Identifying a protein fingerprint for prostate cancer is giving Professor Christopher Landry hope that an effective diagnostic blood test can be developed for the disease.
Landry, chair of the UVM Department of Chemistry, recently co-authored a study, “Spatial Mapping of Protein Adsorption on Mesoporous Silica Nanoparticles by Stochastic Optical Reconstruction Microscopy” in the Journal of the American Chemical Society.
Prepared with UVM alumnus Alden M. Clemments and Dr. Pablo Botella of the Institute for Chemical Technology in Valencia, Spain, the research explains how Stochastic Optical Reconstruction Microscopy (STORM) can be used to develop a quantitative method to study the penetration of proteins within porous silica nanoparticles.
The model presented in the study describes a technique for both imaging and analyzing protein and molecular adsorption in a porous material. An important application of STORM is that it may allow a time-resolved study of protein adsorption to be performed, providing important information about the development of the protein “corona,” the shell of adsorbed proteins that is acquired by a nanoparticle after it is exposed to a protein-containing solution.
The STORM technique was invented in 2006 at Harvard and now used by a handful of universities, including the Larner College of Medicine at UVM. By using this technique, researchers can observe details of nanoscale objects that couldn’t be resolved before. STORM provides three-dimensional, multicolor fluorescence imaging of cells and tissues with sub-diffraction-limit resolution.
Understanding the Protein Corona
While many studies have involved the identification and quantification of the proteins in the corona, very few have investigated the spatial mapping and orientations of the adsorbed proteins. Of those studies, only dense materials have been used, imposing limits on our understanding of how porous materials interact with biological environments.
Landry explains that scientists will often modify the surfaces of nanoparticles with two molecules: a polymer to help nanoparticles penetrate cells, and an antibody to target a protein expressed on the membrane of a specific cell.
“There’s a lot of literature about what happens when the polymer is not present. It turns out that the polymers normally used on the outside completely prevent proteins from absorbing,” Landry says. “Without the polymer, as soon as you put any nanoparticle into a solution that contains proteins, like blood or serum, it immediately acquires a corona. The proteins then prevent the antibody from seeing the receptor on the cell it needs to see.” While Landry says he had success targeting cells with this approach, he and his colleagues set out to learn more about the protein corona.
The protein corona is a dynamic layer in which the arrangement and amount of protein are changeable according to the conditions of physicochemical and biological interaction. It’s divided into “hard corona” and “soft corona.”
In their research, the goal was to understand which proteins are in that corona and what the implications for diagnosis and treatment might be. The technique used to analyze the proteins composing the corona is called Liquid Chromatography-Mass Spectrometry, which identified the proteins by their masses. One key result of their previous work was that porous nanoparticles are particularly good at adsorbing small proteins. Landry hypothesized that is because those proteins fit well into the pores, so the size of the proteins in the corona should be related to the size of the pores.
In the STORM experiments, Landry attached fluorescent dyes to proteins of several sizes and studied the penetration depth of the proteins within porous nanoparticles. “Observing protein adsorption at this resolution is only possible using the STORM technique,” Landry says. He found that indeed, smaller proteins penetrated throughout the particles, while medium and large proteins were present only on the outside and as a result were not present in large amounts.
Ultimately, the adsorption process is likely more complex when multiple proteins are present. To model this process, the researchers also studied protein adsorption from mixtures of two proteins. They found that the smaller protein, particularly proteins that fit well within the pores, always won this competition. Landry says that small proteins quickly fill the pores, limiting the amount of medium and large proteins adsorbed. “This means that the protein corona on a porous particle is different from the corona on a dense particle of the same type,” Landry says.
Landry uses albumin as an example. Albumin, the most prevalent protein of the blood plasma, makes up about 60 percent of the protein in anyone’s blood and is a medium weight protein. “If you did a normal blood draw, albumin would swamp out all the other protein in the sample,” Landry explains. “That is why porous nanoparticle is important because you can screen out the proteins you don’t care about and get a “fingerprint” of the small proteins in a patient’s serum.”
Diagnostic Testing for Prostate Cancer
Prostate cancer is the third leading cause of cancer death in American men. About 1 man in 7 will be diagnosed with prostate cancer during his lifetime. Until recently, many individuals were advised to undergo screening for prostate-specific antigen (PSA), an enzyme produced by the prostate gland. The test, which was developed in the 1980s, looks for elevated PSA levels in the blood, which can indicate cancerous cells in the prostate gland.
The problem was that the PSA does not differentiate between a benign cancer and the malignant and aggressive cancer that requires invasive treatment. Inflammation and other problems unrelated to cancer can also elevate PSA levels. When a PSA test correctly detects cancer, it is often slow-growing or in the earliest stages that it would never have caused death or illness.
Landry says by mapping a protein fingerprint for prostate cancer, researchers can develop a better diagnostic test instead of the unreliable PSA.
In 2018, Landry will develop a protocol to recruit prostate cancer patients at the UVM Medical Center and, with Botella, at the Institute for Chemical Technology to start to their analysis.
“Right now, there is no good marker for detecting prostate cancer with blood. Maybe our technique will be able to give not just one protein that is problematic, but a host of proteins,” Landry says. “You could take serum from a patient you suspect may have prostate cancer, adsorb a protein corona from their serum onto porous nanoparticles, and can compare that against the coronas from control patients.”
Landry is optimistic about developing a successful diagnostic test for prostate cancer with Botella, whom he met an international conference in 2010 about porous materials. The two researchers later worked together in Spain while Landry was on a Fulbright Senior Research Fellowship.
“We are both moving forward, and we believe it’s an important research project,” Landry says. “We’re two researchers from two different hospitals in two different countries with different populations. That alone will add lot of robustness to the statistics in our model and improve its accuracy.”