Quantum Dot Conjugates for Imaging Applications · Slide16: oleic acid ligand exchange:...

Quantum Dot Conjugatesfor Imaging Applications

Sungjee KimDept. of Chemistry

POSTECH

SlideQD 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Quantum dot emission spectra: unpublished data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

Norm

alize

d PL

inte

nsity

Wavelength (nm)

SlideAdvantage 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 coefficientsof biomolecules>

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

SlideImaging Setup for NIR Fluorescence Multiplexed Imaging 3

InGaAs CCD

ZoomLens

ColorCCD

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)

Dichroicmirror

Motorized filter wheel

1000 LPF

1050 BPF1250 LPF

blue arrow: visible light red arrow: infrared light

rotation

Adv. Healthcare Mater. 2018, 7, 1800695.

SlidePbS/CdS QDs for Multiplexed Imaging 4

1000 1200 14000.0

0.2

0.4

0.6

0.8

1.0

Norm

alzie

d FL

inte

nsity

(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


SlidePolymer-encapsulated QDs (PQDs) 5

0 2 4 6 8 100

20

40

60

80

100

120

PQD in DMEM w/ 10% FBS at 25 °C PQD in DMEM w/ 10% FBS at 37 °CRe

lative

FL

inten

sity (

%)

Time (day)

10 20 30 400

10

20

30

popu

latio

n (%

)

hydrodynamic size (nm)

1080-PQD 1280-PQD

Relative FL intensity and HD size change over 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 sizeand the same Zeta potential.


SlideNIR Fluorescence Multiplex Imaging 6

1080-PQD

1080

-PQ

D +

1280

-PQ

D blankagar gel

1280

-PQ

D

S-channel L-channel merged image

O-channelS-channel L-channel1080-PQD 1280-PQD

PQD aqueous solutions

S-channel; 1050 nm band pass filterL-channel; 1250 nm long pass filterO-channel; 1000 nm long pass filter

Nude mouse that was subcutaneously injected agar gel-PQD mixtures


SlideBioconjugation of PQDs 7

100 μm

100 μm 100 μm

100 μm 100 μm 100 μm

100 μm100 μm

FA-P

QD

PQD

Human dermal fibroblast cell(folate receptor-negative)

HeLa (human cervical cancer) cell(folate receptor-positive)

PQD : polymer-encapsulated QDFA-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.


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.

Whole body in vivo NIR-II image


SlideIn vivo Multiplexed NIR-II Imaging 9

tumornormal

L-channelS-channel220

30

125

1080-PQD FA-1280-PQD2.0

2.5

3.0

Tum

or /

Norm

al

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 filterL-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.


Unconjugated1080-PQD

FA-Conjugated1280-PQD

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.

SlideActivatable fluorescent probes 11

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)

EmitterQuencher

Energy/charge transfer

Simple scheme of activatable fluorescent probeLinker

• 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

off-state on-state

SlideDesign of Matrix Metalloproteinase(MMP)-activatable

probe for cancer-microenvironment detection 12

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.

SlideQuenching via photoinduced electron transfer by methylene blue 13

400 600 800 1000 1200 1400 1600

fluorescence intensity (a.u.)

abso

rban

cewavelength (nm)

MB PbS/CdS/ZnS QD

no spectral overlap between QD and MBà no change of FRET based quench

Absorption spectrum of MB andfluorescence 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 bandVB = valence band

SlideSynthesis of NIR-II emitting PbS/CdS/ZnS QD 14

thermolysis

zinc oleate, sulfurPbS

CdS

ZnS

PbS/CdS/ZnS

core/shell/shell QD

cation exchangePbS

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

SlideHAADF EM Image 15

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

20 nm

a

PbS core

CdS shell

5 nm

b

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


oleic acid

ligand exchange

: dihydrolipoic acid,

Ligand exchange from hydrophobic to hydrophilic QD

0 5 10 15 20 25 30 35 400

10

20

30

popu

latio

n (%

)

hydrodynamic size (nm)

average size = 9.7 nm

Hydrodynamic size of PbS/CdS/ZnS QD Color (left) and FL (right) image ofwater-soluble PbS/CdS/ZnS QD

Water-soluble PbS/CdS/ZnS QDs

QD QD


SlideSurface 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


SlideQuenching and activation of QD-MMPCP-MB complex 18

0 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

rela

tive

FL in

tens

ity

time (min)

Time-dependent FL recovery ofQD-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 NaClMMP-I : global MMP inhibitor


SlideHow to design the quencher peptide sequence

1. spacer sequence 19

QD-(-)MMPCP-MBforbidden proteolysis by MMP-2

QD-PEG-(-)MMPCP-MBallowed proteolysis by MMP-2

PEG = polyethylene glycol

0 10 20 30 40 50 601.0

1.5

2.0

2.5

rela

tive

FL in

eten

sity

time (min)

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

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

PbS

CdSZnS

CleavageSite D4-

MB+

PbS

CdSZnS

CleavageSite

PEG8

D4-

MB+


SlideHow to design the quencher peptide sequence

2. charged state of quencher sequence 20

repulsive force

no electrostatic force

0 10 20 30 40 50 60

1.0

1.1

1.2

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tive

fluoe

rsce

nce

inte

nsity

time (min)

(+) (+/-) (-)

0 5 10 15 200.0

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1.0 (+) (+/-) (-)

rela

tive

PLQ

Y

[MMPCP-MB]/[QD]FL intensity of QD-MMPCP-MB

FL recovery of QD-MMPCP-MB with enzyme

PbS

CdSZnS

CleavageSite

PEG8

Dn-

MB+

After enzymatic cleavageD4-

MB+

D2-

MB+no D

MB+

attractive force

(+) charge

(-) charge

(+/-) charge(net zero charge)


Slideex vivo fluorescence cancer imaging

using NIR-II activatable probe 21

AOM : azoxymethaneDSS : 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

SlideColon 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


SlideNormal colon imaging with activatable probes 23


No noticeable 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


0 5 10 15 20 25 30 350.5

1.0

1.5

2.0

2.5

3.0

3.5

rela

tive

FL in

tens

ity

time after probe spray (min)

A1 A3 A6 A2 A5


SlideColon cancer imaging with non-activatable probes 24


non-activatable probe = QD without MMPCP-MB


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


0 5 10 15 20 25 30 350.5

1.0

1.5

2.0

2.5

3.0

3.5

rela

tive

FL in

tens

ity

time after probe spray (min)

N T3 T1 T4 T2 T5


SlideAcknowledgement 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

Slide

Thank you for listening.

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