Download Potential Application of Nanoparticles in Medicine

POTENTIAL APPLICATION OF
NANOPARTICLES IN MEDICINE:
Cancer Diagnosis and Therapy
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 nanomedicine
A. 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
A. Because of their small sizes, nanoparticles are taken by cells
where large particles would be excluded or cleared from the body
1
1) A nanoparticle carries the pharmaceutical
agent inside its core, while its shell is
functionalized with a ‘binding’ agent
2
2) Through the ‘binding’ agent, the ‘targeted’
nanoparticle recognizes the target cell. The
functionalized nanoparticle shell interacts
with the cell membrane
3
3) The nanoparticle is ingested inside the cell,
and interacts with the biomolecules inside
the cell
4
4) The nanoparticle particles breaks, and the
pharmaceutical agent is released
Source: Comprehensive Cancer Center Ohio University
A Drug Delivery Nanoparticle
A. 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)
B.
C.
antibody
lipid
SiRNA
Science 2008, Vol. 316, pp 627-630
Healthy tissue
Sick tissue treated with non-targeted
nanoparticles
Sick tissue treated with targeted nanoparticles
Medical Imaging
A. 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.
B.
C. The quantum dots (QD) can be injected to a
subject, and then be detected by exciting them
to emit light
CdSe nanoparticle (QD) structure
Source: Laurence Livermore Laboratories
Imaging of QD’s targeted on cellular structures
Nano Letters 2008., Vol. 8, pp3887-3892
Solutions of CdSe QD’s of different diameter
Source: Department of immunology, University of Toronto
A Quantum Dot Nanoparticle
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
3
2
3) The shell is functionalized with a
biocompatible material such as PEG or a
lipid layer
1
4
Source: The scientist (2005), Vol. 19, p. 35
2) The shell consist of an insulator material
that protects the light emitting properties of
the QD in the upcoming functionalization
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
Targeting QD’s for intracellular imaging
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
B.
Ligand coated
QDNC
Ingestion
C. QD (red)intracellular uptake is enhanced
Decomposition
when using the QDNC instead of the free QD’s
labeling
QD release
D. Imaging of nucleus (blue) and cytoplasm
Nano Letters 2008., Vol. 8, pp3887-3892
(other) after 30 min (left) and 3 hours after
uptake
Diagnosis and Sensing
A. Diseases can be diagnosed through the (simultaneous) detection
of a (set of) biomolecule(s) characteristic to a specific disease type
and stage (biomarkers).
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
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
D.
Nanoparticle
Coating molecule
specifically attracted to
the molecular signature
molecular signature of sick
cell of infecting agent
(e.g. an antibody)
Cell membrane
Huffman, Nanomedicine and Nanobiotechnology, Vol. 1, 1, 2009
Nanoparticles in action
A. 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
B.
C.
Accumulation of functionalized ferromagnetic
nanoparticles on staphylococcus
Negligible accumulation of nanoparticles in
absence of functionalization
Analytical Chemistry 2004, Vol. 76, pp.7162-7168
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 nanoparticles
PNAS 2009, Vol. 106, pp.10912-10916
A fluorescence reporter polymer
A Chemical Nose (Multiplex Detection)
detached polymer
D.
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
polymer
NP1
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
G.
Fluorescence change
Cell membrane
NP2
Normal cell
PNAS 2009, Vol. 106, pp.10912-10916
Cancerous
cell
Metastatic
cell
Therapy
A. 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
C.
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
Source: John Hopkins Center
Colloidal gold
Source: www.wikipedia.com
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. Alzheimer
Source: Berkeley Lab
A. Alzheimer and other
degenerative diseases are
caused my the clustering of
amyloidal beta (Aβ) protein.
B.
Alzheimer’s brain
Healthy brain
C.
D. Gold nanoparticles
can be functionalized
to specifically attach
to aggregates of this
protein (amyloidosis)
Functionalized nanoparticle
Chemical structure of Aβ-protein
Source: www.internetchemistry.com
Source: wwwthefutureofthings.com
Gold Nanoparticles vs. Alzheimer
A. 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
Nanoletters 2006, Vol. 6, pp.110-115
After irradiation
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.
PREMISE
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
Annu. Rev. Biomed. Eng. 2007. Vol. 9, pp. 257–88
Cancer Facts
A. The second main cause of death in the
US, and certainly the diseases that lower
the life quality of the patient the most
B. Lung cancer is the overwhelming
lead cause of cancer-related deaths.
BEWARE SMOKERS!!!!
Motivation
DIAGNOSIS
A. The only factor that really correlates to the patient
survival is early cancer detection
THERAPY
B. Chemotherapy and radiotherapy kill healthy and
sick cells indiscriminately
IMAGING
C. Cancer resurgence after surgery occurs due to
failure to recognize and remove all cancerous colonies
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 cells
B.
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
?
?
‘biomarkers’
?
?
Hmmm!! I see you have abnormal
PSA levels. You might have some
problems in your prostate. We must
check for cancer
TODAY
PSA
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
BM5
THE FUTURE
BM1
BM2
BM4
BM3
PSA
Nanoprobes: The usual suspects
Quantum Dots
Nanorods
Liposomes
Gold Nanoparticles
functionalized
to achieve
biocompatibility
and cell
targeting
Nanotubes
Polymeric Nanoparticles
QD Localization of a Tumor
A. 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.)
B.
C. 560-QD-Streptadivin targets and images In-vitro
breast cancer cells having the IgG factor characteristic
of chemotherapy responsive cells
Annu. Rev. Biomed. Eng. 2007. Vol. 9, pp. 257–288
Nature Biotechnology 2003. Vol. 9, pp. 41-46
Gold Nanoparticle Tumor Detection
A. 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
Diagnosis
A. 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
C. Different phenotypes show different
aggressiveness on their metastatic
behavior
Blood
vessels
D.
tumor
Cancer cells
metastasis
Source: www.cancernews.com
Multiplex Diagnosis
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
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
Diagnosis using Nanothermometers
A. 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
C.
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
Correlation between emission and temperature
D.
healthy
sick
Angew. Chem. Int. Ed. 2005, Vol. 44, 7439 –7442
Thermal image of a healthy and cancerous breast
Source: 9th European Congress of Thermology, Krakow, Poland
Therapy
A. 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
Based on retention effect of
particle of certain hydrodynamic
size in cancerous tissues
Active Targeting
Based on nanoparticle
functionalization for specific
targeting of cancerous cells
Taking advantage of retention
A. 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 Nanoparticle
A. 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.
Imaging
agent
An imaging agent can
be added as well
Annu. Rev. Biomed. Eng. 2007. Vol. 9, pp. 257–88
Nanotubes
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
CoMoCAT nanoparticles
with grown nanotubes
Source: www.nanotechweb.org
Source: Southwest nanotechnologies
Heat Release Tests
A. Suspensions of nanotubes at
different concentrations were remotely
irradiated with radio waves, resulting in
heating correlated to the concentration
of nanotubes in suspension
Radiowaves
250mg/L
50mg/L
0mg/L
Nanotube suspension
Source:Hamamatsu Nanotechnology
Cancer 2007;Vol.110, pp. 2654–2665
Heat Release Tests
A. There is a linear increase of the heating rate with the source power, and a nonlinear increase with the nanotube concentration. The irradiation frequencies were
previously shown not to cause damage in normal tissues
600W
RF
SWCNT
Cancer 2007;Vol.110, pp. 2654–2665
Cytotoxicity tests
A. 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
A. Despite the lack of cytotoxicity, bright field images clearly shows the accumulation
of nanotube structure inside the cellular structure
Culture without SWCNT’s
B. Also, the optical response of the cultures to
other imaging techniques is shown by this IR
image
Culture with SWCNT’s
nanotubes
nanotubes
Cancer 2007;Vol.110, pp. 2654–2665
Cytotoxic induced effect
A. 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
Control
B. The counts of cells in phases M1,M2, and M3
is negligible indicating the mortality rate of the
cultured cells after irradiation
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!!!
HepG2
Hep3B
Panc-1
Cancer 2007;Vol.110, pp. 2654–2665
In Vivo cytotoxicity test
A. 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
characteristic of live tissues
is
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
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)
A weak effect
Incoming light
hv1
Outcoming light
hv2
hv1
Source: Earth System Research Laboratory
hv1
hv2
Vibrational energy
Raman Enhancement
A. When a molecule is coupled with a metallic surface its Raman signal is
enhanced n orders of magnitude
Microfluid Nanofluid 2009;Vol.6, pp. 285–297
B. The localization of the different peaks constitute the fingerprint of a
molecule. For instance, malachite green isothiocyanite, a ‘raman reporter’.
Design Considerations
A. 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
mixing
mixing
PEG solution
Raman Reporter
solution
Heterofunctional PEG
solution
mixing
mixing
Resulting
nanoparticle
Nature Biotechnology 2008;Vol.26 pp. 83–90
ScFv EGFR antibody
solution
Optical Characterization
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
Gold
QD
Nature Biotechnology 2008;Vol.26 pp. 83–90
In Vitro Test
A. 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
A. The nanoparticle solution is injected to a mouse and after 4h…
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 ~50fold stronger than that of
the skin
D.
After deep injection the
Raman signal is only ~10fold stronger than that of
the skin
E. It is concluded that the technique penetration is about 2cm…
Nature Biotechnology 2008;Vol.26 pp. 83–90
In Vivo Tumor Detection
A. 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
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
Signal penetration
Biomarkers library
3-D spatial resolution
Success in human trials
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.