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Lecture 19 BIOE 498/598 DP 04/14/2014. Nanoparticle-based Optical Contrast Agents. Carbon-based Material. QD Localization of a Tumor.

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Lecture 19 bioe 498 598 dp 04 14 2014

Lecture 19BIOE 498/598 DP04/14/2014

Qd localization of a tumor
QD Localization of a Tumor

It is possible to overlap X-ray images with infrared images to localize a tumor. The X-ray images give the images an anatomical context, while the infrared images detect the QD’s emission, which correlates to the tumor location.

560-QD-Streptadivin targets and images In-vitro breast cancer cells having the IgG factor characteristic of chemotherapy responsive cells

Nature Biotechnology 2003. Vol. 9, pp. 41-46

Annu. Rev. Biomed. Eng. 2007. Vol. 9, pp. 257–288


It must be multiplexed, i.e. multiple biomarkers must be detected simultaneously

Different phenotypes show different aggressiveness on their metastatic behavior

A specific phenotype of cancer cells has a particular combination of biomarkers on its membrane

Blood vessels


Cancer cells


Source: www.cancernews.com

Multiplex diagnosis
Multiplex Diagnosis

Four quantum dots of different diameter (i.e. different color) are respectively functionalized with four different antigens. Allowing for the distinction of two distinct phenotypes

The peak intensity correlates to the concentration of a specific QD

As a result cancer cells of different phenotype are colored differently

Aggressive cancer cells

Each peak correspond to the emission of a specific QD/antigen

Mild cancer cells

Nature Protocols 2007. Vol. 2, pp. 1-15

Diffuse Reflectance Imaging

Utilizes reflectance geometry but with focused excitation and detection of light.

Utilizes the diffuse nature of light propagation in tissues as a means to extend the depth sensitivity.

DRI gave better contrast than planar reflectance systems for imaging at depths greater than 6 mm.

The depth sensitivity of DRI is related to the separation of the excitation source from the detector. This geometry is used in diffuse optical spectroscopy imaging in which a single source-detector pair or multiple source-detector pairs sample the absorption and/or fluorescence over a given region

Combined Xray digital breast tomosynthesis (A) and optical mammography (B) for identification of breast tumor. Incorporation of tumor-selective optical contrast agents could improve the sensitivity and specificity of optical mammography.

de la Zerda, Bodapati et al. 2010

Raman scattering
Raman Scattering

Raman Scattering occurs when incoming light hits a sample. Most of the light scatters elastically (same wavelength as the incoming light), but a small fraction scatters inelastically (changes wavelength/color)

A weak effect

Incoming light


Outcoming light



Source: Earth System Research Laboratory



Vibrational energy

Raman enhancement
Raman Enhancement

When a molecule is coupled with a metallic surface its Raman signal is enhanced n orders of magnitude

MicrofluidNanofluid 2009;Vol.6, pp. 285–297

The localization of the different peaks constitute the fingerprint of a molecule. For instance, malachite green isothiocyanite, a ‘raman reporter’.


  • Metal nanomaterials are not fluorescent, but interact through inelastic Raman scattering.

  • Metal nanomaterials must be coated with pacifying materials to improve bio-distribution characteristics and in vivo stability.

  • Plasmon resonance occurs in nanoparticles made from gold, silver and other metals due to the large radius of electron orbitals confined by the small size of nanoparticles.

  • This phenomenon is not fluorescence, but scattering of light with size-dependent wavelength.

  • Non-spherical particles have different plasmon resonance that is determined by shape.

  • Nanorodshave scattering spectra related to the length and diameter of the particles.

  • More complex shapes can have unique plasmon resonance signatures.

“Labors of the Months” (Norwich, England, ca. 1480). The ruby color is probably due to embedded gold nanoparticles.

Illuminated from outside

The Lycurgus Cup (glass; British Museum; 4th century A. D.)

The red color is due to tiny gold particles embedded in the glass, which have an absorption peak at around 520 nm

Red color is due to very small amounts of gold powder (about 40 parts per million)

Illuminated from inside

What is the origin of the color answer surface plasmons
What is the origin of the color? Answer: “surface plasmons’’

  • An SP is a natural oscillation of the electron gas inside a gold nano sphere.

  • SP frequency depends on the dielectric function of the gold, and the shape of the nanoparticle.

(not to scale)

Ionic background

Electron sphere

Electron cloud oscillates with frequency of SP; ions provide restoring force.

Nanoparticle Surface Plasmon

d <<



Light resonance with the surface plasmon oscillation causes the free electrons in the metal to oscillate.

Eustis and El-Sayed, Chem. Soc. Rev., 2006, 35, 209

Extinction coefficient dilute suspension of au particles in aqueous solution
Extinction coefficient, dilute suspension of Au particles in aqueous solution

Crosses: experiment [Elghanian et al, Science 277, 1078 (1997); Storhoff et al, JACS 120, 1959 (1998). Dashed and full curves: calculated with and without quantum size corrections [Park and Stroud, PRB 68, 224201 (2003)].

Observed absorbance comparison of unlinked and aggregated au nanoparticles

Observed absorbance: comparison of unlinked and aggregated Au nanoparticles

Absorptance of unlinked and aggregated Au nanoparticles, as measured by Storhoff et al [J. Am. Chem. Soc. 120, 1959 (1998)]

Tuning of Surface Plasmon Resonance Au nanoparticles

Theranostics2013; 3(3):167-180.

A multifunctional gold nanoparticle-based platform incorporating multiple receptor targeting, multimodality imaging, and multiple therapeutic entities. Not all functional

moieties will be necessary and only suitably selected components are needed for each individual application.

Gold nanoparticles have been investigated for cell incorporating multiple receptor targeting, multimodality imaging, and multiple therapeutic entities. Not all functionalphantom and in vivo imaging using various techniques.

Gold incorporating multiple receptor targeting, multimodality imaging, and multiple therapeutic entities. Not all functionalnanoshells can destroy cancer cells both in vitro and in vivo. a. Cells incubated with gold nanoshells can be killed by NIR light (dark area). b. Temporal plots of maximum temperature change of NIR-irradiated tumors with and without nanoshells at depths of 2.5 mm and 7.3 mm beneath the tissue surface. c. Gross pathology after in vivo treatment with nanoshells and NIR laser revealed hemorrhaging and loss of tissue birefringence beneath the apical tissue surface. Hematoxylin/eosin (H&E) staining within the same plane confirms tissue damage within the area that contains nanoshells.

Multiplexed in vivo Raman imaging using SERS nanoparticles. incorporating multiple receptor targeting, multimodality imaging, and multiple therapeutic entities. Not all functional

Optical characterization
Optical Characterization incorporating multiple receptor targeting, multimodality imaging, and multiple therapeutic entities. Not all functional

A. Gold nanoparticles and QD’s both emit light after excitation with near infrared light, however, the gold nanoparticle SERS signal is much sharper than the QD fluorescence signal

B. The contrast of SERS gold nanoparticles is much better than that of QD’s



Nature Biotechnology 2008;Vol.26 pp. 83–90

In vitro test
In Vitro Test incorporating multiple receptor targeting, multimodality imaging, and multiple therapeutic entities. Not all functional

A. Targeting mechanism: The ScFv EFGR antibody of the nanoparticle bind to the EFG antigen of the cancer cell


No response

No response

No response

C. Only when the cancer cell had the antigen corresponding to the nanoparticle antibody there was response, which can be compared to the signal of the pure reporter

Nature Biotechnology 2008;Vol.26 pp. 83–90

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.

Technique penetration in vivo
Technique Penetration trafficking in live In vivo

The nanoparticle solution is injected to a mouse and after 4h…

  • The skin spectrum has to be magnified 210-fold to be distinguishable

  • After subcutaneous injection, the Raman signal fo the reporter can be collected and is ~50-fold stronger than that of the skin

  • After deep injection the Raman signal is only ~10-fold stronger than that of the skin

It is concluded that the technique penetration is about 2cm…

Nature Biotechnology 2008;Vol.26 pp. 83–90

In vivo tumor detection
In Vivo Tumor Detection trafficking in live

  • A sick mouse was injected with the targeted nanoparticle solution

  • The illumination of the liver produced a weak Raman signal

  • The illumination of the tumor immediately produces a strong Raman signal, with the signature characteristic of the reporter…the tumor has been detected!!!

Nature Biotechnology 2008;Vol.26 pp. 83–90

Gold nanoparticles vs alzheimer
Gold Nanoparticles vs. Alzheimer trafficking in live

Source: Berkeley Lab

Alzheimer and other degenerative diseases are caused my the clustering of amyloidal beta (Aβ) protein.

Alzheimer’s brain

Healthy brain

Gold nanoparticles can be functionalized to specifically attach to aggregates of this protein (amyloidosis)

Functionalized nanoparticle

Chemical structure of Aβ-protein

Source: wwwthefutureofthings.com

Source: www.internetchemistry.com

Gold nanoparticles vs alzheimer1
Gold Nanoparticles vs. Alzheimer trafficking in live

The functionalized gold nanoparticles selectively attach to the aggregate of amyloidal protein. The microwaves of certain frequency are irradiated on the sample. Resonance with the gold nanoparticles increases the local temperature and destroy the aggregate

Before irradiation

After irradiation

Nanoletters 2006, Vol. 6, pp.110-115

Therapy trafficking in live

Nanometer-sized particles are particularly responsive to electromagnetic and acoustic excitations through a variety of phenomena (e.g. plasmon resonance) that lead to local extreme conditions (e.g. heating). The nanoparticle is able to tolerate this condition, but not so the biological material nearby

Intramuscular injections of colloidal gold, a suspension of gold nanoparticles, has been used for decades to alleviate pain linked to rheumatoid arthritis. The mechanism is still unknown

Source: John Hopkins Center

An infrared beam illuminates two mice specimens. The local temperature increases for the mouse that received and injection of gold nanorods.

Colloidal gold

Adv. Mater. 2009, 21, 3175–3180

Source: www.wikipedia.com

(1) Laser pulse (<ANSI limit: trafficking in live e.g., 20 mJ/cm2)

(2) Light absorption & heating (~ mK)

(3) Ultrasonic emission (~ mbar)

  • Photoacoustic Tomography (PAT)

(4) Ultrasonic detection(optical scatter/100)

1 mK  8 mbar = 800 Pa

Photoacoustic Effect to produce images via the generation of MHz ranged acoustic waves by EM Energy absorption.

PA imaging utilizes nanosecond pulsed light usually in the NIR range

Wang et al. Nature Biotech 21, 803 (2003)

Nanobeacons trafficking in live for Photoacoustic Imaging

GNB technology is in development phase

Technology sub licensed from WASHU

A “particle within particle” approach


Springdale, Arkansas

Inner matrix:

Veg oil / polysorbate



(1) Probe sonication;

(2) Homogenization

(20,000 psi, 4°C, 4 min)

Kd < 10 nM




avb3-Targeted gold nanobeacons

Pan et. al. 2010 FASEB J, 25, 875-882

Pan et. al. ACS Nano. 2012 Feb 28;6(2):1260-7. 

Pan et. al. AngewChemInt Ed 48,4170 (2009)

Pan et al. Nanosci and Nanotech 2010, 10(12):8118-23.

How Early We Are Able to Detect? trafficking in live

Is it possible to detect neo-angiogenic vessels ?

Early detection of immature, nascent angiogenic

vessels with Photoacoustics Imaging and GNB!



720 nm

720 nm

720 nm

The specificity of αvβ3-integrin –NP was demonstrated by inhibiting αvβ3 -transfected K293 cell binding to vitronectin. Effective affinity = 50 pM (per particle).

300 copies/Gold Nanoparticle

14 days


Integrin avb3-Targeted

Gold NanoBeacons (GNB)


Red arrows denote small neovascular sprouts arising from immature angiogenic vessels with only nascent blood flow.

Pan D, et al. Biomaterials 2010; 31 (14): 4088-4093.

Kim, Lanza, Wang and Pan, Adv. Health Mat 2012

Pan et al. 2010 FASEB J, 25, 875-882

Size Dictates trafficking in live in vivo Distribution of Nanoparticles in the Lymph Nodes!

Sentinel lymph node (SLN) imaging is highly relevant in the context of breast cancer staging and may replace fine needle biopsy (FNB)



Real Time IMAGING?


In collaboration with Lihong V Wang, PhD, WashU BME.

Pan D, et al. Biomaterials 2010; 31 (14): 4088-4093.