Quantum Dot Conjugates for Imaging Applications Sungjee Kim Dept. - PowerPoint PPT Presentation

Quantum Dot Conjugates for Imaging Applications Sungjee Kim Dept. of Chemistry POSTECH

QD as Bright & Tunable IR Emitter Slide 1 Lanthanide complexes (2) Organic dye molecules (1) - Low quantum yield at NIR & IR range - Limited emission wavelength tunability because of molecular vibration modes - Small absorption cross-section 1.0 Normalized PL intensity 0.8 0.6 Quantum Dots at NIR & IR 0.4 0.2 Bright and wavelength-tunable nano-emitters 0.0 1000 1200 1400 1600 Wavelength (nm) (1) http://www.komabiotech.com. (2) Angew. Chem. Int. Ed. 2005, 44 , 2508. Quantum dot emission spectra: unpublished data

Advantage of Near-infrared Region Imaging Slide 2 <Effective attenuation coefficients <Tissue penetration depth of lights> of biomolecules> - Biomolecules have lower absorption and scattering in the NIR region. - The NIR optical window can maximize the tissue penetration depth. First optical window (FOW; 700 – 900 nm) Second optical window (SOW; 1000 – 1400 nm) Nat. Nanotechnol. 2009 , 4 , 710

Imaging Setup for NIR Fluorescence Multiplexed Imaging Slide 3 rotation 1000 LPF InGaAs CCD 1250 LPF 1050 BPF Motorized filter wheel Color Dichroic CCD mirror 1000 LPF : 1000 nm long pass filter (open channel) 1050 BPF : 1050 nm band pass filter Zoom (short wavelength channel) Lens 1250 LPF : 1250 nm long pass filter blue arrow: visible light (long wavelength channel) red arrow: infrared light Adv. Healthcare Mater. 2018 , 7, 1800695.

PbS/CdS QDs for Multiplexed Imaging Slide 4 TEM images PbS/CdS QDs for multiplexed imaging Normalized FL spectra of two PQDs 1080-QD 1280-QD 1080-PQD 1280-PQD 1.0 Normalzied FL intensity (a.u.) 0.8 20 nm 0.6 Fabrication of polymer-encapsulated QDs (PQDs) 0.4 0.2 0.0 1000 1200 1400 20 nm Wavelength (nm) PMAO-PEG : poly(maleic anhydride-alt-1-octadecene) conjugated with poly(ethylene glycol) Adv. Healthcare Mater. 2018 , 7, 1800695.

Polymer-encapsulated QDs (PQDs) Slide 5 1080-PQD 1280-PQD 30 population (%) Dynamic light scattering histogram of the 20 hydrodynamic (HD) size of polymer- encapsulated QDs (PQDs). 10 1080-PQD and 1280PQD show the same hydrodynamic size and the same Zeta potential. 0 10 20 30 40 hydrodynamic size (nm) Relative FL intensity and HD size change Relative FL intensity change over time for over time for PQDs in water PQDs in cell growth media 120 Relative FL intensity (%) 100 80 60 40 PQD in DMEM w/ 10% FBS at 25 ° C 20 PQD in DMEM w/ 10% FBS at 37 ° C 0 0 2 4 6 8 10 Adv. Healthcare Mater. 2018 , 7, 1800695. Time (day)

NIR Fluorescence Multiplex Imaging Slide 6 PQD aqueous solutions 1080-PQD 1280-PQD S-channel L-channel O-channel Nude mouse that was subcutaneously injected agar gel-PQD mixtures 1280-PQD 1080-PQD 1080-PQD + 1280-PQD blank agar gel merged image S-channel L-channel S-channel; 1050 nm band pass filter L-channel; 1250 nm long pass filter O-channel; 1000 nm long pass filter Adv. Healthcare Mater. 2018 , 7, 1800695.

Bioconjugation of PQDs Slide 7 HeLa (human cervical cancer) cell Human dermal fibroblast cell ( folate receptor-positive ) ( folate receptor-negative ) FA-PQD 100 μm 100 μm 100 μm 100 μm PQD 100 μm 100 μm 100 μm 100 μm • 300 nM FA-PQDs or unconjugated PQDs were co-incubated with cells for 8 h. • FA-PQDs can specifically target and label cancer cells that overexpress folate receptors. PQD : polymer-encapsulated QD FA-PQD : folic acid-conjugated PQD Adv. Healthcare Mater. 2018 , 7, 1800695.

Whole body in vivo NIR-II image Slide The mouse was intravenously injected with a mixture of two color NIR-II probes: 1080-PQD and folic acid-conjugated 1280- PQD (FA-1280-PQD). NIR-II FL images under L-channel for FA- 1280-PQD signals. The FL images were taken 5 min after the injection. Adv. Healthcare Mater. 2018 , 7, 1800695.

In vivo Multiplexed NIR-II Imaging Slide 9 S-channel L-channel 220 3.0 Tumor / Normal FA-Conjugated Unconjugated 125 1280-PQD 1080-PQD The FL signal ratio of tumor region to normal region for 2.5 1080-PQD and FA-1280-PQD normal tumor 2.0 1080-PQD FA-1280-PQD 30 (taken 140 min after the injection) • This NIR-II whole body imaging with the two PQDs provided precise evaluation of active ligand-assisted tumor-targeting of the folic acid conjugated PQDs that was unmixed from permeation and retention effects in tumors that are typically heavily dependent on the hydrodynamic size and surface properties. S-channel; 1050 nm band pass filter L-channel; 1250 nm long pass filter Adv. Healthcare Mater. 2018 , 7, 1800695.

Switching Quantum Dot (QD) Fluorescence Slide 10 Attaching a switch onto a QD, thus making the QD-Switch conjugate can be turned on and off responding to external stimuli: light, analyte concentrations, (pH, ions, etc), enzymatic activities, and binding events (small molecule or antigen binding). Applications for sensors, in vivo probes, imaging, memory, etc.

Activatable fluorescent probes Slide 11 Activatable fluorescent probe : fluorophore whose signal is amplified by the biological event of interests such as enzymatic activity, pH, nucleic acids Simple scheme of activatable fluorescent probe Linker Emitter Quencher event of interest on-state off-state (ex) protease activity, pH, nucleic acids) Energy/charge transfer • Sensitive detection of protein activity, nucleic acid, pH in in vitro and in vivo with low background signal • Activatable NIR-II QDs were not reported yet Bremer, C.; Tung, C. H.; Weissleder, R. Nat. Med. 2001 , 7 , 743. Lee, S.; Park, K.; Kim, K.; Choi, K.; Kwon, I. C. Chem. Commun. 2008 , 4250.

Design of Matrix Metalloproteinase(MMP)-activatable Slide 12 probe for cancer-microenvironment detection Quenched Photoluminescence Activated Photoluminescence electron transfer hv hv Peptide cleavage CB by MMP-2 e - h + VB Quencher MMP-cleavable peptide sequence S. Jeong et. al. Nano Letters, 2017, 17, 1378−1386 .

Quenching via photoinduced electron transfer by methylene blue Slide 13 Methylene blue (MB) : Energy level diagram of PbS/CdS/ZnS QD and MB Absorption spectrum of MB and fluorescence spectrum of QD fluorescence intensity (a.u.) MB PbS/CdS/ZnS QD absorbance Fluorescence quench via electron transfer was expected CB = conduction band VB = valence band 400 600 800 1000 1200 1400 1600 FRET = Foster resonance energy transfer wavelength (nm) no spectral overlap between QD and MB à no change of FRET based quench

Synthesis of NIR-II emitting PbS/CdS/ZnS QD Slide 14 Energy level diagram of QD PbS CdS ZnS conduction band Energy valence band • Enhanced quantum yield and photostability rather than PbS QDs Scheme for the fabrication of PbS/CdS/ZnS multishell QD ZnS cadmium oleate zinc oleate, sulfur lead oleate PbS PbS PbS sulfur thermolysis thermolysis cation exchange CdS CdS PbS/CdS PbS/CdS/ZnS PbS QD core/shell QD core/shell/shell QD

HAADF EM Image Slide 15 a b CdS shell PbS core 20 nm 5 nm (a) STEM-HAADF image of PbS/CdS/ZnS QDs. (b) Magnified STEM-HAADF image of single PbS/CdS/ZnS QD. STEM : Scanning transmission electron microscopy HAADF : High-angle annular dark-field imaging S. Jeong et. al. Nano Letters, 2017, 17, 1378−1386 .

Water-soluble PbS/CdS/ZnS QDs Slide 16 Ligand exchange from hydrophobic to hydrophilic QD QD QD ligand exchange : dihydrolipoic acid, : oleic acid S. Jeong et. al. Nano Letters, 2017, 17, 1378−1386 . 30 population (%) 20 10 0 0 5 10 15 20 25 30 35 40 hydrodynamic size (nm) Hydrodynamic size of PbS/CdS/ZnS QD Color (left) and FL (right) image of water-soluble PbS/CdS/ZnS QD average size = 9.7 nm

Surface modification for activatable probe Slide 17 MMP-cleavable peptide sequence (MMCP) maleimide-MB Cleavage Site D 4- NH 2 ≡ + ≡ MB + PEG 8 Step 1: Maleimide coupling of methylene blue and MMPCP Step 2: Conjugation of MMPCP-MB with QD S. Jeong et. al. Nano Letters, 2017, 17, 1378−1386 .

Quenching and activation of QD-MMPCP-MB complex Slide 18 3.0 0 m g/mL 30 m g/mL 10 m g/mL 10 m g/mL+MMP-I 20 m g/mL relative FL intensity 2.5 Time-dependent FL recovery of 2.0 QD-PEG-(-)MMPCP-MB with MMP-2 concentration 1.5 1.0 0 10 20 30 40 50 60 time (min) 100 nM QD-(-)MMPCP-MB solution ([MB]/[QD]=40) buffer condition : 20 mM Tris, 0.1 mM Ca(NO 3 ) 2 , 20 μM Zn(NO 3 ) 2 , 100 mM NaCl MMP-I : global MMP inhibitor • Fluorescence activation was proportional to the concentration of MMP-2 • Suppressed activation with MMP inhibitor showed the origin of FL activation comes from the cleavage activity of MMP-2 S. Jeong et. al. Nano Letters, 2017, 17, 1378−1386 .

How to design the quencher peptide sequence Slide 19 1. spacer sequence ZnS ZnS Cleavage Cleavage CdS CdS Site Site D 4- D 4- PbS PbS MB + MB + PEG 8 QD-(-)MMPCP-MB QD-PEG-(-)MMPCP-MB forbidden proteolysis by MMP-2 allowed proteolysis by MMP-2 2.5 PEG = polyethylene glycol QD-(-)MMPCP-MB QD-PEG-(-)MMPCP-MB relative FL inetensity 2.0 1.5 1.0 0 10 20 30 40 50 60 time (min) S. Jeong et. al. Nano Letters, 2017, 17, 1378−1386 . 100 nM QD-MMPCP-MB buffered solution MMP-2 enzyme 20 μg/mL