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OUTLINE

Potential Application of Nanoparticles in THERANOSTICS : Diagnosis and Therapy Targeting Strategies. OUTLINE. Part I Nanomedicine review Part II Application Part III Biological Therapy. Part I Nanomedicine Review. Nanomedicine. Premise :

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OUTLINE

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  1. Potential Application of Nanoparticles in THERANOSTICS: Diagnosis and TherapyTargeting Strategies

  2. OUTLINE • Part I Nanomedicine review • Part II Application • Part III Biological Therapy

  3. Part I Nanomedicine Review

  4. 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 NANOMEDICINE

  5. Facts and Numbers Drug delivery is the most established technology in the nanomedicine market Nature Biotechnology 2006, Vol. 4, pp.1212-1217

  6. Drug Delivery Because of their small sizes, nanoparticles are taken by cells where large particles would be excluded or cleared from the body • 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 1 2 3 4 Source: Comprehensive Cancer Center Ohio University

  7. Revisiting the Importance of Size in Distributive Properties of Probes

  8. Nanoparticles in action 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 Directed accumulation of dangerous bacteria by conjugation with functionalized magnetic nanoparticles National Research Council, Canada Analytical Chemistry 2004, Vol. 76, pp.7162-7168

  9. A Drug Delivery Nanoparticle 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) antibody lipid SiRNA Healthy tissue Sick tissue treated with non-targeted nanoparticles Science 2008, Vol. 316, pp 627-630 Sick tissue treated with targeted nanoparticles

  10. Medical Imaging 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. 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 Solutions of CdSe QD’s of different diameter Nano Letters 2008., Vol. 8, pp3887-3892 Source: Department of immunology, University of Toronto

  11. A Quantum Dot Nanoparticle 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

  12. Targeting QD’s for intracellular imaging 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 Ligand coated QDNC Ingestion QD (red)intracellular uptake is enhanced when using the QDNC instead of the free QD’s Decomposition labeling QD release Imaging of nucleus (blue) and cytoplasm (other) after 30 min (left) and 3 hours after uptake Nano Letters 2008., Vol. 8, pp3887-3892

  13. 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 (see B.) 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

  14. Part II Application in oncology

  15. Cancer Facts The second main cause of death in the US, and certainly the diseases that lower the life quality of the patient the most Lung cancer is the overwhelming lead cause of cancer-related deaths. BEWARE SMOKERS!!!!

  16. 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

  17. Cancer: Too complex to handle? 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 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 The key is full ‘biomarker’ characterization of a different types of cancer

  18. 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’ ? PSA TODAY 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 BM1 BM2 THE FUTURE BM4 PSA BM3

  19. Diagnosis 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 Tumor Cancer cells Metastasis Blood vessels Source: www.cancernews.com

  20. A Chemical Nose (Multiplex Detection) 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. 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.10912-10916

  21. A Chemical Nose (Multiplex Detection) 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 and emit a fluorescence signal detached polymer polymer NP2 Cell membrane NP1 NP3 Fluorescence change 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 Normal cell Cancerous cell Metastatic cell PNAS 2009, Vol. 106, pp.10912-10916

  22. 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

  23. Diagnosis using Nanothermometers 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 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 healthy sick Thermal image of a healthy and cancerous breast Angew. Chem. Int. Ed. 2005, Vol. 44, 7439 –7442 Source: 9th European Congress of Thermology, Krakow, Poland

  24. Targeting Approaches DRUG DRUG DRUG There is a search dual-mode probes that can detect a tumor imaging) and destroy it (therapy) PROBE PROBE Redox-potential Ultrasound PROBE Temperature pH-sensitive Physical Targeting Active Targeting Passive Targeting Based on nanoparticle functionalization for specific targeting of disease cells Based on retention effect of particle of certain hydrodynamic size in cancerous tissues (e.g. Doxil) Proteins (antibodies and their fragments such as TAT), nucleic acids (aptamers), receptor ligands (peptides, vitamins, and carbohydrates

  25. EPR: Taking advantage of retention A. Tumorous tissues suffer of Enhanced Permeability and Retention effect (RES) 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

  26. Clinical Example of EPR Doxil is a polyethylene glycol coated liposomal formulation of doxorubicin. Marketed by Ben Venue Laboratories  of J&J. Outside the US, Doxil is known as Caelyx (Janssen). Approved by the FDA for treatment of ovarian cancer and multiple myeloma and an AIDS-related cancer.

  27. How Doxil works? Doxorubicin interacts with DNA by intercalation and inhibition of macromolecular biosynthesis. This inhibits the progression of the enzyme topoisomerase II, which relaxes supercoils in DNA for transcription. Doxorubicin stabilizes the topoisomerase II complex after it has broken the DNA chain for replication, preventing the DNA double helix from being resealed and thereby stopping the process of replication.

  28. Doxil Side Effects Liposome-encapsulated doxorubicin is less cardiotoxic than free doxorubicin. Polyethylene glycol results in preferential concentration of Doxil in the skin-----a side effect known as hand-foot syndrome. Small amounts of the Dox can leak from capillaries in the palms of the hands and soles of the feet. >50% of patients treated with Doxil developed hand-foot syndrome. Myocet is a non-pegylated liposomal doxorubicin (Phase III) Plenty of room for improvement!

  29. Example of an Approved Anticancer Agent Protein-bound paclitaxel is an injectable formulation of paclitaxel, a mitotic inhibitor drug used in the treatment of breast cancer, lung cancer and pancreatic cancer. Paclitaxel Albumin

  30. ABRAXANE Clinical Trial Results recTLRR (PRIMARY END POINTS) VS. PACLITAXEL INJECTION FOR ALL RANDOMIZED PATIENTS IN THE PHASE III TRIAL IN METASTATIC BREAST CANCER SIGNIFICANTLY SUPERIOR TUMOR RESPONSE RATE IN ALL RANDOMIZED PATIENTS EFFICACY DEMONSTRATED IN SECOND-LINE METASTATIC PATIENTS AND PATIENTS WHO RELAPSED WITHIN 6 MONTHS OF ADJUVANT CHEMOTHERAPY

  31. Is Abraxane Safe? • On Jan, 25, 2014: 1,893 people reported to have side effects when taking Abraxane. Among them, 25 people (1.32%) have Renal Failure. Time on Abraxane when people have Renal failure :

  32. Targeted Nanoparticle A dual Nanoparticle, the targeting ligand allow it to diagnose if a cell is healthy or sick, and bind specifically to the tumorous cell Once inside the cell, the polymeric nanoparticle degrades and the anticancer agent is set free Imaging agent An imaging agent can be added as well Annu. Rev. Biomed. Eng. 2007. Vol. 9, pp. 257–88

  33. Immuno-Toxin and Antibody-Drug Conjugates • Earliest approaches of active drug targeting is a direct coupling of a drug to a targeting moiety. Immunotoxins is an example of this approach (Ab fragments) • mAbrituximab (Rituxan) for treatment of patients with non-Hodgkin’s lymphoma (a type of cancer that originates in lymphocytes), • Trastuzumab (Herceptin), an anti-HER2 mAb that binds to ErbB2 receptors for the treatment of breast cancer Antibody-Toxin

  34. “Holey” Nano-bialys: Targeted MRI agent Mimicking liposomes with a Synthetic Polymer Chemically cross-linked surface to control the release of soluble drugs Mn(III) Contrast agents Fibrin Clot 1.5 T MRI • Derived from amphiphilic lipid-polymer hybrid (PEI) • Optionally Cross-linked membrane to control the release of insoluble drugs and stability • Ionic r1=3.7 (s·mmol [Mn])-1 particulate relaxivities • 866 989 (s·mmol [nanobialy])-1 Biotin Targeted Clots Theranostic Agent

  35. 20% 100 nm Dissolution experiment with soluble and insoluble drugs TEM AFM

  36. The pH stimuli sensitive systems The pH of the pathological tissue is lower than the normal tissue, e.g. at the site of inflammation pH drops from 7.4 to pH 6.5. The same is observed in the case of infarcts. Also, the pH is lower in the tumor mass (pH 6.5) than the surrounding tissue (pH 7.4). The microenvironment of a tumor is acidic because insufficient oxygen in tumors leads to hypoxia and causes production of lactic acid. 

  37. pH Sensitive NanoCarrier As a Theranostic Platform AuNPs for cancer imaging (A) enzyme sensitive AuNPs for NIRF imaging, (B) tumor targeting: AuNPs for NIRF/CT dual imaging, and (C) schematic illustration of PEGylatedDoxAuNP@CaP for theranosis.

  38. Ultrasound Mediated Therapy Subcutaneously implanted rat prostate carcinomas seven minutes after administration of unspecific microbubbles (L) and RGD-coated microbubbles (R)

  39. Ultrasound images of normal rat livers and tumor bearing livers following bolus injection of microbubbles (50 µL) into the tail vein, portal vein, or hepatic artery. Take of the microbubble contrast (green) by the liver is very high if the injection is made through hepatic artery or portal vein, but poor if injection is made through the tail vein.

  40. In vivo US imaging of microbubbles (MBs) in a mouse bearing a MDA-MB-231 xenograft. Contrast-mode images showing the nonlinear contrast from porshe MBs in a cross section of the entire subcutaneous tumor on the mouse hind leg are displayed. The MB signal, shown as bright spots within the tumor after i.v. injection, enabled identification of the tumor vasculature using a 5–12 MHz US transducer. The bright rod-shaped object is an artifact of the system.

  41. Thermal Therapy

  42. Nanotubes Carbon nanotubes have been found to have a very interesting property, they release heat when exposed to radio frequencies Chemical properties of nanotubes allow them to be easily functionalized For this studies the nanotubes were produced by the CoMoCAT procedure, and functionalized with the polymer Kentera Source: www.nanotechweb.org CoMoCAT nanoparticles with grown nanotubes Source: Southwest nanotechnologies

  43. Heat Release Tests 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

  44. Cytotoxicity tests The following human cells were grown with 24h contact with 500mg/L nanotube solutions: Hepatocellular carcinoma Hep3B Hepatocellular carcinoma HepG2 Panc-1 pancreatic adenocarsinoma 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 CYTOTOXICITY Cancer 2007;Vol.110, pp. 2654–2665

  45. Intracellular Accumulation of Nanotubes Despite the lack of cytotoxicity, bright field images clearly shows the accumulation of nanotube structure inside the cellular structure Culture without SWCNT’s 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

  46. Cytotoxic induced effect 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 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

  47. 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 HepG2 Hep3B Panc-1 Cancer 2007;Vol.110, pp. 2654–2665

  48. 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

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