Talk:20.109(F12) Pre-Proposal: Engineering Viral Magnetic Nanoparticles for Magnetic Hyperthermic Cancer Therapy

This is a brainstorming page.
You are very welcome to write any crazy / non-crazy / inventive / conventional / knowledgeable ideas or information you may have about our project.

Some key words: Magnetic Nanoparticles (MNP), Viruses, Magnetic Hyperthermia, Bioengineering

What is Magnetic Hyperthermia?

Definition

How it works?

Under an alternating magnetic field, MNP releases heat due to relaxation of magnetic moments (hysteresis). This can cause an increase in temperature to the range of 41C to 47C. Since tumor cells are more heat sensitive than normal cells, they will be killed by this thermal dissipation.

Here is an interesting tidbit from a paper I was reading: "In addition to the expected tumor cell death, hyperthermia treatment has also induced unexpected biological responses, such as tumor-specific immune responses as a result of heat-shock protein expression. These results suggest that hyperthermia is able to kill not only local tumors exposed to heat treatment, but also tumors at distant sites, including metastatic cancer cells." (Kobayashi)

Current Research

• Clinical trials in prostate cancer
• Shows promising results when coupled with irradiation on breast cancer (mouse)

Current Limitations (This information will help us shape and define the problem.)

(1) To achieve the necessary rise in temperature with minimal dose of MNP.

In other words, this means:

• High specific loss power / specific absorption rate (SLP) of the MNP.
• why is higher applied dosage bad? > leads to unnecessary heat dissipation

(2) Lack of knowledge about the metabolism, clearance, and toxicity of MNP.

Biomedical potentials of MNP

• Could be used as early detection for the following using MRI:
  • Inflammation
  • Cancer
  • Diabetes
  • Atherosclerosis

• Drug Delivery
• Cellular labeling and tissue targeting
• Purifying and separating cells and DNAs
• Hyperthermia
• Transfection by magnetic nanoparticles
• Tissue repair
• Magnetic resonance imaging (MRI)

Types of Relevant Viruses

1. Tobacco Mosaic Virus (TMV)

• 18nmx300nm, helical
• Can withstand high temperatures up to 50C for 30mins (conventional hyperthermia involves heating up to 50C from an external source
• Safe for human consumption
• Mann group has active research on it
• 2130 molecules of coat protein

2. M13 Bacteriophage

• 6.6nmx880nm, helical (Length is too long - pose an issue in targeting cells)
• Lots of research done by the Belcher group, including attaching MNPs to M13 for imaging purposes
• We are familiar with the system

3. Cowpea chlorotic mottle virus (CCMV)

• 26nm, icosahedral

4. Cowpea mosaic virus (CPMV)

• 27nm, icosahedral

5. Brome mosaic virus (BMV)

• 28nm, icosahedral

6. Turnip yellow mosaic virus (TYMV)

• 30nm, icosahedral

7. SIRV2? MS2? Q-beta? Ad? CPV? PVX? (Viral Nanoparticles: Tools for Material Science and Biomedicine 2011)

Current Work in Viral MNP Attachment

Attachment of MNPs to M13 phage for in vivo imaging of prostate cancer (Belcher group)

What we propose to do

See flowchart sketch.

Specific Aims

1. Identifying / Screening for appropriate virus vehicles and tumor-specific anchoring sequencse
2. Developing / Engineering viral MNPs
3. in vivo testing for efficacy of engineered vMNPs in mouse tumor cells.

We will start with using ferritin (Fe3O4) as the MNP.

Steps

• Stage 1: Virus Hunt
  

We need to investigate how the selected virus (likely one of the following: TMV, M13, CCMV, CPMV, BMV or TPMV) interacts with mammalian cells in vivo.

• Stage 2: Screening for MNP binding site on virus
  

We will start by using Fe₃O₄ as our MNP of interest. With this, a protein coat screen of the selected virus for a protein coat that can bind with our MNP is necessary.

• Stage 3: Screening for tumor-specific sequence binding site on virus
  

We need to do a protein coat or RNA screen of the virus for a region that can bind with a tumor-specific peptide sequence. If necessary, we might need to screen tumors for unique short sequences on their cell surfaces.

• Enhanced Permeability and Retention (EPR) effect: nanoparticles tend to accumulate more in tumor tissue than in normal tissue
• Antibodies against tumor markers:

1. folic acid (folate) receptor: folic acid coated nanoparticles improves its internalization - FRA can be overexpressed by a number of epithelial-derived tumors including ovarian, breast, renal, lung, colorectal, and brain.
2. EGFR-2 (erbB2/HER2): anti-HER2 improves cell-internalization of gelatine/albumin and Au nanoparticles (van Vlerken 2006)

• Stage 4: Virus engineering
  

We can now engineer wild-type viruses using specific protein coats or RNA regions isolated in Stage 2 and 3 to produce the viral MNP of interest.

• Stage 5: in vivo testing
  

Perform an in vivo experiment by injecting the engineered viral MNPs into the circulatory system of mice that have developed tumors. By subjecting these mice to an alternating magnetic field under standard hyperthermia conditions and measuring the change in tumor size, we will be able to quantify the efficacy of using viral MNPs in magnetic hyperthermia.

Future directions:

• Experimenting with double layer MNP to increase response
• Target other cancerous cells
• Experiment with other types of viruses

Quantitative Goals (We can quantify with IC50 value)

• Size of MNP: This says 18nm Iron Oxide Nanoparticle work really well: http://www.ruf.rice.edu/~rau/phys600/JPCM2006s2919.pdf So it seems that the size of MNP does not really matter, because we can control the size we want to make.
• Currently, with the aid of 15Gy radiation, the hyperthermia treatment successfully accumulated less than 0.3mg Fe/g tissue. Dosage: 0.2mg Fe per gram of mouse. Say mouse is 25g, so 5mg total dosage injected. so 1% efficiency with the aid of radiation. (MNP sizes used: 70nm and 120nm; murine flank breast tumors were 150mm3)

Calculations:
From http://manalis-lab.mit.edu/publications/grover%20PNAS%202011.pdf, we estimated that a typical cell has an average density of 1.1g/mL. Since the murine flank breast tumors were 150mm3, and 0.25mg Fe/g of tumor was detected in the tumors, we can calculate that only a total of 0.0495mg of Fe is accumulated in the tumors. This gives a % efficacy of 1%.

• South Korean experiment: 75ug of MNPs were injected.
• From Belcher lab's paper, what is the % efficacy of using M13?

Potential Issues

• "The actual rotations of the nanoparticles are disordered because the microviscosity of the local environment in cancer cells is not constant, and effective elasticity depends on the binding conditions between nanoparticles and membranes."

but this is actually present because when treatment is done with individual MNPs, one side of the MNP is always bound to the targeted cell, so direction is never constant!

Useful Resources

1. Gupta AK, Naregalkar RR, Vaidya VD, and Gupta M. Recent advances on surface engineering of magnetic iron oxide nanoparticles and their biomedical applications. Future Medicine. 2007. 2(1), 23-39.
2. Bakoglidis KD, Simeonidis K, Sakellari D, G. Stefanou, and Angelakeris M. Size-Dependent Mechanisms in AC Magnetic Hyperthermia Response of Iron-Oxide Nanoparticles. IEEE Transactions on Magnetics. 2012. 48:1320-1323.
3. Great layman's way of explaining magnetic hyperthermia http://trialx.com/curetalk/2012/11/cancer-treatment-multifunctional-magnetic-nanoparticles-for-molecular-imaging-and-hyperthermia/
4. A.J. Giustini, A.A. Petryk, S.M. Cassim, J.A. Tate, I. Baker, P.J. Hoopes. Magnetic nanoparticle hyperthermia in cancer treatment. Nano LIFE 2010; 01: 17.
5. D. Ghosh, Y. Lee, S. Thomas, A. G. Kohli, D. S. Yun, A. M. Belcher, K. A. Kelly. M13-templated magnetic nanoparticles for targeted in vivo imaging of prostate cancer. Nat. Nanotechnol. 2012; 7 (10): 677–82.
6. L. E. van Vlerken, M. M. Amiji. Multi-functional polymeric nanoparticles for tumour-targeted drug delivery. Informa healthcare. 2006; 3 (2): 205-216 (http://informahealthcare.com/doi/abs/10.1517/17425247.3.2.205%20)
7. A review on FR accumulation http://www.sciencedirect.com/science/article/pii/S0169409X04000146
8. Manchester, M.; Steinmetz, N. F. In Viral nanoparticles: Tools for Material Science and Biomedicine; Pan Stanford Publishing: 2011; . http://www.crcnetbase.com/isbn/9789814267458
9. Literature on attaching folic acid to viruses via NHS esters: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2293326/pdf/nihms33395.pdf (Folic acid-mediated targeting of cowpea mosaic virus particles to tumor cells), but the actual chemical reactions for making NHS-folate, and then conjugate with liposomes (but we are going to use viral proteins): http://www.jbc.org/content/269/5/3198.full.pdf
10. Synthesizing Iron Oxide MNPs in lab: Laurent S, Forge D, Port M, Roch A, Robic C, Elst LV, Muller RN. Magnetic Iron Oxide Nanoparticles: Synthesis, Stabilization, Vectorization, Physicochemical Characterizations, and Biological Applications. Chemical Reviews. 2008. 108 (6): 2064-2110
11. Add more references as deem appropriate

Budget

http://www.bsdpostdoc.uchicago.edu/downloads/Seminars/GrantBudeting031706.pdf NIH proposal budget sample
http://law.niu.edu/osp/proposal/nihbudgetprep.shtml#detailed another example

• Personnel
• Travel (usually $.55/mile)
• Equipment and Supplies (BRAND Inverted Microscope, Gloves, Petri dishes, Pipette tips)
• Institutional overhead
• Subcontracts
• Other eligible costs
• Animal Purchase Costs: For maintaining and backcrossing lines: 60 lines x twenty C57BL/J females x

$11/female = $1,320/year for 120 mice.

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1. TEM at the Koch Institute: JEM-2100F TEM (In Belcher's paper, they used JEOL 2010F TEM)
2. CMSE TEM facilities: http://web.mit.edu/cmse/facilities/electron.shtml (we can use JEOL 2010 FEG Analytical Electron Microscope, also equipped for elemental analysis)

Feedback

11/29 from Professor Angela Belcher:

1. Look at Nature Nano Belcher lab paper
2. Need to do very good characterization of materials using TEM, elemental analysis, etc.