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Nanoparticle Enrichment Could Enhance Plasma Protein Biomarker Discovery

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NEW YORK (GenomeWeb) – Researchers at the University of Manchester are using nanoparticles to aid in protein biomarker discovery.

In a pair of recent papers, one published last month in Advanced Materials and the other in October in Biomaterials, the scientists demonstrated that circulating nanoparticles can enrich low molecular weight, low abundance plasma proteins that would typically go undetected by standard proteomic workflows.

This suggests these nanoparticles could be useful tools in protein biomarker discovery, said Marilena Hadjidemetriou, a postdoctoral fellow at the University of Manchester and first author on both studies. She and her colleagues are currently testing various types of nanoparticles to determine which work best in particular biofluids or in combination with each other.

The project originated in questions around the plasma proteins that collect on the surface of nanoparticles used in drug delivery. Nanoparticles like liposomes are in some cases used to encapsulate drugs, which can improve their targeting and uptake and extend the length of time they circulate in a patient's blood. Upon injection into the patient bloodstream, these nanoparticles accumulate circulating proteins on their surface, forming what is termed the "protein corona."

These accumulated proteins can interfere with the nanoparticle-drug complex's desired effects, and Hadjidemetriou noted that research in the field has traditionally focused on trying to reduce the amount of protein accumulation.

However, analysis of the contents of these coronas indicates that many of proteins present are low abundance and low molecular weight species that are difficult to detect in plasma, which means the nanoparticles' protein corona could be a potential source of disease biomarkers.

Blood is one of the most commonly investigated biofluids in biomarker research, but it is a challenging sample for proteomics researchers to work with due to its large dynamic range and the fact that a handful of proteins like albumin and immunoglobulins constitute the vast majority of blood protein content and therefore mask the presence of lower abundance proteins that could provide information on patient health and disease states.

The field has developed a variety of approaches to address this problem, such as immuno-depletion of high abundance proteins or extensive chromatographic fractionation prior to mass spec analysis. These methods have their downsides, though. Immuno-depletion is expensive and can remove lower abundance proteins bound to the highabundance species being depleted. Fractionation is time consuming, making it difficult to run large numbers of samples.

Nanoparticle-based enrichment potentially offers researchers a way to access low abundance plasma proteins without using depletion or fractionation methods.

This enrichment can take place either in vivo, in which nanoparticles are injected into the subject's bloodstream and then collected and analyzed, or ex vivo, in which nanoparticles are incubated with blood that has been drawn from the subject and then analyzed.

Hadjidemetriou said that analysis of nanoparticle protein coronas was done using ex vivo analysis until a 2016 study she and her colleagues published in ACS Nano.

"Everything in the literature until then was done ex vivo," she said. "So we decided to develop a protocol to do this in vivo where we injected nanoparticles in mice and then recovered them and purified them from the unbound proteins and only characterized what was bound to the surface of the nanoparticles."

In that work, the researchers studied the corona that formed both ex vivo and in vivo on Doxil, a liposome-doxorubicin complex that is sold by Janssen for the treatment various cancers. They found that while both the ex vivo and in vivo coronas contained roughly the same amount of protein, the in vivo corona contained a significantly wider variety of proteins.

The Manchester team followed this study with the Advanced Materials study published last month in which they looked at in vivo corona formation in humans, analyzing the corona formed on Doxil in six advanced ovarian cancer patients. As in the mouse study, the researchers found that the in vivo corona had a richer variety of proteins than ex vivo coronas. They also identified proteins in the Doxil corona that were not detected by mass spec analysis of patient blood samples.

"You cannot completely mimic what is going on in vivo when you incubate ex vivo, Hadjidemetriou said, explaining why the in vivo corona might contain more proteins than the ex vivo corona. "You have the blood flow dynamics, you have a more dynamic environment, and there is a response to the nanoparticle injection, as well. So I think that is why we are getting a molecularly richer proteome."

She said that in their biomarker discovery work, the researchers plan to do initial discovery work in mouse models analyzing in vivo coronas but then move to ex vivo corona analysis in humans, which will allow them to use nanoparticles that have not been approved for injection into humans.

She acknowledged that the greater richness of the in vivo corona proteome means the researchers may identify some markers in their mouse models that they are not able to identify in their ex vivo human samples, but she said that in such cases they could move to targeted assays like ELISAs to measure those proteins.

The Manchester team tested the approach in the October Biomaterials study using mass spec to identify liposome corona proteins that could distinguish between healthy mice and those with melanoma or lung carcinoma. In this analysis, they found a total of 384 proteins that were differentially expressed in the corona of healthy versus tumor-bearing mice. A mass spec analysis of conventional blood samples taken from the same mice found only six differentially expressed proteins, suggesting the potential usefulness of nanoparticle-based enrichment in protein biomarker discovery.

Hadjidemetriou noted, though, that these efforts are still in their infancy. Currently, she said, the researchers are focused on developing nanoparticles with different sizes, shapes, and functionalizations and exploring how those particles behave in different biofluids and how they might be used in combination to further enhance protein biomarker discovery.

She and her colleagues have patented their nanoparticle technology, she said, but she declined to say whether they had any specific commercialization plans.

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