POTENTIAL APPLICATION OF NANOPARTICLES IN MEDICINE: Cancer Diagnosis and Therapy

Diego A. Gomez-GualdronMidterm Project

Nanotechnology; CHEN 689-601Texas A&M University

March 11th, 2010

OUTLINE

• SECTION INanomedicine overview• SECTION IINanotechnology potential in oncology• SECTION IIIPromising works• SECTION IVAssessment

SECTION INanomedicine Review

Nanomedicine• Premise:Nanometer-sized particles have optical, magnetic,

chemical and structural properties that set them apart from bulk solids, with potential applications in medicine.

• Potential applications

DRUG DELIVERY MEDICAL IMAGING

DIAGNOSIS & SENSING THERAPY

Interesting facts about nanomedicineA. Interest in the area has grown exponentially B. Drug delivery is the most productive area

C. Drug delivery is the most established technology in the nanomedicine market

Nature Biotechnology 2006, Vol. 4, pp.1212-1217

Drug Delivery

1

3

2

4

1) A nanoparticle carries the pharmaceutical agent inside its core, while its shell is functionalized with a ‘binding’ agent

2) Through the ‘binding’ agent, the ‘targeted’ nanoparticle recognizes the target cell. The functionalized nanoparticle shell interacts with the cell membrane

3) The nanoparticle is ingested inside the cell, and interacts with the biomolecules inside the cell

4) The nanoparticle particles breaks, and the pharmaceutical agent is released

Source: Comprehensive Cancer Center Ohio University

A. Because of their small sizes, nanoparticles are taken by cells where large particles would be excluded or cleared from the body

A Drug Delivery NanoparticleA. Nanoparticles for drug delivery can be metal-, polymer-, or lipid-based. Below (left) an example of the latter, containing SiRNA encapsulated, and functionalized with an specific antibody. SiRNA can control often lethal inflammatory body responses, as shown in the microscopic images below (right)

Science 2008, Vol. 316, pp 627-630

antibodylipid

SiRNA Healthy tissue Sick tissue treated with non-targeted nanoparticles

Sick tissue treated with targeted nanoparticles

B. C.

Medical ImagingA. Optical properties of nanoparticles depend greatly on its structure. Particularly, the color (wavelength) emitted by a quantum dot (a semiconductor nanoparticle) depends on its diameter.

C. The quantum dots (QD) can be injected to a subject, and then be detected by exciting them to emit light

Source: Department of immunology, University of Toronto

Solutions of CdSe QD’s of different diameter

CdSe nanoparticle (QD) structureSource: Laurence Livermore Laboratories

Imaging of QD’s targeted on cellular structures

Nano Letters 2008., Vol. 8, pp3887-3892

B.

A Quantum Dot Nanoparticle

1

2

3

4

A. The quantum dot itself (the semiconductor nanoparticle) is toxic. Therefore some typical modifications has to be made for it to become biocompatible.

1) The core consist of the semiconductor material that emits lights

2) The shell consist of an insulator material that protects the light emitting properties of the QD in the upcoming functionalization

3) The shell is functionalized with a biocompatible material such as PEG or a lipid layer

4) Additional functionalization can be done with several purposes (e.g. embed a drug for drug delivery, or assemble an antibody to become the QD target-specific

Source: The scientist (2005), Vol. 19, p. 35

Targeting QD’s for intracellular imaging

Ligand coated QDNC

QD release

Ingestion

Decomposition

labeling

Nano Letters 2008., Vol. 8, pp3887-3892

A. Using a drug-delivery-like mechanism, a targeted lipid-based nanoparticle (TNP) encapsulating QD’s specifically ‘attacks’ a cell having the receptors that pair with its ligand coating. Upon ingestion and destruction of the TNP, the QD’s are set free and accumulate on intracellular structures

C. QD (red)intracellular uptake is enhanced when using the QDNC instead of the free QD’s

B.

D. Imaging of nucleus (blue) and cytoplasm (other) after 30 min (left) and 3 hours after uptake

Diagnosis and SensingA. Diseases can be diagnosed through the (simultaneous) detection of a (set of) biomolecule(s) characteristic to a specific disease type and stage (biomarkers).

Huffman, Nanomedicine and Nanobiotechnology, Vol. 1, 1, 2009

D.

molecular signature of sick cell of infecting agent

(e.g. an antibody)

Cell membrane

NanoparticleCoating molecule

specifically attracted to the molecular signature

C. A nanoparticle can be functionalized in such a way that specifically targets a biomarker. Thus, the detection of the nanoparticle is linked to the detection of the biomarker, and to the diagnosis of a disease

B. Each cell type has unique molecular signatures that differentiate healthy and sick tissues. Similarly, an infection can be diagnosed by detecting the distinctive molecular signature of the infecting agent

Nanoparticles in actionA. Modifying a ferromagnetic nanoparticle with human immunoglobulin G (IgC), which specifically binds the protein A in the cellular wall of staphylococcus, the bacteria can be detected through a MRI test

Accumulation of functionalized ferromagnetic nanoparticles on staphylococcus

Negligible accumulation of nanoparticles in absence of functionalization

B.

Analytical Chemistry 2004, Vol. 76, pp.7162-7168

C.

Directed accumulation of dangerous bacteria by conjugation with functionalized magnetic nanoparticles

National Research Council, Canada

A Chemical Nose (Multiplex Detection)

A. Determining if a an apple is rotten or not, doing a thorough chemical analysis can be a very frustrating job. Due to the complex chemistry of the membrane, so can it be determining if a cell is sick or healthy.

B. As well as our noses response to the overall chemistry of the apple, we can device an experiment that responses to the overall chemistry of the cell using the elements below

C. D.

Three sets (NP1,NP2,NP3) of functionalized gold nanoparticlesA fluorescence reporter polymer

PNAS 2009, Vol. 106, pp.10912-10916

A Chemical Nose (Multiplex Detection)

D.

PNAS 2009, Vol. 106, pp.10912-10916

E. The polymer fluorescence is turned off while conjugated to the nanoparticle. Due to the interaction with the cell, the polymeric traces detach from the nanoparticle an emit a fluorescence signal

NP1

NP2

NP3

F. The responses from a NP1, NP2 and NP3 are different due to the different functional group. Thus, the combination of the three signals is characteristic of each cell

polymer

detached polymer

G.Ce

ll m

embr

ane

Fluo

resc

ence

cha

nge

Metastatic cell

Normal cell Cancerous cell

TherapyA. 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 no so the biological material nearby

B. 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

Colloidal gold

Source: www.wikipedia.com

Source: John Hopkins Center

C.

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

Adv. Mater. 2009, 21, 3175–3180

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

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

Functionalized nanoparticle

Source: www.internetchemistry.com

Chemical structure of Aβ-protein

Source: wwwthefutureofthings.com

C.

B.

Alzheimer’s brain Healthy brain

Source: Berkeley Lab

Gold Nanoparticles vs. AlzheimerA. 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

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

Before irradiation After irradiation

SECTION IINanotechnology potential in oncology

Cancer Nanotechnology A. It is an interdisciplinary area merging science, engineering and medicine with the sole purpose of provide humanity new tools to fight cancer

B. C.

Annu. Rev. Biomed. Eng. 2007. Vol. 9, pp. 257–88

Cancer nanotechnology, as a particular area of nanomedicine, is based upon the same premise that nanoparticles display unique properties potentially useful in medical (oncological) applications.

Nanoparticles in the size range of 5-100nm have enough surface area to be properly functionalized to bind specific targets, with a variety of ulterior purposes

PREMISE

Cancer Facts

B. Lung cancer is the overwhelming lead cause of cancer-related deaths.

BEWARE SMOKERS!!!!

A. The second main cause of death in the US, and certainly the diseases that lower the life quality of the patient the most

Motivation

A. The only factor that really correlates to the patient survival is early cancer detection

B. Chemotherapy and radiotherapy kill healthy and sick cells indiscriminately

C. Cancer resurgence after surgery occurs due to failure to recognize and remove all cancerous colonies

DIAGNOSIS

THERAPY

IMAGING

Cancer: Too complex to handle?

A. If you are an engineer, you can think of cancer as a living organism finally succumbing to entropy. Therefore, cancer is not one disease but million of diseases characterized by the disordered an uncontrolled growth of cellsB. entropy C. There are a myriad of

metabolic/biological events that can unleash the growth of cancer cells. We must completely understand all the complex biochemistry of cancer to improve both diagnosis and treatment

D. The key is full ‘biomarker’ characterization of a different types of cancer

Biomarker Research Status

TODAY

PSA

‘biomarkers’Hmmm!! I see you have abnormal PSA levels. You might have some problems in your prostate. We must check for cancer

Oh!! You have abnormal PSA levels. Also, your levels of BM1,BM2,BM3 are off, and BM4 levels are subnormal. You are starting to develop prostate cancer of the A phenotype. But don’t worry your BM5 is fine, so metastasis hasn’t occurred yet. Let’s start treatment

PSA

? ?

? ?

BM1 BM2

BM3

BM4

BM5THE FUTURE

Nanoprobes: The usual suspects

Quantum Dots Gold Nanoparticles

Liposomes Polymeric Nanoparticles

functionalized to achieve

biocompatibility and cell

targeting

NanotubesNanorods

QD Localization of a TumorA. 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 (see B.)

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

B. C. 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

Gold Nanoparticle Tumor DetectionA. The common strategy to detect the tumor is the functionalization of the nanoparticle with an antibody specific to the tumor antigens, and then detect the nanoparticle by some spectroscopic technique

B. Tumor photograph

Imaging with gold nanoparticles as contrast agent

Nanotechnology 2009. Vol. 20, 395102

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

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

D.

C. Different phenotypes show different aggressiveness on their metastatic behavior

tumor

Blood vessels

Cancer cells

metastasis

Source: www.cancernews.com

Multiplex Diagnosis

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

A. 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

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

The peak intensity correlates to the concentration of a specific QD

Aggressive cancer cells

Mild cancer cells

As a result cancer cells of different phenotype are colored differently

Diagnosis using NanothermometersA. Cancer cells appears to have a more elevated temperature than normal cells. Therefore, a local temperature mapping can be used to determine the spread of a tumor

B. A gold nanoparticle is functionalized with a PEG coating, which itself is assembled to a layer of smaller QD’s. The emission properties of the nanoparticle change with temperature due to the stretching/contraction of the PEG

C.

Correlation between emission and temperatureD.

Thermal image of a healthy and cancerous breast

healthy sick

Source: 9th European Congress of Thermology, Krakow, Poland

Angew. Chem. Int. Ed. 2005, Vol. 44, 7439 –7442

TherapyA. There is a search dual-mode nanoparticle that can detect a tumor (imaging)and destroy it (therapy)

B. There is two action modes for therapeutical nanoparticles

Passive Targeting Active Targeting

Based on nanoparticle functionalization for specific targeting of cancerous cells

Based on retention effect of particle of certain hydrodynamic

size in cancerous tissues

Taking advantage of retentionA. Tumorous tissues suffer of Enhanced Permeability and Retention effect

B. Nanoparticles injected in the blood stream do not permeate through healthy tissues

C. Blood vessels in the surrounding of tumorous tissues are defective and porous

D. Nanoparticles injected in the blood permeate through blood vessels toward tumorous tissues, wherein they accumulate

Annu. Rev. Biomed. Eng. 2007. Vol. 9, pp. 257–88

A Targeted Polymer NanoparticleA. A dual Nanoparticle, the targeting ligand allow it to diagnose if a cell is healthy or sick, and bind specifically to the tumorous cell

B. Once inside the cell, the polymeric nanoparticle degrades and the anticancer agent is set free

C.An imaging agent can be added as wellImaging

agent

Annu. Rev. Biomed. Eng. 2007. Vol. 9, pp. 257–88

A commercial Anticancer NanoparticleA. The nanoparticle drug ABRAXANE is one of the fruits of nanomedicine applied to cancer therapy. It consist in nanoparticles carrying an agent interfering with the feeding mechanism of cancerous cell. Click on the video to see action mechanism

SECTION IIIPromising work

Nanotubes

Source: www.nanotechweb.org

A. Carbon nanotubes have been found to have a very interesting property, they release heat when exposed to radio frequencies

B. Chemical properties of nanotubes allow them to be easily functionalized

C. For this studies the nanotubes were produced by the CoMoCAT procedure, and functionalized with the polymer Kentera

Source: Southwest nanotechnologies

CoMoCAT nanoparticles with grown nanotubes

Heat Release Tests

Nanotube suspensionSource:Hamamatsu Nanotechnology

Radiowaves

Cancer 2007;Vol.110, pp. 2654–2665

250mg/L50mg/L0mg/L

A. Suspensions of nanotubes at different concentrations were remotely irradiated with radio waves, resulting in heating correlated to the concentration of nanotubes in suspension

Heat Release Tests

Cancer 2007;Vol.110, pp. 2654–2665

A. There is a linear increase of the heating rate with the source power, and a non-linear increase with the nanotube concentration. The irradiation frequencies were previously shown not to cause damage in normal tissues

600W

SWCNTRF

Cytotoxicity testsA. The following human cells were grown with 24h contact with 500mg/L nanotube solutions:

Hepatocellular carcinoma Hep3B Hepatocellular carcinoma HepG2

Panc-1 pancreatic adenocarsinoma

B. The results shown correspond to fluorescence cytometric results, the segments represent stages of cellular growth, which appear unaltered despite the presence of the nanotubes. NO CITOTOXICITY

Cancer 2007;Vol.110, pp. 2654–2665

Intracellular Collection of Nanotubes

nanotubes

nanotubes

A. Despite the lack of cytotoxicity, bright field images clearly shows the accumulation of nanotube structure inside the cellular structure

Culture without SWCNT’s

Culture with SWCNT’s

B. Also, the optical response of the cultures to other imaging techniques is shown by this IR image

Cancer 2007;Vol.110, pp. 2654–2665

Cytotoxic induced effectA. Now, the cytotoxic effect of the SWCNT’s during the irradiation of with radio waves on carcinoma cultures is tested

Hepatocellular carcinoma Hep3B

No Irradiation

2 min Irradiation

B. The counts of cells in phases M1,M2, and M3 is negligible indicating the mortality rate of the cultured cells after irradiation

Control

Cancer 2007;Vol.110, pp. 2654–2665

In vitro induced cytotoxicity A. The cytotoxicity correlates with

the nanotube concentration

B. Some carcinomas are more susceptible to death (HepG2) after radiation

C. Remarkably, the control (the polymer alone) showed some degree of cytotoxicity

D. In vitro test successful!!!

HepG2Hep3B

Panc-1Cancer 2007;Vol.110, pp. 2654–2665

In Vivo cytotoxicity testA. In the top panel, the

photomicrograph of a hepatic tumor on a rabbit. The black stains correspond to nanotube accumulation on the tumorous cell

B. The purple staining is characteristic of live tissues

C. In the bottom panel, the photomicrograph of the same hepatic tumor after 2 min. radio frequency waves irradiation.

D. The brownish color is indicative of necrosis (tissue death)

Cancer 2007;Vol.110, pp. 2654–2665

Raman Scattering

Incoming light

Outcoming light

hv1

hv2

hv1

A weak effect

hv2hv1 Vibrational energy

Source: Earth System Research Laboratory

A. 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)

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

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

Microfluid Nanofluid 2009;Vol.6, pp. 285–297

Design ConsiderationsA. Raman Reporter (malachite green) with a characteristic Raman signal

B. A 60nm gold nanoparticle that enhances the reporter Raman signal 14 orders of magnitude

C. A PEG polymer to coadsorb on the gold nanoparticle (together with the reporter) and improves biomobility of the nanoparticle

D. A Hetero-PEG polymer to coadsorb with the PEG and the reporter, and easily functionalized

E. A ScFv EGFR antibody functionalized on the hetero-PEG to become the nanoparticle target specific

Synthesizing the nanoparticle

Colloidal gold solution Raman Reporter solution

mixing

Heterofunctional PEG solution

mixing

PEG solutionmixing

ScFv EGFR antibody solution

mixing

Resulting nanoparticle

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

Optical CharacterizationA. 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

Gold QD

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

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

B.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

Technique Penetration In vivo

B. The skin spectrum has to be magnified 210-fold to be distinguishable

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

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

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

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

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

In Vivo Tumor DetectionA. A sick mouse was injected

with the targeted nanoparticle solution

B. The illumination of the liver produced a weak Raman signal

C. 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

SECTION IVAssesment

What have we learned?

• Nanoparticles have very special properties that make them attractive for nanomedicine

• Nanoparticles can be functionalized with antibodies to target their binding toward specific cells

• Nanoparticles can be used in diagnosis through the detection of biomarkers

What have we learned?• Nanoparticles can respond to external radiation

and release heat, killing cells around them

• Nanoparticles can be made of lipids or polymers than decompose once a target is reached and deliver a pharmaceutical agent

• Quantum dots are special nanoparticles that emit light of different colors according to its diameter, and can be used for complex diagnosis

What have we learned?• PEG is the most used polymer to coat

nanoparticles due to the biocompatibility and biomobility that confers to the nanoparticle

• Targeted nanoparticles offer a light of hope for the fight against cancer

• An ideal nanoparticle is three-modal: detects, diagnoses and attacks tumorous cells

Unsolved issues

Long-term toxicity

Biomarkers library

Success in human trials

3-D spatial resolution

Signal penetration

Challenges

• Multiple modality and functional nanoparticles• Fight against the tendency of nanoparticles to be

adsorbed by reticuloendothelial system• Avoid aggregation of nanoparticles for in vivo

viability• Improve retention times of the nanoparticles

inside the body to allow the therapeutic effect• Substitute potentially toxic elements

Challenges

• Compromise between coating and hydrodynamic radius

• Eliminate the inflammatory and immune response triggered by some polymer coatings

• Avoid undesired degradation exposing toxic elements (QD) or untimely delivering cargo

• Increase contrast for human medical imaging (tissues are naturally fluorescent)

Challenges

• Real-time monitoring of drug distribution, action mechanism and patient’s response

• Fast detection of biomarkers at lower limits• Understanding the mechanism of cancer• Diagnosis leading to personalized treatments• Detection of deep tumors• Selective targeting in extremely heterogeneous

tissues.

Thanks!