20.109(S09)/TRBlue

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Overall Goal

Use quantum dots as a tumor-imaging device as well as a therapeutic tool to promote more efficient delivery of siRNA to silence survivin, an apoptosis inhibitor that is present in tumor cells

Background

Quantum dots

What are they?

   * Fluorescent semiconductor nanoparticles (nanocrystals) that can be used as a labeling device

Structure:

   * Usually around 2-20 nm in diameter (10 - 50 atoms)
   * Quantum confined semiconductor
   * Semiconductor: external stimulus changes electrical conductivity
   * Generally composed of groups II and VI elements or groups III and V elements of periodic table
   * Surfactant-coated particles are not soluble in water
         o Polar surfactant head group attached to inorganic core of QD
         o Hydrophobic chain protruding into organic solvent


How do they work?

   * Quantum confinement effect: Causes the energy difference to increase between energy states and band gap
         o QD physical dimensions smaller than the exciton Bohr radius
   * Larger quantum dot = lower energy = more red fluorescence
   * Smaller quantum dot = higher energy = more blue fluorescence
   * Wavelength of fluorescence of QD depends on energy gap (diff in energy between ground and excited state), 
     which is determined by its size


Biological Applications:

   * Fluorescent labeling or tracking device, improved photostability
   * Substitute for organic dyes --> 10-100x more stable (less photobleaching) and higher quantum yield
   * 3D imaging: quantum dots allows for reconstruction of 3D image after multiple consecutive confocal imaging
   * Real-time tracking (hours at a time, up to 4 months)
   * Making QDs water soluble: encapsulated in ampiphilic polymers and bound to tumor-targeting ligands and drug 
     delivery vesicles for targeting, imaging, and treating tumor cells (Vashist)
         o Surfactant layer replaced or coated with additional layer such as hydrophilic or amphipathic polymers
     


Quantum Dots in Early Diagnosis of Cancer (Vashist):

   * Current methods for diagnosis: medical imaging, tissue biopsy, bioanalytical assay of body fluids by ELISA
         o Insufficiently sensitive and specific to detect most types of early-stage cancers
         o Labor-intensive, time consuming, expensive, no multiplexing capability
   * QD based detection
         o Rapid, easy, economical
         o What makes them ideal for detecting tumors:
               + Intense and stable fluorescence for longer time
               + Resistance to photobleaching
                     # Photobleaching: irreversible photooxidation process which makes fluorophores non-fluorescent
                     # Fluorophores can be optically excited only within narrow range of wavelengths vs. QDs can be 
                       excited with a single light source having wavelength shorter than wavelength of fluorescence
               + Large molar extinction coefficients
               + High sensitivity due to ability to absorb and emit light very efficiently
               + Large surface area:volume ratio --> can be conjugated to various molecules for multiplex approach
                     # Fluorescence spectra of QDs are narrow, symmetric, and can be observed and ditinguished without 
                       any spectral overlap-->lots of colors!
                     # Multicolor labelling of different structures
               + In the past, QD have been covalently linked to antibodies, peptides, nucleic acids, and other fluorescence 
                 probes
   * In most cases, functional QD conjugates for cancer detections are composed of:
         o Semiconductor core (CdSe, CdTe)
         o Additional shell (ex ZnS) in the case of CdSe QDs have a higher band gap than CdSe to increase quantum yield
         o Water soluble hydrophilic coating
         o Functionalized antibodies/other biomolecules complementary to the target cancer markers at tumor sites
   * Overall process for employing QDs to diagnose cancer in vivo:
         o Form QD bioconjugates (attach antibodies against Cancer markers, PEG)
         o Inject QD bioconjugates into mouse intravenously
         o QD bioconjugates actively target tumor cells


Overcoming the Toxic Nature of Quantum Dots (Vashist):

   * CdSe QDs are highly toxic to cells exposed to UV for long periods of time
   * Polymer-coated QDs are non-toxic in the absence of UV
   * QDs are normally encapsulated inside the outer coating of amphiphilic polymers to make them water-soluble and 
     resistant to chemical/enzymatic degradation
   * Typically synthesized in organic solvents having long alkyl chains and high bp to prevent aggregation
   * Most commonly linked to polyethylene glycol (PEG) or similar ligands to make them biocompatible and to reduce 
     nonspecific binding
   * Make specific to target site by conjugating to various bioaffinity ligands: peptides, antibodies, oligonucleotides


Active and Passive Quantum Dot Targeting Mechanisms (Vashist):

   * Passive Targeting
         o QD bioconjugates accumulate preferentially at tumor sites due to enhanced permeability and retention effect
         o Possible because angiogenic tumors:
               + Produce vascular endothelial growth factors--> enhanced permeability
               + Lack effective lymphatic drainage system--> QD bioconjugate accumulation
   * Active Targeting
         o Antibody-conjugated QDs are employed
         o Antibody attached to specific tumor biomarkers (ex: prostate specific membrane antigen present on tumor cell)


Quantum Dot-Peptide Conjugates Shown to Target Tumour Cells (Vashist):

   * Akerman et al.: showed targeting capabilities of WDs coated with different peptides
         o Case 1:  QDs coated with a lungh-targeting peptide accumulated in lungs of mice after intravenous injection
               + Peptide bound to membrane dipeptidase on endothelial cells in lung blood vessels
         o Case 2: QDs coated with targeting peptide got bound to blood vessels and tumor cells in certain tumors
         o Case 3: QDs coated with targeting peptide bound to lymphatic vessels and tumor cells
         o Adding PEG to outer coating of QDs prevents nonselective accumulation of QDs in reticuloendothelial tissues
   * Quantum Dot Corporation & Genentech: Quantum Dots Able to Idnetify Live Breast Cancer Cells
         o QDs linked to immunoglobulin G (IgG) and streptavidin used to label Her2 cancer markers on surface of live 
           breast cancer cells
         o Simultaneous labelling of Her2 on cell surface and in nucleus
         o Simultaneously detected two cellular targets with single excitation wavelength
         o Different colored QDs could be used together to distinguish diff parts of a single cell-->multiplex detection
   * Gao et al.: Multifunctional Quantum Dots Simulatenously Taret and Image Tumors in Living Animals
         o Highly stable QD conjugate made of:
               + Amphiphilic triblock copolymer (for in vivo protection)
               + Targeting ligands (tumor antigen recognition)
               + PEG molecules (improved biocompatibility/circulation)
         o QD probes accumulated at targeted tumor sites by passive AND active targeting mechanisms
         o Within small period of time, QDs accumulated in cell nuclei
         o Treated cells having QDs can be tracked inside live animals by virtue of fluorescence


Quantum Dot Based Drug Delivery System to Target Cancer (Vashist):

   * Nie et al.: modified QD with an impermeable coating of polymer to prevent leaking out of highly toxic Cd ions from 
     QD conjugate and to provide a means to chemically attach tumor-targeting molecules and drug delivery functionality 
     to QD conjugate
   * QDs tuned to radiate in IR region to prevent tissue damage from QD energy emissions
   * QDs conjugated to peptide/antibodies specific against cancer maker on surface of target cancer cells
   * Made to release drug only when hit with laser light
         o allows control of cells that will receive the toxin
         o minimizes side effects
   * Efforts to extend wavelength of fluorescence of QDs above 900 nm since there are hardly any biomolecules which emit 
     about this wavelength

siRNA

What is it?

   * Usually 18-25 nucleotides long, with 2-3 nucleotides as overhangs on both ends
   * Targets complementary sequence 

What is it used for?

   * RNA interference tool to silence gene expression, by either complete knockdown of the gene or downregulation

Survivin

Structure:

   * Mr ~ 16,500 cytoplasmic protein
   * Single BIR, no RING finger
   * Human Survivin Gene (Zaffaroni):
         o Spans 14.7kb on telomeric position of chromosome 17
         o Made of 3 introns + 4 exons
         o Splicing variants:
               + Suvivin-△Ex3 : removal of exon 3
                     # Heterodimerization of surviving with this variant is essential for inhibition of mitochondrial-dependent
                       apoptosis
               + Surivin 2alpha : exon1+exon2+ 3' 197bp region of intron 2
                     # Attenuates anti-apoptotic activity of survivin


Function (Zaffaroni)

   * Apoptosis mediated by caspases:
         o Intrinsic apoptotic pathway
               + Mitochondria release cytochrome c
               + Cytochrome c binds and activates Apoptotic Protease Activating Factor-1 (Apaf-1)
               + Causes assembly of a multiprotein caspase-activating complex (apoptosome)
               + Leads to activation of caspase-9 and initiation of protease cascade
               + This pathway primarily governed by proteins of Bcl-2 family
                     # Includes both pro- and anti-apoptotic molecule
                     # Differentially affect mitochondrial homeostasis and cytochrome c release
         o Survivin is member of inhibitor of apoptosis proteins (IAPs)
               + Block a common step downstream of mitochondrial cytochrome c release
               + Inhibit terminal effector caspase-3 and caspase-7
               + Interfere with caspase-9 activity and processing
   * Surviving exists in 2 immunohistochemically distinct pools
         o Nuclear pool: localized to kinetochores of metaphase chromosomes and to central spindle midzone at anaphase
         o Cytosolic pool: associated with interphase microtubules, centrosomes, spindle poles, and mitotic spindle 
           microtubules at metaphase and anaphase
               + Microtubule-associated pool appears to be quantitatively predominant and functionally relevant
   * Survivin downregulation causes pleiotropic  cell-division defects
   * Forced expression of survivin in HeLa epithelial carcinoma cells influenced microtubule dynamics and stabilized microtubules
     against nocodazole-induced depolymerization
         o Promotes resistance to drugs targeting the mitotic spindle!
   * Participates in regulation of chromosome segregation
   * In response to cell death stimulation, mitochondrial survivin rapidly discharged in cytosol
         o Prevents caspase activation
         o Inhibits apoptosis


Role in cancer

   * Cannot be detected in 'terminally differentiated adult tissues' (Tanaka)
   * Expressed in many cancers: stomach, colorectal, lung, breast, pancreatic, prostate, etc.
   * No correlation with p53 mutations, but is associated with bcl-2
   * Survivin expression in human tumors (Zaffaroni):
         o Survivin strongly expressed in embryonic and fetal organs, but has not been reported in differentiated normal tissues
               + Exceptions: thymus, basal colonic epithelium endothelial cells, neural stem cells during angiogenesis
         o Survivin expression in majority of human tumor types:
               + Lung, breast, colon, gastric, oesophageal, pancreatic, liver, bladder, uterine, ovarian,
               + Large-cell non-Hodgkin's lymphomas, leukaemias, nuroblastoma, brain tumors, pheochromocytoma, soft tissue 
                 sarcomas, melanomas
         o Re-expression of survivin may occur early during malignant transformation or following disturbance in the balance 
           between cell proliferation and death
               + Also found in variety of preneoplastic and/or benign lesions
   * Upregulation of survivin at transcriptional level in human tumors confirmed
         o Molecular abnormalities
               + Neuroblastoma: gain of 17q25 sequence containing survivin locus
               + Ovarian cancers: suvivin exon 1, normally silenced by methylation, becomes unmethylated
         o Linked to loss of wt p53
               + Accumulation of wt p53 in ovarian cancer cells induced survivin transcriptional repression
   * High levels of protein were predictive of tumor progression
         o Negative prognostic factor in patients with acute myeloid leukemia
         o Significantly shorter survival observed in large-B lymphoma/mantle cell lymphoma patients with high survivin expression

Resources

Quantum Dots

1. Review of Quantum Dot Technologies for Cancer Detection and Treatment: http://www.azonano.com/details.asp?ArticleID=1726

2. Gene Silencer and Quantum Dots Reduce Protein Production to a Whisper: http://newswise.com/articles/view/542018/

3. Targeted Quantum Dot Conjugates for siRNA Delivery: http://pubs.acs.org/doi/full/10.1021/bc060367e

4. Biological Applications of Quantum Dots: http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6TWB-4PC90K0-2&_user=501045&_rdoc=1&_fmt=&_orig=search&_sort=d&view=c&_acct=C000022659&_version=1&_urlVersion=0&_userid=501045&md5=4fa96764e93f1eeb4f7ec8ccd6907564

5. Noninvasive Imaging of Quantum Dots in Mice: http://pubs.acs.org/doi/pdf/10.1021/bc034153y?cookieSet=1


Survivin/siRNA

1. Inhibitors of Apoptosis Proteins (IAPs) expression and their Prognostic Significance in Hepatocellular Carcinoma: http://www.ncbi.nlm.nih.gov/pubmed/19397802?ordinalpos=8&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DefaultReportPanel.Pubmed_RVDocSum

2. Expression of Survivin in Lung Esoinophils is Associated With Pathology in a Mouse Model of Allergenic Asthma: http://www.ncbi.nlm.nih.gov/pubmed/19395375?ordinalpos=9&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DefaultReportPanel.Pubmed_RVDocSum

3. Anti-survivin Antibody Responses in Lung Cancer: http://www.ncbi.nlm.nih.gov/pubmed/19380192?ordinalpos=15&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DefaultReportPanel.Pubmed_RVDocSum

4. Effects of 2-methoxyestradiol on the Expression of Caspase-3 and Survivin in Chronic Myelocytic Leukemia K562 Cells: http://www.ncbi.nlm.nih.gov/pubmed/19379562?ordinalpos=17&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DefaultReportPanel.Pubmed_RVDocSum

5. Expression of survivin and its relationship to loss of apoptosis in breast carcinomas.

Tanaka K, Iwamoto S, Gon G, Nohara T, Iwamoto M, Tanigawa N. Expression of surviving and its relationship to loss of apoptosis in breast carcinomas. Clinical Cancer Research January 2000. Vol. 6, 127-134.

6. Kruppel-like Factor 4 represses the transcription of survivin gene in esophageal cancer cell lines: http://www.reference-global.com/doi/abs/10.1515/BC.2009.060

7. Endogenous Survivin modulates survival and proliferation in UVB-treated human keratinocytes: http://www3.interscience.wiley.com/journal/122241817/abstract?CRETRY=1&SRETRY=0

8. Knockdown of survivin expression by siRNAs enhances chemosensitivity of prostate cancer cells and attenuates its tumorigenicity: http://www.ncbi.nlm.nih.gov/pubmed/19280061?ordinalpos=9&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DefaultReportPanel.Pubmed_RVDocSum

9. Induction of apoptosis of human colon cancer cells by siRNA recombinant expression vector targeting survivin gene: http://www.ncbi.nlm.nih.gov/pubmed/19224161?ordinalpos=12&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DefaultReportPanel.Pubmed_RVDocSum

10. Survivin gene RNA interference inhibits proliferation, induces apoptosis, and enhances radiosensitivity in HeLa cells: http://www.ncbi.nlm.nih.gov/pubmed/19224161?ordinalpos=12&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DefaultReportPanel.Pubmed_RVDocSum

11. Survivin as a target for new anticancer interventions: http://www3.interscience.wiley.com/journal/119920977/abstract (Vashist)