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Diego A. Gomez-Gualdron Midterm Project Nanotechnology; CHEN

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1 Potential Application of Nanoparticles in Medicine: Cancer Diagnosis and Therapy
Diego A. Gomez-Gualdron Midterm Project Nanotechnology; CHEN Texas A&M University March 11th, 2010

2 OUTLINE SECTION I Nanomedicine overview SECTION II
Nanotechnology potential in oncology SECTION III Promising works SECTION IV Assessment

3 SECTION I Nanomedicine Review

4 Nanomedicine Premise: Potential applications
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

5 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

6 Source: Comprehensive Cancer Center Ohio University
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 A nanoparticle carries the pharmaceutical agent inside its core, while its shell is functionalized with a ‘binding’ agent Through the ‘binding’ agent, the ‘targeted’ nanoparticle recognizes the target cell. The functionalized nanoparticle shell interacts with the cell membrane The nanoparticle is ingested inside the cell, and interacts with the biomolecules inside the cell The nanoparticle particles breaks, and the pharmaceutical agent is released 2 3 4 Source: Comprehensive Cancer Center Ohio University

7 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 Healthy tissue Sick tissue treated with non-targeted nanoparticles Science 2008, Vol. 316, pp Sick tissue treated with targeted nanoparticles

8 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, pp Solutions of CdSe QD’s of different diameter Source: Department of immunology, University of Toronto

9 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. The core consist of the semiconductor material that emits lights The shell consist of an insulator material that protects the light emitting properties of the QD in the upcoming functionalization The shell is functionalized with a biocompatible material such as PEG or a lipid layer 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 3 2 1 4 Source: The scientist (2005), Vol. 19, p. 35

10 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 when using the QDNC instead of the free QD’s Decomposition labeling QD release D. Imaging of nucleus (blue) and cytoplasm (other) after 30 min (left) and 3 hours after uptake Nano Letters 2008., Vol. 8, pp

11 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). Nanoparticle 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 D. Coating molecule specifically attracted to the molecular signature molecular signature of sick cell of infecting agent (e.g. an antibody) 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 Cell membrane Huffman, Nanomedicine and Nanobiotechnology, Vol. 1, 1, 2009

12 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 Directed accumulation of dangerous bacteria by conjugation with functionalized magnetic nanoparticles Analytical Chemistry 2004, Vol. 76, pp National Research Council, Canada

13 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 A fluorescence reporter polymer PNAS 2009, Vol. 106, pp

14 A Chemical Nose (Multiplex Detection)
detached polymer 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 D. polymer NP2 NP1 Cell membrane NP3 G. 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 Fluorescence change Normal cell Cancerous cell Metastatic cell PNAS 2009, Vol. 106, pp

15 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 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:

16 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: Source:

17 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 After irradiation Nanoletters 2006, Vol. 6, pp

18 SECTION II Nanotechnology potential in oncology

19 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 Vol. 9, pp. 257–88

20 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!!!!

21 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

22 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 entropy B. 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

23 Biomarker Research Status
Hmmm!! I see you have abnormal PSA levels. You might have some problems in your prostate. We must check for cancer ? ? ‘biomarkers’ 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 THE FUTURE BM5 BM1 BM2 BM4 PSA BM3

24 Nanoprobes: The usual suspects
Quantum Dots Gold Nanoparticles functionalized to achieve biocompatibility and cell targeting Nanorods Nanotubes Liposomes Polymeric Nanoparticles

25 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.) C. 560-QD-Streptadivin targets and images In-vitro breast cancer cells having the IgG factor characteristic of chemotherapy responsive cells B. Annu. Rev. Biomed. Eng Vol. 9, pp. 257–288 Nature Biotechnology Vol. 9, pp

26 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 Tumor photograph B. Imaging with gold nanoparticles as contrast agent Nanotechnology Vol. 20,

27 Source:
Diagnosis A. It must be multiplexed, i.e. multiple biomarkers must be detected simultaneously C. Different phenotypes show different aggressiveness on their metastatic behavior B. A specific phenotype of cancer cells has a particular combination of biomarkers on its membrane Blood vessels D. tumor Cancer cells metastasis Source:

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

29 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

30 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 Active Targeting Based on retention effect of particle of certain hydrodynamic size in cancerous tissues Based on nanoparticle functionalization for specific targeting of cancerous cells

31 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 Vol. 9, pp. 257–88

32 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. An imaging agent can be added as well Imaging agent Annu. Rev. Biomed. Eng Vol. 9, pp. 257–88

33 A commercial Anticancer Nanoparticle
A. 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

34 SECTION III Promising work


36 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: Source: Southwest nanotechnologies

37 Source:Hamamatsu Nanotechnology
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 250mg/L 50mg/L 0mg/L Radiowaves Nanotube suspension Source:Hamamatsu Nanotechnology Cancer 2007;Vol.110, pp. 2654–2665

38 Heat Release Tests 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 RF SWCNT Cancer 2007;Vol.110, pp. 2654–2665

39 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

40 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

41 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

42 In vitro induced cytotoxicity
The cytotoxicity correlates with the nanotube concentration Some carcinomas are more susceptible to death (HepG2) after radiation Remarkably, the control (the polymer alone) showed some degree of cytotoxicity In vitro test successful!!! HepG2 Hep3B Panc-1 Cancer 2007;Vol.110, pp. 2654–2665

43 In Vivo cytotoxicity test
In the top panel, the photomicrograph of a hepatic tumor on a rabbit. The black stains correspond to nanotube accumulation on the tumorous cell The purple staining is characteristic of live tissues In the bottom panel, the photomicrograph of the same hepatic tumor after 2 min. radio frequency waves irradiation. The brownish color is indicative of necrosis (tissue death) Cancer 2007;Vol.110, pp. 2654–2665


45 Source: Earth System Research Laboratory
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) Incoming light Outcoming light hv1 hv2 A weak effect Vibrational energy Source: Earth System Research Laboratory

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

47 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

48 Synthesizing the nanoparticle
Colloidal gold solution Raman Reporter solution mixing mixing Heterofunctional PEG solution PEG solution mixing ScFv EGFR antibody solution mixing Resulting nanoparticle Nature Biotechnology 2008;Vol.26 pp. 83–90

49 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

50 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

51 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 ~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 E. It is concluded that the technique penetration is about 2cm… Nature Biotechnology 2008;Vol.26 pp. 83–90

52 In Vivo Tumor Detection
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

53 SECTION IV Assesment

54 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

55 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

56 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

57 Success in human trials
Unsolved issues Long-term toxicity Signal penetration Biomarkers library 3-D spatial resolution Success in human trials

58 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

59 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)

60 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.

61 Thanks!

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