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

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

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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

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SLIDE 1

Quantum Dot Conjugates for Imaging Applications

Sungjee Kim

  • Dept. of Chemistry

POSTECH

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SLIDE 2

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QD as Bright & Tunable IR Emitter

1

(1) http://www.komabiotech.com. (2) Angew. Chem. Int. Ed. 2005, 44, 2508.

Quantum dot emission spectra: unpublished data

Lanthanide complexes(2)

  • Limited emission wavelength tunability
  • Small absorption cross-section

Organic dye molecules(1)

  • Low quantum yield at NIR & IR range

because of molecular vibration modes

Quantum Dots at NIR & IR

Bright and wavelength-tunable nano-emitters

1000 1200 1400 1600 0.0 0.2 0.4 0.6 0.8 1.0

Normalized PL intensity Wavelength (nm)

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SLIDE 3

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Advantage of Near-infrared Region Imaging

2

  • Nat. Nanotechnol. 2009, 4, 710
  • Biomolecules have lower absorption and scattering in the NIR region.
  • The NIR optical window can maximize the tissue penetration depth.

<Tissue penetration depth of lights> <Effective attenuation coefficients

  • f biomolecules>

First optical window (FOW; 700 – 900 nm) Second optical window (SOW; 1000 – 1400 nm)

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SLIDE 4

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Imaging Setup for NIR Fluorescence Multiplexed Imaging

3

InGaAs CCD

Zoom Lens

Color CCD 1000 LPF : 1000 nm long pass filter (open channel) 1050 BPF : 1050 nm band pass filter (short wavelength channel) 1250 LPF : 1250 nm long pass filter (long wavelength channel) Dichroic mirror Motorized filter wheel 1000 LPF 1050 BPF 1250 LPF blue arrow: visible light red arrow: infrared light rotation

  • Adv. Healthcare Mater. 2018, 7, 1800695.
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SLIDE 5

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PbS/CdS QDs for Multiplexed Imaging

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1000 1200 1400 0.0 0.2 0.4 0.6 0.8 1.0 Normalzied FL intensity (a.u.) Wavelength (nm) 1080-PQD 1280-PQD

20 nm 20 nm

Fabrication of polymer-encapsulated QDs (PQDs)

PMAO-PEG : poly(maleic anhydride-alt-1-octadecene) conjugated with poly(ethylene glycol)

1080-QD 1280-QD TEM images PbS/CdS QDs for multiplexed imaging Normalized FL spectra of two PQDs

  • Adv. Healthcare Mater. 2018, 7, 1800695.
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SLIDE 6

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Polymer-encapsulated QDs (PQDs)

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2 4 6 8 10 20 40 60 80 100 120 PQD in DMEM w/ 10% FBS at 25 °C PQD in DMEM w/ 10% FBS at 37 °C

Relative FL intensity (%) Time (day)

10 20 30 40 10 20 30

population (%) hydrodynamic size (nm)

1080-PQD 1280-PQD Relative FL intensity and HD size change

  • ver time for PQDs in water

Relative FL intensity change over time for PQDs in cell growth media Dynamic light scattering histogram of the hydrodynamic (HD) size of polymer- encapsulated QDs (PQDs). 1080-PQD and 1280PQD show the same hydrodynamic size and the same Zeta potential.

  • Adv. Healthcare Mater. 2018, 7, 1800695.
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SLIDE 7

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NIR Fluorescence Multiplex Imaging

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1080-PQD 1080-PQD + 1280-PQD blank agar gel 1280-PQD

S-channel L-channel merged image O-channel S-channel L-channel

1080-PQD 1280-PQD

PQD aqueous solutions

S-channel; 1050 nm band pass filter L-channel; 1250 nm long pass filter O-channel; 1000 nm long pass filter

Nude mouse that was subcutaneously injected agar gel-PQD mixtures

  • Adv. Healthcare Mater. 2018, 7, 1800695.
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SLIDE 8

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Bioconjugation of PQDs

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100 μm 100 μm 100 μm 100 μm

100 μm 100 μm 100 μm 100 μm

FA-PQD PQD Human dermal fibroblast cell (folate receptor-negative) HeLa (human cervical cancer) cell (folate receptor-positive)

PQD : polymer-encapsulated QD FA-PQD : folic acid-conjugated PQD

  • 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.
  • Adv. Healthcare Mater. 2018, 7, 1800695.
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SLIDE 9

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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.

Whole body in vivo NIR-II image

  • Adv. Healthcare Mater. 2018, 7, 1800695.
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SLIDE 10

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In vivo Multiplexed NIR-II Imaging

9 tumor normal L-channel S-channel

220 30 125

1080-PQD FA-1280-PQD 2.0 2.5 3.0 Tumor / Normal

The FL signal ratio of tumor region to normal region for 1080-PQD and FA-1280-PQD

(taken 140 min after the injection) S-channel; 1050 nm band pass filter L-channel; 1250 nm long pass filter

  • 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.

  • Adv. Healthcare Mater. 2018, 7, 1800695.

Unconjugated 1080-PQD FA-Conjugated 1280-PQD

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SLIDE 11

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Switching Quantum Dot (QD) Fluorescence

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.

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SLIDE 12

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Activatable fluorescent probes

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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.

Activatable fluorescent probe : fluorophore whose signal is amplified by the biological event of interests such as enzymatic activity, pH, nucleic acids

event of interest (ex) protease activity, pH, nucleic acids) Emitter Quencher Energy/charge transfer

Simple scheme of activatable fluorescent probe

Linker

  • 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
  • ff-state
  • n-state
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SLIDE 13

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Design of Matrix Metalloproteinase(MMP)-activatable probe for cancer-microenvironment detection

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hv e-

h+

Quenched Photoluminescence Activated Photoluminescence

CB hv VB

Peptide cleavage by MMP-2

Quencher MMP-cleavable peptide sequence

electron transfer

  • S. Jeong et. al. Nano Letters, 2017, 17, 1378−1386.
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SLIDE 14

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Quenching via photoinduced electron transfer by methylene blue

13

400 600 800 1000 1200 1400 1600

fluorescence intensity (a.u.) absorbance wavelength (nm)

MB PbS/CdS/ZnS QD no spectral overlap between QD and MB à no change of FRET based quench Absorption spectrum of MB and fluorescence spectrum of QD Methylene blue (MB) : Energy level diagram of PbS/CdS/ZnS QD and MB

FRET = Foster resonance energy transfer

Fluorescence quench via electron transfer was expected

CB = conduction band VB = valence band

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SLIDE 15

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Synthesis of NIR-II emitting PbS/CdS/ZnS QD

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thermolysis zinc oleate, sulfur

PbS CdS ZnS

PbS/CdS/ZnS core/shell/shell QD

cation exchange

PbS CdS

PbS/CdS core/shell QD

cadmium oleate

PbS

PbS QD

lead oleate sulfur

thermolysis PbS CdS ZnS conduction band valence band Energy

Energy level diagram of QD Scheme for the fabrication of PbS/CdS/ZnS multishell QD

  • Enhanced quantum yield and photostability rather than PbS QDs
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SLIDE 16

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HAADF EM Image

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(a) STEM-HAADF image of PbS/CdS/ZnS QDs. (b) Magnified STEM-HAADF image of single PbS/CdS/ZnS QD.

20 nm

a

PbS core CdS shell

5 nm

b

STEM : Scanning transmission electron microscopy HAADF : High-angle annular dark-field imaging

  • S. Jeong et. al. Nano Letters, 2017, 17, 1378−1386.
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SLIDE 17

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: oleic acid

ligand exchange

: dihydrolipoic acid, Ligand exchange from hydrophobic to hydrophilic QD

5 10 15 20 25 30 35 40 10 20 30

population (%) hydrodynamic size (nm)

average size = 9.7 nm Hydrodynamic size of PbS/CdS/ZnS QD Color (left) and FL (right) image of water-soluble PbS/CdS/ZnS QD

Water-soluble PbS/CdS/ZnS QDs

QD QD

  • S. Jeong et. al. Nano Letters, 2017, 17, 1378−1386.
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SLIDE 18

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Surface modification for activatable probe

17 Step 1: Maleimide coupling of methylene blue and MMPCP Step 2: Conjugation of MMPCP-MB with QD MMP-cleavable peptide sequence (MMCP)

+

≡

Cleavage Site PEG8 D4- MB+

NH2

≡

maleimide-MB

  • S. Jeong et. al. Nano Letters, 2017, 17, 1378−1386.
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SLIDE 19

Slide

Quenching and activation of QD-MMPCP-MB complex

18 10 20 30 40 50 60 1.0 1.5 2.0 2.5 3.0 0 mg/mL 30 mg/mL 10 mg/mL 10 mg/mL+MMP-I 20 mg/mL

relative FL intensity time (min)

Time-dependent FL recovery of QD-PEG-(-)MMPCP-MB with MMP-2 concentration

  • 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

100 nM QD-(-)MMPCP-MB solution ([MB]/[QD]=40) buffer condition : 20 mM Tris, 0.1 mM Ca(NO3)2, 20 μM Zn(NO3)2, 100 mM NaCl MMP-I : global MMP inhibitor

  • S. Jeong et. al. Nano Letters, 2017, 17, 1378−1386.
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SLIDE 20

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How to design the quencher peptide sequence

  • 1. spacer sequence

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QD-(-)MMPCP-MB forbidden proteolysis by MMP-2 QD-PEG-(-)MMPCP-MB allowed proteolysis by MMP-2

PEG = polyethylene glycol

10 20 30 40 50 60 1.0 1.5 2.0 2.5

relative FL inetensity time (min)

QD-(-)MMPCP-MB QD-PEG-(-)MMPCP-MB

100 nM QD-MMPCP-MB buffered solution MMP-2 enzyme 20 μg/mL

PbS CdS ZnS

Cleavage Site D4- MB+

PbS CdS ZnS

Cleavage Site PEG8 D4- MB+

  • S. Jeong et. al. Nano Letters, 2017, 17, 1378−1386.
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SLIDE 21

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How to design the quencher peptide sequence

  • 2. charged state of quencher sequence

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repulsive force no electrostatic force

10 20 30 40 50 60 1.0 1.1 1.2 1.3 1.4 1.5

relative fluoerscence intensity time (min)

(+) (+/-) (-) 5 10 15 20 0.0 0.2 0.4 0.6 0.8 1.0 (+) (+/-) (-)

relative PLQY [MMPCP-MB]/[QD]

FL intensity of QD-MMPCP-MB FL recovery of QD-MMPCP-MB with enzyme

PbS CdS ZnS

Cleavage Site PEG8 Dn- MB+

After enzymatic cleavage

D4- MB+ D2- MB+ no D MB+ attractive force (+) charge (-) charge (+/-) charge (net zero charge)

  • S. Jeong et. al. Nano Letters, 2017, 17, 1378−1386.
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SLIDE 22

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ex vivo fluorescence cancer imaging using NIR-II activatable probe

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AOM : azoxymethane DSS : dextran sulfate sodium salt

  • colorectal cancer model (AOM/DSS-treated mouse) is known for high

upregulation of MMPs in cancer microenvironment Scheme for ex vivo fluorescence imaging of colon cancer model

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SLIDE 23

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Colon cancer imaging with activatable probe

22 Time-dependent signal activation Cancer microenvironment-specific fluorescence activation

Probe : 1 μM QD-PEG-(-)MMPCP-MB in PBS buffer at pH 7.4 ([MB]/[QD]=40) excited by 910 nm laser with 200 mW/cm2 exposure time = 90 ms

Time-dependent fluorescence image

  • S. Jeong et. al. Nano Letters, 2017, 17, 1378−1386.
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SLIDE 24

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Normal colon imaging with activatable probes

23 Time-dependent signal activation No noticeable fluorescence activation Time-dependent fluorescence image

Probe : 1 μM QD-PEG-(-)MMPCP-MB in PBS buffer at pH 7.4 ([MB]/[QD]=40) excited by 910 nm laser with 200 mW/cm2 exposure time = 90 ms

5 10 15 20 25 30 35 0.5 1.0 1.5 2.0 2.5 3.0 3.5

relative FL intensity time after probe spray (min)

A1 A3 A6 A2 A5

  • S. Jeong et. al. Nano Letters, 2017, 17, 1378−1386.
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SLIDE 25

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Colon cancer imaging with non-activatable probes

24 Time-dependent signal activation non-activatable probe = QD without MMPCP-MB Time-dependent fluorescence image

Probe : 1 μM QD in PBS buffer at pH 7.4 excited by 910 nm laser with 200 mW/cm2 exposure time = 90 ms

5 10 15 20 25 30 35 0.5 1.0 1.5 2.0 2.5 3.0 3.5

relative FL intensity time after probe spray (min)

N T3 T1 T4 T2 T5

  • S. Jeong et. al. Nano Letters, 2017, 17, 1378−1386.
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SLIDE 26

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Acknowledgement

25 Alumni: Junhyuck, Park, Sungwook Jung, Sanghwa Jeong Group members: Yebin Jung, Youngju Kwon, Junhwa Lee, Wonsuk Lee, Yunmo Sung, Jihye Lee, Woojin Lee, Eunjae Lee, Seunghwa Hong, Sungbin Yang, Jeongmin Kim, Soomin Lee, Doowon Choi, Sujin Lee, Anastasia Agnes, Eunjeong Kim

Nanophotonics and Nanomedical Research Group

Collaborator (partial list):

  • Prof. Nam Ki Lee, SNU
  • Prof. Jong Bong Lee, POSTECH
  • Prof. Seung-Jae Myung, AMC
  • Prof. Chan Ki Pack, AMC
  • Prof. G-One Ahn, POSTECH
  • Prof. Junsang Doh, POSTECH
  • Prof. Jung-Joon Min, Chonnam Univ.
  • Prof. Chulhong Kim, POSTECH
  • Prof. Ki Hean Kim, POSTECH
  • Prof. Euiheon Chung, GIST
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SLIDE 27

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Thank you for listening.

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