XPCS proves to be a powerful tool for nanoparticles analysis in complex biological media

Article published by CNPEM researchers used the Cateretê beamline to analyze the corona formation process in silica nanoparticles

An article published by CNPEM researchers was featured on the Nano Letters scientific journal’s cover and explores how the X-ray Photon Correlation Spectroscopy (XPCS) technique can distinguish protein corona formation from nanoparticle aggregation in complex biological media.

The innovative work, carried out at Sirius, expands analysis capacity in nanomedicine and highlights the XPCS potential to characterize nanoparticle interactions in biological environments in real time, providing a valuable resource for nanobiotechnology research and new biomedical materials development. 

The innovative nanoparticles applications in biomedicine

Nanoparticles are tiny structures, with dimensions generally between 1 and 100 nanometers. Due to its size, they can interact with cells, proteins and molecules in a highly precise way, which allows driven delivery of medicines and therapeutic agents. This allows, for example, for cancer treatments to be more effective, by releasing drugs directly into tumor cells, minimizing side effects on healthy tissues.

Furthermore, nanoparticles can be designed for responding to specific stimuli, such as pH, temperature or biological signs, allowing a controlled release of medicines only when necessary.

In the diagnosis area, nanoparticles offer new ways ​​to prematurely detect diseases. They can be linked to specific biomarkers that bind to molecular targets, making it easier to identify cancerous cells or the presence of viruses and bacteria, for example. 

The interaction between nanoparticles and proteins in biological systems

These applications, however, are conditioned to a predictable behavior of these nanoparticles in complex biological systems. In some cases, by coming into contact with biological fluids, such as blood, a protein coating can be formed around nanoparticles, a phenomenon known in biomedicine by the English term “protein corona”. 

This happens because nanoparticles attract proteins present in the biological environment, forming a “corona” or “crown” around its surface. The formation of this protein corona strongly influences how do nanoparticles interact with cells and tissues in the organism, which can affect its efficacy and safety in medical applications, such as drug therapies, diagnostics, and vaccine development. 

For these reasons, studying the protein corona formation and characteristics is crucial for the development of nanoparticles that are safe and effective for biomedical use. 

Limitations of optical techniques for analyzing these samples

Optical techniques, such as Fluorescence Correlation Spectroscopy (FCS) and Dynamic Light Scattering (DLS), face significant limitations when analyzing nanoparticles in complex biological environments. One of the main limitations is the need for diluted and transparent samples, which makes it difficult to analyze nanoparticles in highly concentrated media, such as blood and body fluids. In complex media, particles and biomolecules can interfere with light propagation, causing spreading and excessive absorption, which compromises the accuracy of nanoparticle size and concentration measurements. 

Furthermore, optical techniques rely on nanoparticle specific properties, which limits its application to particles that present these specific characteristics. For example, in the FCS case, it is necessary that nanoparticles show fluorescence in order to be detected, restricting the technique’s use to fluorescent materials. This is one of the limitations that makes optical techniques less suitable to characterize nanoparticles under realistic conditions and in real time, as in unprocessed samples of biological fluids. 

XPCS: A powerful technique for nanoparticles analysis in complex media

The X-ray Photon Correlation Spectroscopy (XPCS) technique appears as a good alternative by offering significant advantages for nanoparticle analysis in complex biological environments, overcoming many of the optical techniques limitations. One of its main advantages is the ability to analyze highly concentrated and complex samples, such as blood and other bodily fluids, without need for dilution or transparency.

The use of coherent X-rays allows particles direct analysis in its native conditions, minimizing interferences that arise with visible light-based methods. Recently, this capacity was explored in a study by Federal University of Rio de Janeiro (UFRJ), where researchers used XPCS at the Cateretê beamline to investigate the biophysical mechanisms of the prion protein aggregation process, whose results were published in the Science Advances journal. 

Furthermore, XPCS does not require nanoparticles to have fluorescent characteristics or specific optical properties, which broadens its application scope to a variety of materials and nanoparticle sizes. The technique enables the particle dynamics observation, such as Brownian motion, aggregate formation and structural changes, In real time and with high accuracy. This is particularly useful for distinguishing critical phenomena, such as protein corona formation around nanoparticles and the aggregation in complex media, offering valuable insights into the biomedical nanomaterials development.   

XPCS Capabilities on Sirius

In the study published in the Nano Letters journal, the researchers used the Cateretê beamline to perform XPCS measurements with silica nanoparticles (SiO₂) exposed to different biological environments, from simple solutions to complex media containing proteins such as bovine serum albumin (BSA) and fetal bovine serum (FBS).

This technique made it possible to observe how nanoparticles behaved in each environment, including monitoring of Brownian motions, aggregate formation and structural alterations. Measurements were performed on samples with different nanoparticle sizes, functionalized and non-functionalized with polyethylene glycol (PEG), analyzing how size and functionalization influence their interactions in the biological environment.

Non-functionalized nanoparticles are those that do not have any modification or additional coating on their surface. They are in their “pure” form, without the addition of chemical groups, specific molecules or polymers that can alter its properties or increase its compatibility in certain environments. 

Nanoparticle functionalization, on the other hand, involves the addition of molecular groups, such as polyethylene glycol (PEG), to provide greater stability, biocompatibility or to direct them to a specific target within biological systems. Non-functionalized nanoparticles generally interact more directly with their environment, which can lead to phenomena such as protein corona formation or aggregation, especially in complex biological media. 

The results of the measurements carried out at Sirius showed that non-functionalized nanoparticles tend to form a protein corona by interacting with the BSA-containing media and,  in more complex environments such as FBS, end up aggregating (diagram [f] of the previous figure). In contrast, PEG-functionalized nanoparticles maintained their stability, without forming protein corona or aggregates, due to the hydration layer around the particles, which prevents the adhesion of proteins (diagram [g] of the previous figure). 

“This distinction between protein corona and aggregation, made possible by the use of XPCS, is a significant advance for the characterization of nanoparticles under realistic biological conditions, reinforcing the potential of this technique to improve understanding of fundamental interactions in nanomedicine”, says Mateus Cardoso, chief of the Soft and Biological Matter Division and one of the article’s authors.

The research carried out at Sirius opens the way for a deeper understanding of interactions among nanoparticles and biomolecules in complex environments, demonstrating XPCS’s potential to become a powerful analysis tool in this field and fostering the development of advanced nanomaterials, more effectively, for use in medical therapies.

Find out more about the Cateretê beamline

About CNPEM

The Brazilian Center for Research in Energy and Materials (CNPEM) is home to a state-of-the-art, multi-user and multidisciplinary scientific environment and works on different fronts within the Brazilian National System for Science, Technology and Innovation. A social organization overseen by the Ministry of Science, Technology and Innovation (MCTI), CNPEM is driven by research that impacts the areas of health, energy, renewable materials, and sustainability. It is responsible for Sirius, the largest assembly of scientific equipment constructed in the country, and is currently constructing Project Orion, a laboratory complex for advanced pathogen research. Highly specialized science and engineering teams, sophisticated infrastructure open to the scientific community, strategic lines of investigation, innovative projects involving the productive sector, and training for researchers and students are the pillars of this institution that is unique in Brazil and able to serve as a bridge between knowledge and innovation. It is responsible for operating the Brazilian Synchrotron Light (LNLS), Biosciences (LNBio), Nanotechnology (LNNano), and Biorenewables (LNBR) National Laboratories, as well as the Ilum School of Science, which offers a bachelor’s degree program in science and technology with support from the Ministry of Education (MEC).