Era of Hope 2011 Investigator Vignette

Title: Surface Functionalized Nanoparticles for Proximity Activated Targeting of Dual-Mode Therapy for Multidrug Resistant Breast Cancer and Breast Cancer Metastases

Investigator: Craig L. Duvall, PhD, Vanderbilt University

The focus of our research is breast cancer metastatic disease. And we’re taking a nano-approach to improving drug delivery to treat metastatic breast cancer.

So we know that around a third of the breast cancer patients will eventually form some kind of dispersed or metastatic disease.

Many of the drug-targeting approaches that are currently used are based upon receptor-specific targeting cells for example, the HER2 or the estrogen receptor positive patients in general respond quite well to some classes of therapeutics. But what is called the triple negative breast cancer phenotype, we have really no good means to target that specific subset of breast cancers.

And so the type of targeting that we’re using here is dependent upon an external environmental factor or matrix metalloproteinase that actually activates this pro-drug into its active form. And so we have sort of a dual-pronged approach, one, where we’re trying to get better drug targeting specifically to metastases, to minimize nonspecific toxicities which we all know is a very predominant side effect for breast cancer therapy. And then the second part is actually to achieve a better bioactivity of the drugs that we deliver.

And so we actually propose to simultaneously deliver the chemotherapeutic agent while silencing what’s called P-glycoprotein which is a pump that’s known to impart multi-drug resistance in breast cancer cells. So we want to simultaneously deliver the drug and an agent that sensitizes the cells to that drug.

So the way that we’re doing this is we’re developing a nano-technological approach or nano-biomedical approach for this problem.

Here you can see the general concept of our nanoparticle design.

The targeting mechanism that we’re using here is called proximity-activated targeting. This does not target a specific receptor on the surface of the cells. And it actually targets the environment of the breast cancer. And so we know that one of the enzymes prevalent in breast cancer metastases is matrix metalloproteinase 7, MMP-7, and so we know that MMP-7 is involved in degradation of breast cancer extracellular matrix and an invasive and metastatic phenotype of those cells.

And so, the way our nanoparticle is designed is such that when it’s initially delivered it’s highly inert so there should be no nonspecific cytotoxic side effects from this carrier alone until it is activated in proximity to MMP-7. So that’s the proximity-activated targeting component of the drug. And once it’s activated, what you actually get is a shedding of this outer layer of the nanoparticle. So you can hear—you can see this layering effect of red, blue, green, and yellow in the nanoparticle. MMP-7 acts on this little green linker which is cleavable by that enzyme’s activity. When it cleaves off this outer shell it essentially activates the nanoparticle to be taken in by the cells. You’ll get simultaneous silence of PGP, which makes the cancer cells more susceptible to the chemotherapeutic agent, while also simultaneously delivering the chemotherapeutic agent itself.

We find that these nanoparticles are actually pH responsive, which is a very important mechanism for intracellular delivery of the biologic short interfering RNA that is one of the really important components for the silencing of the PGP or the p-glycoprotein. And so it’s this pH responsiveness that allows us to effectively deliver the siRNA inside the cells. So these things are taken up by an endosomal pathway that has a lower pH than the extracellular environment. And it’s that lower pH that activates a second smart function of this nanoparticle to enable endosomal disruption and release of the siRNA into the cell.

So what you see here is just some dynamic light scattering, sizing data from these nanoparticles, so we see a physiologic pH; you can see it around 100 nanometers or slightly less. This is where you see this peak, so our nanoparticles again are around 100 nanometers. What we see is when we lower the pH to something that mimics that environment in the endosome; you see that the nanoparticles start to fall apart. And so that’s really important because that disassembly is actually mediating intercellular delivery of the drugs to the inside of the cell. And then finally, this is really the punch line that we have at this point in our research…

So what we’ve done in this very preliminary experiment is we’ve looked at delivery of fluorescent-labeled siRNA to breast cancer cells. We’ve treated these—the full nanoparticle. What you can see here is the blue line. The red line is a no-treatment or negative control.

And so what you see is the no-treatment as you’d expect, there’s no fluorescence in these cells. When we deliver the charred shielded mice cell or this we do get some degree of uptake, but however when we pre-treat it with MMP-7 we get significantly more uptake. So you can see that in the orange line here, as you see this—this curve shifting to the right. So that’s indicating that we are getting significantly better uptake by the breast cancer cells after the proximity-activated targeting mechanism is enabled in these—in these nanoparticulate carriers.

So again a very promising result and we really need to now bring these things together and do some more optimizations, and then I think we’ll be ready to take this into some—some preclinical models and really test our—test our mettle so to speak.

This is—this is definitely a high-risk, high-reward project, which is one of the best things about the BCRP is they’re really willing to take—take a chance on a new investigator, on an as-yet unproven idea in hopes of really coming upon that really next transformative discovery. And so this—this project really fits that bill quite well.