By Deborah Borfitz
November 4, 2021 | Researchers in Denmark are pursuing development of a diagnostic approach pairing a liquid biopsy test with nanoparticle tracking analysis to measure disease severity in patients with autoimmune diseases. While at least five years away from making an appearance in hospital labs, their patented Nanoscale Immunoactive Protein Quantification (NIP-Q) technique has already been used to discover patients with systemic lupus erythematosus (SLE) have a higher concentration of certain DNA–protein complexes in their blood, according to postdoc Kristian Juul-Madsen, Ph.D., in the department of biomedicine at Aarhus University.
The spread between patients, from extremely low to significantly elevated levels of the nanoparticles, also appears to correlate with disease severity, he adds. The number of particles present in samples closely parallel scores produced by the global Systemic Lupus Erythematosus Disease Activity Index.
Moreover, the size of the particles—on the order of 200 to 300 nanometers (nm), many times the size of most proteins in the blood—is connected to endothelial inflammation, among the most important causes of morbidities in lupus, says Thomas Vorup-Jensen, Ph.D., biomedicine professor at Aarhus University. Particle size can consequently serve as an additional disease biomarker.
These were key findings of a study recently published in PNAS (DOI: 10.1073/pnas.2106647118), highlighting the role of protein size as an explanation for autoimmune diseases. NIP-Q was used to track and analyze nanoparticles formed by mannose-binding lectin (MBL), an oligomeric plasma protein that is part of the innate immune system, with DNA.
It was previously established that inflammatory diseases lead to the release of DNA in the blood and that the process can activate the immune system. MBL had also been identified as a promising marker of SLE and believed to activate the “complement” pathway protecting the body from the destructive effects of pathogens, Vorup-Jensen explains. But technical challenges have heretofore limited the application of nanotechnology to accurately detect and characterize MBL in blood plasma.
Owing to the size of the nanoparticles being tracked with NIP-Q, they follow the laws of physics, including their motion in a blood vessel, he continues. They therefore have convective-driven distribution in the bloodstream and can potentially end up in the vessel wall and create inflammation.
The physical size of the nanoparticles themselves also changes their distribution in the bloodstream, adds Juul-Madsen, further contributing to the formation of atherosclerotic plaques. “This is also part of our explanation for their [role in] the pathology of lupus.”
Road To The Clinic
Findings of the PNAS study are based on a relatively small cohort, he notes, and SLE is a clinically heterogeneous disease. “Even though we analyzed 40 patients with multiple different manifestations of the disease, we weren’t able to look at those that specifically correlated with this increase in particles in the blood.”
Whether the detected DNA-protein complexes might be associated with atherosclerosis, arthritis, or neurological symptoms remains an open question—but one he and Vorup-Jensen plan to address soon. Forthcoming studies with a larger patient cohort are intended to validate the marker against various clinical observations to make the measurements interpretable at the individual level.
That should also clear up the question about whether some level of the nanoparticles may be present in healthy people. But given their extreme size and direct ties to lupus, Vorup-Jensen says, they could well be a marker highly selective for inflammatory diseases.
“We have a small spread inside our control group that suggests there could be some background level in some individuals,” says Juul-Madsen. “This could be due to these people having a simple infection, with a turnover of immune cells that combine with cellular DNA to form these complexes, but not at the same rate as patients suffering from the disease.”
For NIP-Q to reach the clinic will require a larger, longitudinal study where it is shown to predict disease progression based on different treatments, he says. But for now, advanced training is required to employ the technique and interpret the results, its utility is envisioned primarily in the hospital setting.
NIP-Q is a completely novel technique developed at Aarhus University to measure the newly discovered large protein particles in the blood, says Juul-Madsen. Most critically, the process involves coupling antibodies that recognizes MBL to “quantum dots”—semiconductive metals with very bright fluorescent intensity—allowing the nanoparticles to be illuminated for up to several minutes while they get measured using NanoSight NS300 system of Malvern Panalytical.
When antibody conjugates bind to the protein particles, the researchers track their diffusion coefficient and the distribution pattern can then be plotted, he explains. “We do this on a single particle basis, which is different from what most people do [i.e., dynamic light scattering].”
“Quantum dots is one of the most powerful demonstrations of what nanoscience can be used for,” notes Vorup-Jensen. The wavelength of the light quantum dots emit is dependent on the particle size and, in this instance, they were particularly large (about 20 nm) with a “very distinct” fluorescent signal.
Fluorescent detection is useful when working with heterogenous samples and was the rationale for developing new protocols for serum preparation, and how to run and machine and do the analysis, Juul-Madsen says. The entire measurement process takes just five minutes, which is one of the advantages of the NIP-Q technique on a single-sample basis.
One of its disadvantages is, unlike ELISA, detection is still on a serial basis, he adds. “If you want to analyze 1,000 samples, then you have to do 1,000 measurements,” which would take about three and a half days of continuous measurement.
Analysis of protein-DNA complexes were validated using ultracentrifugation, which separates different classes of proteins in solution by their rate of sedimentation, says Juul-Madsen. “Analytical ultracentrifugation is able to help us quantify or characterize these particles we were looking at in the blood in a way our nanoparticle tracking analysis is not able to do.”
But unlike analytical ultracentrifugation, which is used mostly on purified proteins, nanoparticle tracking analysis can be done on complex blood samples, he adds. “It takes about 24 hours to do a single measurement using analytical ultracentrifugation, but you get some different data that can validate findings using the nanoparticle tracker.”
While Vorup-Jensen and Juul-Madsen invented the technology, the patent is held by Aarhus University and is specific to the application as a biomarker for disease severity in SLE as well as rheumatoid arthritis. Longer term, they expect that their NIP-Q technique will prove useful in detecting a multitude of inflammatory diseases.