational energy levels

  15. Major optical features of Qdots • Large molar extinction coefficient • Highly sensitive fluorescent agents (or fluorescent tags) for labeling cells and tissues • Large molar extinction coefficient: 0.5-5 x 106 M-1 cm-1, absorption rate is approximately 10-50x faster than organic dyes • This higher rate of absorption is directly correlated to the Qdot brightness, 10-20x brighterthan organic dyes, allowing highly sensitive fluorescence imaging • Excellent photo-stability • Much longer lifetime • Allowing effective separation of Qdot fluorescence from background fluorescence • This will improve the image contrast by reducing signal-to-noise ratio dramatically in time-delayed data acquisition mode • Large stokes shift

  16. QdotSynthesis • Made out of hundreds to thousands of atoms that typically belong to group II and VI elements or group III and V elements in the periodic table. • CdSe, CdTe, and ZnSe are group II-VI semiconductor Qdots • InP and InAsQdots: group III-V semiconductors • Qdot emission can be continuously tuned from 400 to 2000 nm by changing both the particle size and chemical composition • two robust synthesis techniques • Hot Solution-phase Mediated Qdot Synthesis • This is the most popular technique of synthesizing high quality Qdots • Qdots are synthesized at elevated temperature in high boiling point non-polar organic solvent • Bawendi’s group have reported the synthesis of highly crystalline and andmonodisperse (size distribution 8-11%) CdSeQdots using high-temperature growth solvents/ligands • (mixture of trioctylphosphine/trioctylphophine oxide, TOP/TOPO) • A combination of TOPO and hexadecylamine can also be used

  17. In vivo luminescence imaging of Panc-1 tumor-bearing mice injected with non-bioconjugated QDs (a) and ~4 mg of FA conjugated QDs (b), respectively. All images were acquired under the same experimental conditions. The autofluorescence from mice is coded green and the unmixed QD signal is coded red.

  18. Immunofluorescent labeling of cancer marker Her2 and other cellular targets with Qdots Her2/neu: human epidermal growth factor receptor 2- Overexpressed in breast cancer cell Detection of cancer marker Her2 with QD-IgG (A) Her2 labeled with QD 535−IgG. (C) Her2 labeled with QD 630−IgG. (B, D) Incubated with normal mouse IgG and QD-IgG. No signal. Nuclei: stained with Hoechst 33342 (blue) Nature Biotechnology21, 41 - 46 (2002)

  19. Protein: IgG (Immunoglobin G ) QD-IgG Well-dispersed and primarily single QDs were detected in the presence of BSA Polyclonal antibody recognized the Fab fragments of the Immunoglobinand led to extensive aggregation of the QDs This simple experiment shows that QD-IgG complex is capable of binding specific antibodies to form aggregates in solution

  20. Detection of Her2 with QD-streptavidin (A) QD 560−streptavidin on the surface of fixed cells. (B) QD 608−streptavidin on the surface of unfixed live cells. (C) Unfixed cells were incubated with anti-human IgG−biotin and QD 608−streptavidin. No signal. (D) Sections of mouse mammary tumor tissue with QD 630-streptavidin. (D) Combination with a rabbit antiserum recognizing the cytoplasmic domain of the antigen

  21. Upconversion Nanoparticles • Normal fluorescence converts higher-energy (shorter wavelength) light to lower-energy (longer wavelength) emitted light. • Upconversionluminescence is based on the absorption of two or more low-energy (longer wavelength, typically infrared) photons by a nanocrystal followed by the emission of a single higher-energy (shorter wavelength) photon. • This energy transfer is most often accomplished using a combination of rare-earth lanthanides as dopants on ceramic microparticles. Upconversion luminescence is a unique process and does not occur in nature.

  22. An Example of Upconversion Process Nd3+ → Yb 3+ → Er 3+ (Tm 3+ ) tri-dopants system with 800 nm excitation

  23. Upconversionbioimaging has significantly lower autofluorescenceand a higher signal to noise ratio when compared to single-photon excitation for fluorescence detection. Comparison of Sunstone UpconversionNanocrystals (left) to fluorescein (right) for imaging of prostate serum antigen (PSA) in human prostate cancer immunohistology sample. Dr. Sam Niedbala, Lehigh University

  24. The ability to adjust the size, morphology, absorption, emission, rise time, decay time, power density, and other properties of UCP enables the formation of materials with an infinite amount of distinctive signatures. UCP with sizes ranging from 5 nm to 400 microns have been prepared, and the morphology of the crystals can be spherical, hexagonal, cubic, rod-shaped, diamond-shaped, and random. UCP do not photobleach and allow permanent excitation with simultaneous signal integration. They can be stored indefinitely without a decrease in light emitting efficiency and thus allow repeated irradiation and analysis. Transmission electron microscope images of rod-shaped ~30-nm NaYF4 Sunstone UpconvertingNanocrystals.

  25. A Few Applications of UCP Nanoparticles Simultaneous dual-label imaging of CD-4 and CD-3 human lymphocytes using antibodies conjugated to different Sunstone Nanocrystals.     CD-3:Yb:Er doped, green emission CD-4, Yb:Tm doped, blue emission. Luminescent in vivo and in situ lymphatic imaging with infrared Sunstone Upconvertingnanocrystals obtained with both spectral and single shot imaging. The nanocrystals depicted the draining lymph nodes during in vivo and in situ imaging in mice. Due to the minimal background, single shot luminescence images obtained at 800 nm were comparable to the spectrally unmixed images which were post processed to remove autofluorescence4. (Dr. Hisataka Kobayashi, National Institutes of Health)

  26. Gold nanoparticles for dynamic imaging of EGF receptor trafficking in live cells. Dark-field and transmission electron microscopy images of cells labeled with 25-nm anti-EGF receptor (EGFR)-targeted gold nanoparticle conjugates at (A & D) 4°C, (B & E) 25°C and (C & F) 37°C. Labeling at these temperatures arrests the EGFR regulatory process at critical points, with receptors located on the cell membrane at 4°C, endosomal internalization at 25°C and MVB sorting at 37°C. (G) The relationship between EGFR regulation state and the optical signature of the gold nanoparticles in that arrangement. MVB: Multivesicular body.

  27. Oil Dye and H2O Reverse Micelles + Dye Silane Washing NH4OH Silane Extraction Hydrolysis Dye doped nanoparticles Dye-doped Silica Nanoparticle Reverse microemulsion method: down to a size of 15 nm

  28. IR Dye 800 and SWNT Conjugates

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