Helen He, Alice Chen WF Pink Research Proposal

Research Proposal

Helen He and Alice Chen

General idea of project: HDL nanoparticles used as a strategy for B cell lymphoma inhibition and its modification for other therapeutic uses (inspired by Yang, Shuo, Marina G. Damiano, Heng Zhang, Sushant Tripathy, Andrea J. Luthi, Jonathan S. Rink, Andrey V. Ugolkov, Amareshwar T. K. Singh, Sandeep S. Dave, Leo I. Gordon, and C. Shad Thaxton, Biomimetic, synthetic HDL nanostructures for lymphoma, PNAS (2013).)

• Nanoparticle enabled therapeutic approach to B-cell lymphoma using synthetic high density lipoprotein particles (HDL-NPs)

• NDL-NPs were synthesized with gold nanoparticle template to control conjugate size and ensure spherical shape of the particles. Gold template also promotes cellular cholesterol efflux and limits cholesterol delivery

• NDL-NPs target scavenger receptor type B1, a high affinity HDL receptor expressed by lymphoma cells. It binds to B1 and starves the cells of cholesterol, which induces apoptosis. It was shown to inhibit B-cell lymphoma growth in mice.

Motivation

• 70,000 new cases of lymphoma each year, B-cell lymphomas constitute of 90% of these cases. ~18,940 deaths annually

• Recent studies show that enhanced uptake of cholesterol through HDL carriers could result in lymphoma cell proliferation and the inhibition of cholesterol ester formation was shown to inhibit cell growth.

• HDL is generally biocompatible and could present a way to treat cancer without the risk of bio-incompatibility

Project Overview: Researchers used synthetic HDLs, which are cholesterol transporters, to bind to SR-B1 receptors on cancer cells to starve them and eventually kill them. In our proposed study, we will:

1. Test the nanoparticles for their toxicity, specificity to cancer cells, biodegradability and other important qualities (the paper does fairly complete tests, but we would perform more complete and comprehensive tests on these nanoparticles)

2. Look for other potential uses for these nanoparticles

3. Ways to modify the nanoparticles to enhance performance and for use in other areas (ex. Drug delivery)

Background

• HDLs: natural nanoparticles that solubilize and transport cholesterol; inverse correlation between circulating blood HDL and cardiovascular disease onset

• SR-B1: high affinity receptor that HDLs bind to and mediates cholesterol uptake and efflux. Binding also facilitates movement of free cholesterols from the cell and the particle. Other cancers use cholesterol to maintain cell membrane integrity and promote proliferation.

• Researchers in this paper developed a biomimetic spherical nanoparticle, HDL-NP, with surface chemical properties similar to natural HDL, particularly the ability to sequester cholesterol

     o 	5nm diameter gold nanoparticle was used as template to assemble surface chemical components of natural HDLs (ex. Phospholipids, apolipoprotein)
     
     o 	Gold occupies the place in natural HDLs that would be occupied by esterified cholesterol, limiting HDL-NP’s ability to deliver cholesterol

• Their hypothesis: Cancer cells could be targeted by HDL-NPs through SR-B1 binding to prevent cholesterol delivery to the cancer cells that need them

• Data shows that HDL-NPs are selectively toxic to B-cell lymphoma cells in vitro and in vivo through biomimetic binding of SR-B1

• B-cells with lymphoma have 9-16x higher expression of SR-B1 than healthy B-cells. They are expressed in multiple B-cell lymphoma cell lines, but not in normal human lymphocytes.

• MTS: colorimetric assay where magnitude of absorbance is proportional to cell viability

• Treatment of cells with normal HDL increased absorbance for B-lymphoma cells, but treatment with HDL-NPs resulted in dose dependent decrease in absorbance in cells

      o	HDL-NPs reduced viability of B lymphoma cells but didn’t affect absorbance of other cells. 

• Using annexin V and propidium iodide cell labeling and flow cytometry, HDL-NPs induced dose and time dependent apoptosis in B cell lymphoma cell lines

• Toxicity of HDL-NPs to normal hepatocytes and macrophages: No apoptosis in hematocytes and lymphocytes after exposure to HDL-NPs (10nM, a dose toxic to B lymphocyte cells)

• • • Paper performed some other tests with testing cholesterol flux; this will also be incorporated into our final wiki/presentation

Testing and Modifying HDL-NPs

• We will test for toxicity of HDL-NPs to normal cells. Gold is tested to be biocompatible, but we could perform viability tests to make sure that applying doses, including therapeutic doses, of this

compound does not decrease the viability of normal cells.

• Biocompatibility tests, biodegradability tests, specificity, etc.

• SR-B1 receptors are specifically expressed in cancer cells and liver cells. Although the study says that it should be specific to cancer cells, it could also potentially affect the cells in the liver.

      o	We would do an immunoblotting/gene expression check to look at SR-B1 expression in liver cells. Depending on the amount, we would see if there are any toxic side effects of NDL-NPs to liver cells.

• In terms of drug delivery, how can we divert these nanoparticles away from liver cells and direct them towards cancer cells? Potentially modifying HDL-NPs in a way that will increase specificity. [1]

• SR-B1 receptors have been found in other cancers besides lymphoma, specifically in breast cancer. We can do similar tests that the paper does to see if the nanoparticles have the same effect.

• How can we increase the efficiency of targeting cancer cells?

Other potential health benefits of HDL-NPs

• By limiting uptake of cholesterol into the cell, this strategy would increase blood HDL levels, so it is important to measure whether this would have health side effects and see if it reduces risk of coronary heart disease, etc.

      o	We can do in vivo studies on mice by giving them high cholesterol diet and injecting these nanoparticles. We can then dissect the mouse and look at whether plaques formed in the arteries. 

      o	Can this strategy also be used as a method to increase the concentration of HDLs in the blood to get rid of LDLs? Can this potentially be a treatment for high cholesterol?

HDL-NPs used for drug delivery

• We can potentially use this strategy as a method for drug delivery. Instead of a gold nanoparticle or in addition to the gold nanoparticle, we can put anti-cancer drugs in the middle and deliver it with the HDL exterior.

      o	This allows for increased targeting for drugs and it will delivery them directly to the cancer cells, preventing any toxic effects to normal cells. 

      o	This method of delivery is useful in delivering drugs that are easily biodegradable by encapsulating them into a HDL particle.

References

Yang, Shuo, Marina G. Damiano, Heng Zhang, Sushant Tripathy, Andrea J. Luthi, Jonathan S. Rink, Andrey V. Ugolkov, Amareshwar T. K. Singh, Sandeep S. Dave, Leo I. Gordon, and C. Shad Thaxton, Biomimetic, synthetic HDL nanostructures for lymphoma, PNAS (2013).

Mian MK Shahzad, Lingegowda S Mangala, Hee Dong Han, Chunhua Lu, Justin Bottsford-Miller, Masato Nishimura, Edna M Mora, Jeong-Won Lee, Rebecca L Stone, Chad V Pecot, Duangmani Thanapprapasr, Ju-Won Roh, Puja Gaur, Maya P Nair, Yun-Yong Park, Nirupama Sabnis, Michael T Deavers, Ju-Seog Lee, Lee M Ellis, Gabriel Lopez-Berestein, Walter J McConathy, Laszlo Prokai, Andras G Lacko, Anil K Sood, Targeted Delivery of Small Interfering RNA Using Reconstituted High-Density Lipoprotein Nanoparticles, Neoplasia. 2011 April; 13(4): 309–319.