Volume 3, Issue 1 
1st Quarter, 2008


Imaging and Treatment of Cancer through Combinations of Nanoparticles and Hormones

Carola Leuschner, Ph.D.

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There are a lot of cancers which have specifically over-expressed these receptors, and these cancers could be targeted with our approach, which I will explain. The luteinizing hormone-releasing hormone receptor is a receptor that binds to LHRH, which is a decapeptide, ten amino acids long, and naturally released from the brain and regulates reproductive function.  However, cancer cells also over-express LHRH receptors, therefore, can be targeted.

We have synthesized and characterized super-paramagnetic iron oxide nanoparticles. These nanoparticles are the basis for contrast agents for the magnetic resonance imaging. Superparamagnetic iron oxide nanoparticles are produced through adding chemicals to iron chloride.  These nanoparticles have been characterized and it has been shown that they are not toxic, super-paramagnetic, and through physical and chemical measurements, these particles have been thoroughly analyzed in Dr. Challa Kumar's lab [1].


Image 3

In summary, it is important that these particles are super-paramagnetic and, therefore, they increase the contrast in magnetic resonance imaging.

These particles, in order to use them as specific targeting molecules or entities for magnetic resonance imaging, have to be connected with a targeting agent which is, in this case, the luteinizing hormone-releasing hormone. That is binding directly to the cancer cells, that is called the LHRH.

Here you see in this panel, the LHRH iron oxide nanoparticle that has (in the peptide-bound stage), a small reduction in electronic saturation, and it changes its features.  The ion oxide nanoparticle is positively charged, whereas the LHRH-SPION is almost neutral, which is a very important finding.

If we are looking at the accumulation of iron oxide nanoparticles, there are LHRH conjugated nanoparticles in breast cancer cells, which over express LHRH receptors.


Image 4

You can already see by looking at the different shades of blue, which in this case is an iron detection, there's free iron oxide nanoparticles. It's a fairly light blue.  There is much darker blue, which means much more iron from the nanoparticles are inside the cells in the case when we have specifically targeted the LHRH-SPIONs to the cancer cells.

That is a direct receptor-mediated endocytosis [2] has been proven by co-incubating these cancer cells with the ligand LHRH, blocking the receptor for LHRH and basically preventing the LHRH-SPIONs to enter into the cell, which shows in the lighter blue stain at the lower row of the panel.

Another important part is that we do not want nanoparticles to be taken up by macrophages [3].  Depending on charge and surface, and also size, macrophages take up injected nanoparticles, which would basically be removed from the circulation and therefore, would be unable to reach the target organ or the target cells.

If you look here at the free SPIONs, the SPIONs are very easily taken up by macrophages.  Looking at the conjugated LHRH-SPIONs, we have very poor uptake, meaning we have a very extended circulation time and increase the chance to deliver the nanoparticles to the cancer cells, to their target.

image 5
Image 5

The next part of preclinical development involves in vivo experiments.  In most in vivo experiments we use a cancer xenograft mouse model in which we have implanted the human breast cancer cell line. These cells also proliferate well, so we can always find the metastases in each organ of the mouse. The mice were intravenously injected with the iron oxide nanoparticles.

After twenty hours, the mice are necropsied; each individual organ is investigated.  In histology procedures iron is detected by a prussian blue staining. We also homogenized each individual organ in order to determine how many tumor cell are in each organ and how much iron is detectable in those tumor cells.  

Looking at the relative iron distribution in mice after injection of free iron oxide nanoparticles (SPIONs) and conjugated iron oxide nanoparticles (LHRH-SPIONs), if you look at the tumor, we have a very high accumulation of LHRH-SPIONs in the tumor itself, and  fairly low accumulation of iron oxide nanoparticles (SPIONs).

Compared to injection of just the free iron oxide particle, we can show that specific targeting through LHRH-SPIONs is certainly improving the accumulation of the contrast agent.

Now if we have a look at the lungs, we have a fairly high amount of LHRH-SPIONs accumulating in the lung tissue.  Looking at free iron oxide nanoparticles, there is a very
small amount of iron detectable in the lung tissue.

image 6
Image 6

Both of these animal groups had lung metastases.  Looking at the lung tissue in animals without any tumor, there is absolutely no accumulation of LHRH-SPIONs. Therefore, we have again, a very specific accumulation of nanoparticles conjugated to LHRH, which are directly related to the extent of metastatic disease.

 

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Footnotes

1. Challa S. S. R. Kumar, Ph.D. - 2002 - Present   Group Leader, Nanofabrication, Center for Advanced Microstructures and Devices, Louisiana State University, Baton Rouge, LA.
http://www.camd.lsu.edu/staff/ckumar.html  January 3, 2008 11:06AM EST

2. Apoptosis – n. A natural process of self–destruction in certain cells that is determined by the genes and can be initiated by a stimulus or by removal of a repressor agent. Also called programmed cell death.
The American Heritage STEDMAN’S Medical Dictionary. Boston, New York: Houghton Mifflin Company, 2004: 58.

3. Macrophage – n. Any of the large phagocytic cells found in the reticuloendothelial system.
The American Heritage STEDMAN’S Medical Dictionary. Boston, New York: Houghton Mifflin Company, 2004: 478.

 

 

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