Immuno ICG Nanocapsules and Scanning Nonlinear Optical Endomicroscopy

for Translational Biophotonics Imaging

Xingde Li, PhD

(1994-1998 PhD at JF)

Department of Biomedical EngineeringJohns Hopkins University

BC Legacy Workshop, June 4, 2011

Thank you!

Joining the Lab: June 1994

Fluorescence Imaging in Turbid Media?

Source

rsDetector

rd

phase shift and demodulation due tofluorophore concentration and lifetime

Point Fluorophore

r

ex em

( r )

1 - i ( r )L (r, ) =

Source Intensity

Near-infrared Fluorescence Contrast Agents

In vivo Fluorescence SignalsTumor versus Control

0.5

1

1.5

2

2.5

3

3.5

0

0.5

1

1.5

2

0 5 10 15 20 25 30 35 40

Ratio of Max Fluorescenceand Tumor Size

Ra

tio

(T

um

or

/ C

on

tro

l)

Tu

mo

r Size

(cm 3)

Days After Injection of Tumor Cells

Tumor size

Theory

Ratio (T/N)

0

2

4

6

8

10

0 2 4 6 8

Typical Fluorescence Signal

Flu

ore

sc

en

ce

(A

rb.

Un

its

)

Time (min)

Tumor

Normal

Ratio of max fluorescence signal: tumor / normal ~2.5 foldRatio of initial clearance velocity: tumor / normal ~4 fold.

Theoretical contrast: Nobj/Nbg ~7-fold

S-D separation: 8 mmTumor depth: 2 mm (subcutaneous)

Summer 1994

Preliminary Clinical Study

Patient with breast tumor palpable, ~2cm deep, 0.8 cm in diameterRatio of max fluorescence signal: tumor / normal ~1.5 foldRatio of initial clearance velocity: tumor / normal ~2.0 fold

0.0

0.5

1.0

1.5

2.0

2.5

0 2 4 6 8 10 12

Patient (M84)

Flu

ore

scen

ce

(Arb

. U

nit

s)

Time (min)

Tumor

Normal

Dosage: 0.4 mg / Kg BW( limit ~2.0 mg / Kg BW)

Summer 1994

Fluorescent Polymeric Micelles

Encapsulate ICG into its hydrophobic core.Enhancing fluorescence emission and stabilityPotentially bioconjugatible, enhanced permeation and retention (EPR) FAD approval?

V. Rodriguez, S. Henry, A. Hoffman, P. Stayton, X. D. Li, and S. H. Pun, Journal of Biomedical Optics (2008)

(pluronic micelles)

ICG in Chloroform

Stirring 3h Evaporation

Polymer (Aqueous Solution)

OH OH

OHOH

ICG Encapsulation by Pluronic Micelle

(Emulsification and Evaporation)

~30 nm in diameter

ATM Image of ICG Nanocapsules

Size is small, suitable for in vivo tumor delivery.

ICG Fluorescence Stability of in Micelles

T. H. Kim et al., Pharmaceutical Research 27 (9), 1900-1913 (2010).

Excellent ICG fluorescence stability micellar nanocapsules.

Tumor

mA

b-m

icel

le/IC

G

Tumor Tumor

Strong ICG fluorescence from targeted tumor with immunomicelles.

In vivo Tumor Imaging with ICG-Immunomicelles

(3 days post injection of ICG micelles with an ICG dose of 0.5 mg/Kg )

XENOGEN IVIS System Tomography System

Tumor

Mic

elle

/ICG Tumor Tumor

nM

In-tumor ICG Concentration vs Time (Initial Pharmacokinetics Study)

3.0 3.5 4.0 4.5 5.0

4

8

12

16

20

24

Dye

Co

nce

ntr

atio

n (

nM

)

Time (Days)

Immuno-Micelle/ICG Micelle/ICG

Scanning Nonlinear Endomicroscopy:Enabling in vivo Two-photon Fluorescence (TPF)& Second Harmonic Generation (SHG) Imaging

About Graduation

Chatting with BC about what to do next: Metabolic/Functional Imaging, in vivo

Two-photon Fluorescence Imaging

Mucosal mast cells: Green: plasma membraneBlue: nucleiRed: mitochondria

www.biorad.com

Nature Biotechnology  21, 1369 - 1377 (2003)

Single-photon Flr Two-photon Flr

Distal End

Motivation: Nonlinear Optical Microscope Endomicroscope

Accessory Port(2.8 mm)

TPF Endomicroscope

(distal end)

Photo of Scanning TFP Endomicroscope

Distal End

Report in Biophotonics International, June 2006.M. T. Myaing, D. J. MacDonald, and X. D. Li, Optics Letters 31(8):1076-1078 (2006).

2.4 mm DiameterStandard Endoscope

Endomicroscope

Visualization of Histopathology in situ

Concept Barrett’s

Cancer

R. Kiesslich and M. I. Canto, GI Endoscopy Clinics N Am 19(2):261-272(2009).

(Active) Neuron / Brain Function Imaging(ultimately on freely walking animals)

20um

Vessel

Astrocyte?

Astrocyte (star-shaped glial cells): provision of nutrients, maintenance of extracellular ion balance,long-distance propagation of Ca+2 wave, etc.

Endomicroscope

Freely-walking animal

Basic Building Blocks

Core

Inner Cladding

Outer Cladding

Double-clad Fiber: Delivery and Collection

Photonic Crystal Fiber (Dispersion Management)

Miniature Scanning Head

DCF with MMFPZT(b)

5 mm

Compound lens

Resonant PZT Scanner (1mm, kHz)Miniature Compound Lens

Rapid Spiral Scanning Pattern

X. M. Liu, M. J. Cobb, M. B. Kimmey, and X. D. Li, Optics Letters (2004)Y. C. Wu, Y. Leng, J. F. Xi, and X. D. Li, Optics Express (2009).L. Huo, J. F. Xi, Y. C. Wu, and X. D. Li, Optics Express (2010).

Microendoscope

fs Fiber Laser

12”

18”

7”

Photo of TPF/SHG Endomicroscope

The whole system is very compact!

TPF Images of GFP Mouse Tissues (ex = 870nm)

Pancreas20um 20um

Liver20um

Salivary Gland

20umKidney

20umBrain

20umSpleen

Collaboration with Dr. R. Weigert at NIH

20µm

Intrinsic TPF Endomicroscopy of Fresh Tissues

Fresh Pig Esophagus

Fresh Rat Oral Cavity Fresh Rat LipFresh Mouse Dorsal Skin

Fresh Mouse Xenograft TumorFresh Mouse Colon

20µm

20µm20µm

20µm20µm60mW 60mW

60mW

60mW

50mW 50mW

Further Comparison of Endomicroscopy with Microscopy for SHG Imaging of Breast Tissues

10 μm 10 μm10 μm 10 μm

10 μm10 μm 10 μm10 μm

Endo EndoMicroscopy Microscopy

No

rmal

Tu

mo

r

Thank you!